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WO2018059581A1 - Sonde permettant la détection de génotypage du papillomavirus humain au moyen d'un kit, utilisation et technique efirm - Google Patents

Sonde permettant la détection de génotypage du papillomavirus humain au moyen d'un kit, utilisation et technique efirm Download PDF

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Publication number
WO2018059581A1
WO2018059581A1 PCT/CN2017/104829 CN2017104829W WO2018059581A1 WO 2018059581 A1 WO2018059581 A1 WO 2018059581A1 CN 2017104829 W CN2017104829 W CN 2017104829W WO 2018059581 A1 WO2018059581 A1 WO 2018059581A1
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Prior art keywords
probe
seq
capture probe
base sequence
hpv
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English (en)
Chinese (zh)
Inventor
廖玮
莫亚勤
林晓燕
张晨光
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Guangzhou Ezlife Sci & Tech Co Ltd
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Guangzhou Ezlife Sci & Tech Co Ltd
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Priority claimed from CN201610877670.0A external-priority patent/CN106282413B/zh
Priority claimed from CN201610877702.7A external-priority patent/CN106399589B/zh
Priority claimed from CN201610881088.1A external-priority patent/CN106367536A/zh
Application filed by Guangzhou Ezlife Sci & Tech Co Ltd filed Critical Guangzhou Ezlife Sci & Tech Co Ltd
Publication of WO2018059581A1 publication Critical patent/WO2018059581A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

Definitions

  • the invention enjoys the invention entitled "probe combination, kit and method for genotyping detection of HPV high-risk strain” submitted on September 30, 2016, the application number is 201610877670.0; the invention name is “an HPV virus based on EFIRM technology” Detected probe combination, kit and method” application number is 201610881088.1 and the invention name is "a probe combination, kit and method for virus genotyping detection", and three invention patent applications with application number 201610877702.7 Priority of the Chinese Utility Model Patent Application No. 201620769829.2, entitled “Detection Electrode Structure and Detection Orifice", submitted on December 26, 2016, the entire contents of the above four Chinese patent applications This is hereby incorporated by reference in its entirety for all purposes.
  • the invention relates to the field of gene detection technology, in particular to a probe kit, a kit and a method based on the EFIRM technology HPV virus detection.
  • Cervical cancer is one of the most common malignant tumors in the female genital tract.
  • HPV human papillomavirus
  • multiple infections are one of the important causes of cervical cancer, a worldwide study.
  • the results showed that the presence of high-risk HPV DNA was detected in 99.7% of cervical cancer patients.
  • HPVs there are more than 200 types of HPVs, including low-risk and high-risk types. Different types have different correlations with different diseases.
  • Low-risk HPV infection may cause genital warts and high-risk HPV infection is associated with cervical cancer and vaginal cancer.
  • there is still a lack of effective treatments for HPV so early detection and early prevention of cervical HPV is the key to blocking cancer.
  • HPV nucleic acid detection technology mainly includes hybridization capture method, PCR-fluorescence probe method, transcription-mediated nucleic acid amplification technology and PCR-hybridization method.
  • the early screening method for cervical cancer is to use Pap smear and improved TCT technology. These methods can only evaluate the incidence of cervical cancer from the perspective of cytological lesions, that is, it needs to wait until the cells infected with the virus begin to appear. It can be detected when there is a significant change, and the specificity and sensitivity are not ideal.
  • HPV gene detection has become more widely used as a screening method for cervical cancer.
  • HPV typing detection technology is mainly based on polymerase chain reaction, such as fluorescence quantification, reverse dot blot hybridization and gene chip technology.
  • HC2 is a nucleic acid hybridization detection method using microplate chemiluminescence for signal amplification, and is recognized as the gold standard for evaluating new HPV detection technology.
  • the HC2 detection method is expensive, and the method is cumbersome to operate, and it is easy to cross-contamination during operation and false positives occur.
  • a contribution of an aspect of the present invention is to provide a probe kit based on EFIRM technology HPV virus detection, which can detect genotype of virus in a sample to be tested, has strong specificity, high sensitivity and Low false positive rate, and the detection results are accurate and reliable.
  • the second aspect of the present invention is to provide a kit and a detection plate for detecting HPV virus based on EFIRM technology, which is capable of genotyping detection of a virus in a sample to be tested, which has strong specificity and high sensitivity. Sensitivity, and the detection results are accurate and reliable, the false positive rate is low, and the operation is simple and convenient.
  • the contribution of the three aspects of the present invention is to provide a method for genotyping detection of HPV virus based on EFIRM technology, which can detect genotype of virus in a sample to be tested, has strong specificity and high sensitivity, and is detected. The result is accurate and reliable, and the detection method is simple and easy to operate.
  • a probe kit for HPV virus genotyping detection characterized in that
  • each of the capture probes being used to bind a target sequence on a HPV viral genotype
  • the plurality of capture probes include: a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes; ,among them,
  • a first capture probe for detecting a HPV 16 subtype the base sequence of which is set forth in SEQ ID NO.
  • a second capture probe for detecting a HPV 18 subtype the base sequence of which is set forth in SEQ ID NO.
  • a third capture probe for detecting a HPV 31 subtype the base sequence of which is set forth in SEQ ID NO.
  • a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype the base sequence is shown in SEQ ID NO.
  • a fifth capture probe for detecting the HPV 35 subtype the base sequence of which is set forth in SEQ ID NO.
  • a sixth capture probe for detecting a subtype of HPV 39 the base sequence of which is set forth in SEQ ID NO.
  • a 7th capture probe for detecting the HPV 45 subtype the base sequence of which is set forth in SEQ ID NO.
  • An 8th capture probe for detecting a HPV 51 subtype the base sequence of which is set forth in SEQ ID NO.
  • a ninth capture probe for detecting a HPV 56 subtype the base sequence of which is set forth in SEQ ID NO.
  • a 10th capture probe for detecting a HPV 59 subtype the base sequence of which is set forth in SEQ ID NO.
  • An 11th capture probe for detecting the HPV 68 subtype the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
  • the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
  • the base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 10th capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe to which the 16th capture probe is ligated is shown in SEQ ID NO.
  • a probe kit for genotyping detection of high-risk HPV viruses characterized in that
  • each of the capture probes being used to bind a target sequence on an HPV viral genotype
  • a third capture probe for detecting a HPV 31 subtype the base sequence of which is set forth in SEQ ID NO.
  • a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype the base sequence is shown in SEQ ID NO.
  • a fifth capture probe for detecting the HPV 35 subtype the base sequence of which is set forth in SEQ ID NO.
  • a sixth capture probe for detecting a subtype of HPV 39 the base sequence of which is set forth in SEQ ID NO.
  • a 7th capture probe for detecting the HPV 45 subtype the base sequence of which is set forth in SEQ ID NO.
  • An 8th capture probe for detecting a HPV 51 subtype the base sequence of which is set forth in SEQ ID NO.
  • a ninth capture probe for detecting a HPV 56 subtype the base sequence of which is set forth in SEQ ID NO.
  • the 10th capture probe was used to detect the HPV 59 subtype, and the base sequence is shown in SEQ ID NO.
  • it also includes
  • a first capture probe for detecting the HPV 16 subtype the base sequence is shown in SEQ ID NO. 1; and a second capture probe for detecting the HPV 18 subtype, the base sequence is SEQ ID NO. Shown.
  • the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
  • the base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the third capture probe or the fifth capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the fourth capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the sixth capture probe or the 11th capture probe capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the seventh capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the eighth capture probe or the ninth capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 10th capture probe is shown in SEQ ID NO.
  • a probe kit for high-risk HPV virus genotyping detection comprising at least one selected from the group consisting of 12th to 16th capture probes, each of which is used to bind an HPV virus a target sequence on a genotype;
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the 16th capture probe was used to detect the HPV 82 subtype, and the base sequence is shown in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO. 1; and a second capture probe for detecting the HPV 18 subtype, the base sequence is as SEQ ID NO. 3 is shown.
  • the method further comprises a plurality of detection probes that cooperate with the capture probe and can bind the corresponding target sequence, as follows:
  • the base sequence of the detection probe complexed with the first capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the second capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 12th capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 13th capture probe or the 14th capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 15th capture probe is as shown in SEQ ID NO.
  • the base sequence of the detection probe complexed with the 16th capture probe is shown in SEQ ID NO.
  • a probe kit for HPV virus genotyping detection characterized in that
  • a first capture probe for detecting a HPV 16 subtype the base sequence of which is set forth in SEQ ID NO.
  • the detection probe for detecting type 16 HPV is further included, and the base sequence is as shown in SEQ ID NO.
  • the detection probe of type 18 HPV was detected, and the base sequence is shown in SEQ ID NO.
  • the 3' or 5' end of the detection probe is labeled with an affinity for binding to a catalytic enzyme for catalyzing the chemical reaction of the substrate to form a stream of electrons.
  • a kit for detecting HPV virus based on EFIRM technology characterized in that it comprises a probe in any of the above probe kits.
  • the kit further comprises a fixture for fixing the capture probe to a detection orifice plate, the fixture comprising a conductive polymer and an ionic compound;
  • the conductive polymer is any one selected from the group consisting of pyrrole, aniline and thiophene;
  • the ionic compound is any one selected from the group consisting of sodium chloride and potassium chloride.
  • the kit further comprises the catalytic enzyme, the catalytic enzyme is a labeled horseradish peroxidase or alkaline phosphatase, and the label is used for binding to the affinity,
  • the marker is any one of a digoxin antibody, a fluorescein isothiocyanate antibody, and streptavidin;
  • the affinity labeled with the 3' or 5' end of the detection probe is one of digoxin, fluorescein isothiocyanate and biotin corresponding to the label.
  • the kit further comprises the substrate of the catalytic enzyme
  • the substrate is any one of TMB, ABTS and OPD;
  • the substrate is a combination of BCIP and NBT, p-nitrophenyl phosphate, disodium 4-nitrobenzene phosphate, naphthol AS-BI phosphate, Any of naphthol-AS-MX-phosphate.
  • the kit further includes a cleaning solution comprising a lotion A and a lotion B, the lotion A being an SDS-containing SSC buffer, and the lotion B is a Tween 20-containing PBS buffer.
  • the kit further includes the detection well plate, wherein the capture probes are respectively fixed in different reaction wells of the detection well plate according to their corresponding HPV virus genotypes; There is a working electrode and is configured to apply a voltage to form an electric field.
  • the bottom of the reaction hole is further provided with an opposite electrode, and the opposite electrode is disposed on the bottom plate of the reaction hole and configured to acquire a detection signal and output the detection signal;
  • the working electrode includes at least one uniform width a first linear portion, the opposite electrode includes at least one second linear portion having a uniform width, the first linear portion and the second linear portion are alternately arranged at a bottom of the reaction hole; at least two The working electrodes in the adjacent reaction wells are electrically connected.
  • a detection plate for HPV virus genotyping detection based on EFIRM technology is characterized in that a bottom of a reaction well of the detection plate is provided with a working electrode and is configured to apply a voltage to form electric field;
  • a capture probe is dispensed and fixed in the reaction well of the detection well plate, and the dispensed and fixed capture probe is selected from any one of the following groups:
  • Group 1 comprising a first capture probe and a second capture probe, at least one selected from the group consisting of the third to 11th capture probes, and at least one selected from the group consisting of the 12th to 16th capture probes;
  • Group 2 comprising at least one of the third to eleven capture probes
  • Group 3 comprising at least one of the 12th to 16th capture probes
  • Group 4 comprising at least one of a first capture probe and a second capture probe, and a third to eleven capture probe;
  • Group 5 comprising at least one of a first capture probe and a second capture probe, and a 12th to 16th capture probe;
  • a first capture probe for detecting a HPV 16 subtype the base sequence of which is set forth in SEQ ID NO.
  • a second capture probe for detecting a HPV 18 subtype the base sequence of which is set forth in SEQ ID NO.
  • a third capture probe for detecting a HPV 31 subtype the base sequence of which is set forth in SEQ ID NO.
  • a fourth capture probe for detecting HPV 33 subtype or 52 subtype or 58 subtype the base sequence is shown in SEQ ID NO.
  • a fifth capture probe for detecting the HPV 35 subtype the base sequence of which is set forth in SEQ ID NO.
  • a sixth capture probe for detecting a subtype of HPV 39 the base sequence of which is set forth in SEQ ID NO.
  • a 7th capture probe for detecting the HPV 45 subtype the base sequence of which is set forth in SEQ ID NO.
  • An 8th capture probe for detecting a HPV 51 subtype the base sequence of which is set forth in SEQ ID NO.
  • a ninth capture probe for detecting a HPV 56 subtype the base sequence of which is set forth in SEQ ID NO.
  • a 10th capture probe for detecting a HPV 59 subtype the base sequence of which is set forth in SEQ ID NO.
  • An 11th capture probe for detecting the HPV 68 subtype the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the base sequence is set forth in SEQ ID NO.
  • the dispensing means that each of the capture probes is immobilized in a different one of the reaction wells.
  • the bottom of the reaction well is further provided with an opposite electrode, and the opposite electrode is disposed on the reaction well bottom plate and configured to acquire a detection signal and output the detection signal;
  • the working electrode includes at least one first linear portion having a uniform width, the opposite electrode including at least one second linear portion having a uniform width, and the first linear portion and the second linear portion are in the reaction The bottoms of the holes are alternately arranged;
  • the working electrodes of at least two adjacent ones of the reaction wells are electrically connected.
  • the capturing probe is mixed with a conductive polymer and an ionic compound to form a mixed solution, and then added to the reaction hole, and then the first square wave electric field is applied through the working electrode.
  • a conductive polymer and an ionic compound to form a mixed solution, and then added to the reaction hole, and then the first square wave electric field is applied through the working electrode.
  • the parameters of the first electric field are: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles are performed.
  • a method for detecting HPV virus genotyping based on EFIRM technology characterized in that the kit according to any one of claims 13-19 is used, the steps are as follows:
  • the detection orifice plate according to any one of claims 20-22, or the capture probe is added to the blank detection orifice plate, and the bottom of the reaction well is provided with an electrode for turning on the EFIRM detector. Applying an electric field to the solution in the reaction well to carry out polymerization reaction; after the EFIRM detector is turned on, a first electric field is applied to the solution in the reaction well to carry out polymerization reaction; after the electric field treatment is completed, the detection orifice plate is cleaned.
  • the parameters of the first electric field processing are: voltage 200-500 mV, 1-5 s; voltage 800-1500 mV, 1-5 s; 3-10 cycles;
  • the concentration of the detection probe in the detection probe solution is 0.5-1.5 ⁇ mol/L;
  • the beneficial effects of the probes, kits and methods based on the EFIRM technology HPV virus detection provided by the present invention are that the probe combination based on the EFIRM technology HPV virus detection provided by the present invention comprises a plurality of probe pairs, each probe pair comprises There is a capture probe for binding to a target sequence and a detection probe corresponding to the capture probe and capable of binding the target sequence, and the capture probe binds to the target sequence through the principle of base complementation, and the target sequence is captured and fixed for the first time.
  • the detection probe binds specifically to the target sequence through the principle of base complementation, and the detection probe is bound and immobilized; the labeled affinity of the 3' or 5' end of the detection probe binds the catalytic enzyme
  • the catalytic enzyme catalyzes the release of the current signal from the substrate, detects the released current signal, and detects the viral genotype. Since the target sequence can be signaled only after two specific bindings simultaneously with the capture probe and the detection probe, which greatly increases the specificity of the detection, making the detection result accurate and reliable, false positive The rate is very low.
  • the present invention provides a base sequence of a capture probe of each probe pair, which is capable of separately capturing, for example, a common type of HPV 16 subtype, 18 subtype, and a medium and high risk type of HPV 26, 53, 66, 73, 82.
  • the target sequence in the subtype which in turn can detect whether the sample to be tested contains one of the common type viruses such as HPV 16 subtype, 18 subtype, and high risk type viruses such as HPV HPV26, 53, 66, 73, 82 subtypes. Or a variety of viral subtypes.
  • Embodiment 1 is an exemplary detection result of Embodiment 1 of the present invention.
  • Embodiment 2 is an exemplary detection result of Embodiment 2 of the present invention.
  • Embodiment 3 is an exemplary detection result of Embodiment 3 of the present invention.
  • Embodiment 4 is an exemplary detection result of Embodiment 4 of the present invention.
  • FIG. 5 is an exemplary detection result according to Embodiment 5 of the present invention.
  • Embodiment 6 is an exemplary detection result of Embodiment 6 of the present invention.
  • FIG. 7 is a schematic plan view showing the structure of a detecting electrode in the detecting orifice plate of the present invention.
  • FIG. 8 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention.
  • FIG. 9 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention.
  • FIG. 10 is a schematic plan view showing another structure of a detecting electrode in the detecting orifice plate of the present invention.
  • Figure 11a is a perspective view of a detection orifice plate according to the present invention.
  • Figure 11b is a schematic plan view of a detecting orifice plate according to the present invention.
  • FIG. 12 is a partial perspective view of a detecting orifice plate according to the present invention.
  • Figure 13 is a partial perspective view of a detecting orifice plate according to the present invention.
  • Figure 14 is a partial perspective view of a detecting orifice plate according to the present invention.
  • Figure 15 is a partial side elevational view of the detection orifice plate of the present invention.
  • the invention is illustrated by the following examples, which are not indicated in the examples, and are carried out according to conventional conditions or conditions recommended by the manufacturer.
  • the reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained by commercially available purchase.
  • the EFIRM detector used in the present invention is produced by Guangzhou Yihuo Biotech Co., Ltd., and is described in "Felvi Chemical Sensor for Multiplex Biomarkers Detection, Clin Cancer Res. 2009 Jul 1; 15 (13) published by Fang Wei et al. In 4446–4452, the electrochemical detector used therein.
  • a square wave (csw E-field) can be applied to the reaction well by a general square wave-generating instrument, and the living biotechnology limited can also be adopted.
  • the company's pre-developed EFIRMY instruments and supporting software are implemented.
  • HPV16, 18 subtypes are the most common clinical, and the two types of viruses have the highest probability of occurrence in cervical cancer patients; HPV 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68 subtypes are high Risk type (collectively referred to as high risk group), HPV26, 53, 66, 73, 82 subtypes are medium risk type (collectively referred to as medium and high risk groups), and the present invention is based on the above virus subtypes.
  • the probe sequence is designed and the resulting probe combination can be used to detect the above viral subtypes.
  • the invention provides a probe combination based on EFIRM technology HPV virus detection, which comprises a plurality of probe pairs.
  • Each probe pair includes a capture probe for binding to a target sequence and a detection probe corresponding to the capture probe and conjugateable to the target sequence.
  • the 3' or 5' end of the detection probe of each probe pair is labeled with an affinity for binding to a catalytic enzyme for catalyzing the chemical reaction of its substrate to form a stream of electrons.
  • the plurality of probe pairs include a first probe pair, a second probe pair, and at least one selected from the group consisting of the 12th to 16th probe pairs.
  • the base sequence of the capture probe and the detection probe detected by each probe pair, and the HPV subtype thereof detected will be described in detail below.
  • the first probe pair for detecting the HPV16 subtype has a base sequence of the capture probe as shown in SEQ ID NO. 1, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the second probe pair for detecting the HPV18 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 3, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 12th probe pair for detecting the HPV26 subtype is shown in SEQ ID NO. 20, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the base sequence of the capture probe is shown in SEQ ID NO. 21
  • the base sequence of the detection probe is shown in SEQ ID NO.
  • the 14th probe pair for detecting the HPV66 subtype is shown in SEQ ID NO. 22, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 15th probe pair for detecting the HPV73 subtype is shown in SEQ ID NO. 23, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 16th probe pair for detecting the HPV82 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 24, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the plurality of probe pairs may further comprise probe pairs for detecting high risk HPV:
  • a third probe pair for detecting the HPV31 subtype the base sequence of the capture probe is shown in SEQ ID NO. 5, and the base sequence of the detection probe is shown in SEQ ID NO.
  • a fourth probe pair for detecting HPV33 subtype or 52 subtype or 58 subtype the base sequence of the capture probe is shown in SEQ ID NO. 6, and the base sequence of the detection probe is SEQ ID NO. As shown in .15, the three subtypes share the fourth probe pair.
  • the 5th probe pair for detecting the HPV35 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 7, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 6th probe pair for detecting the HPV39 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 8, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 7th probe pair for detecting the HPV45 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 9, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 8th probe pair for detecting the HPV51 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 10, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the ninth probe pair for detecting the HPV 56 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 11, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 10th probe pair for detecting the HPV59 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 12, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the 11th probe pair for detecting the HPV68 subtype has the base sequence of the capture probe as shown in SEQ ID NO. 13, and the base sequence of the detection probe is shown in SEQ ID NO.
  • the detection probe corresponds to the capture probe, and one capture probe and one detection probe form a probe pair, which can be used for detecting a certain type of virus.
  • Both the capture probe and the detection probe are based on the same type of virus of the same type. Designing a target sequence or a conserved DNA fragment, the capture probe and the detection probe can bind to different regions or binding sites of the same conserved gene or conserved DNA fragment of the virus type through the principle of base complementation, but capture The probe is not complementary to the detection probe, and the base sequence is also different, and the binding sites of the two and the target sequence do not overlap or overlap. Further, the binding region of the capture probe and the detection probe to the target sequence may be adjacent or may be separated by a plurality of bases. As long as the capture probe and the detection probe are capable of binding to the same conserved gene of the same type of virus or the same conserved DNA fragment by the principle of base complementation.
  • the capture probe sequence of each probe pair and the base sequence of the detection probe sequence may not correspond to each other, that is, in other embodiments, the capture probe of each probe pair
  • the sequence may be the base sequence described above, and the base sequence of the corresponding detection probe may be complementary to other sequences taken from the same DNA conserved region of the same detection subtype virus; or in other embodiments,
  • the detection probe sequence of each probe pair may be the above-mentioned base sequence, and the base sequence of the corresponding capture probe may be another sequence selected from the same DNA conserved region of the same detection subtype virus.
  • the base sequences of the capture probes and the detection probes of the probe pairs provided by the present invention have the advantages of stronger specificity, higher sensitivity, etc. when they are detected, and the detection results can be seen in the examples. .
  • group detection can be performed, for example, only the virus containing a certain risk level (for example, a high-risk group or a medium-high-risk group) is detected, and it is not necessary to detect that it contains the group.
  • a certain risk level for example, a high-risk group or a medium-high-risk group
  • Specific type of virus for example, specific subtypes can also be detected, for example, by detecting a subtype of a particular high-risk group, such as the HPV53 subtype virus, in the sample.
  • the specific detection method is used to detect the degree to which the detection result is obtained, and the tester can select according to the specific situation to provide a more reasonable guiding significance.
  • the specific group detection is described in the embodiment.
  • the probe combination for viral gene analysis detection may include only one or two or three or more probe pairs of the first to 16th probe pairs, as long as Any combination selected from the first to 16th probe pairs is within the scope of the present invention.
  • the affinity of the 3' or 5' end of each of the detection probes is biotin, and the biotin acts to bind to the streptavidin-labeled catalytic enzyme and catalyze the catalytic end.
  • the current generated by the object releases the detection signal.
  • the detection probe may bind to the catalytic enzyme without the recognition system of biotin-streptavidin, and may be combined with other catalytic systems such as antibody/antigen, ligand/receptor, etc.
  • the enzyme therefore, the affinity may be one of an antigen/antibody.
  • the affinity may be digoxin or fluorescein isothiocyanate, and correspondingly, the catalytic enzyme may be labeled with a high-octane antibody or a fluorescein isothiocyanate antibody.
  • the affinity may be labeled at the 3' end of the detection probe or at the 5' end, either.
  • the kit for detecting HPV virus based on EFIRM technology comprises any one of the probe combinations described above.
  • each probe is independently present in the form of a solution, for example, the capture probe is present in the form of a capture probe solution containing a capture probe, and the detection probe is present in the form of a test probe solution containing the detection probe.
  • the concentration of the probe contained in each probe solution can be set according to actual conditions.
  • each probe solution contains a probe having a final concentration of 0.5 to 1.5 ⁇ M.
  • the kit for detecting HPV virus based on EFIRM technology may further comprise a fixture for fixing the capture probe of the probe pair to the detection well plate.
  • the fixture includes a conductive polymer and an ionic compound.
  • the conductive polymer is selected from one of pyrrole, aniline and thiophene.
  • the conductive polymer may also be other conductive polymer materials.
  • the ionic compound is selected from any one of sodium chloride and potassium chloride.
  • the conductive polymer is positively charged, and forms a network cross-linked structure under the action of an electric field, and is deposited at the bottom of the reaction hole.
  • the mesh cross-linked structure can stably fix the capture probe at the bottom, which helps to improve the capture probe. Needle stability and capture ability.
  • the kit for detecting HPV virus based on EFIRM technology may further comprise a catalytic enzyme, the catalytic enzyme is a horseradish peroxidase with a label, preferably, the catalytic enzyme is labeled with streptavidin. Horseradish peroxidase.
  • the catalytic enzyme may also be a labeled alkaline phosphatase, and the label is any one of a digoxin antibody, a fluorescein isothiocyanate antibody or streptavidin, a label and an affinity. Correspondingly, it can be selected based on the type of affinity on the detection probe.
  • the affinity is biotin
  • the marker is streptavidin
  • the affinity is digoxin
  • the marker is a digoxin antibody
  • the affinity is fluorescein isothiocyanate
  • the marker is a fluorescein isothiocyanate antibody.
  • kit for detecting HPV virus based on EFIRM technology may further comprise a substrate, and the class of the substrate is selected according to the type of catalytic enzyme.
  • the substrate is TMB (Tetramethylbenzidine, tetramethylbenzidine), ABTS (2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate, 2,2-diazepine) - bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and OPD (o-Phenylenediamine, o-phenylenediamine).
  • TMB Tetramethylbenzidine, tetramethylbenzidine
  • ABTS 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate, 2,2-diazepine) - bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt
  • OPD o-Phenylenediamine, o-phenylenediamine
  • TMB, ABTS and OPD are both horseradish peroxidation
  • the substrate of the enzyme under the catalysis of horseradish peroxidase, undergoes a color reaction and is accompanied by a current generation, which contributes to an increase in the release of the detection signal.
  • the substrate is BCIP (5-Bromo-4-Chloro-3-Indolyl Phosphate, 5-bromo-4-chloro-3-indolyl-phosphate) and NBT (Nitrotetrazolium). Blue chloride, tetrazolium nitroblue) composition, nitrophenyl phosphate, disodium 4-nitrobenzene phosphate, naphthol AS-BI phosphate, naphthol-AS-MX-phosphate .
  • kit for detecting HPV virus based on EFIRM technology may further include a cleaning solution including washing liquid A and washing liquid B, washing liquid A is SDS buffer containing SDS, and washing liquid B is containing Tween20. PBS buffer.
  • kit for detecting HPV virus based on EFIRM technology may further comprise a diluent, which is a casein-containing PBS buffer.
  • the invention provides a method for detecting HPV virus based on EFIRM technology, which comprises:
  • Providing a plurality of probe pairs comprising: a first probe pair, a second probe pair, and at least one selected from the group consisting of 3rd to 11th probe pairs; each probe pair including for binding a capture probe of the target sequence and a detection probe corresponding to the capture probe and capable of binding to the target sequence, and the 3' or 5' end of the detection probe is labeled for binding to catalyze the formation of a chemical reaction of the corresponding substrate.
  • base sequence of the capture probe of the first probe pair is as shown in SEQ ID NO. 1
  • base sequence of the capture probe of the second probe pair is as shown in SEQ ID NO. 3, and 12 to 16
  • the base sequences of the capture probes of the probe pair are shown in SEQ ID NO. 20 to 24, respectively.
  • Capture probe immobilization step The capture probe for binding to the target sequence is first fixed to the reaction well of the detection well by an electric field. The capture probe moves to the bottom of the reaction well under the action of an electric field and is fixed at the bottom of the reaction well.
  • Sample hybridization step The sample to be tested is added to the reaction well.
  • the target sequence and the capture probe in the sample to be tested are captured and fixed at the bottom of the reaction well by the principle of base complementation.
  • Detection probe binding step adding a detection probe corresponding to the capture probe and binding the target sequence to the reaction well, and the 3' end or the 5' end of the detection probe is labeled for binding to catalyze the corresponding substrate production
  • the chemical reaction forms an affinity for the catalytic enzyme of the electron flow.
  • the detection probe is immobilized in the reaction well by binding to the target sequence through the principle of base complementation.
  • Enzyme catalytic reaction detection step a catalytic enzyme and a substrate are added to the reaction well, and the current in the reaction well is detected by a current detecting device.
  • the detection probe binds to the catalytic enzyme through the affinity, and the catalytic enzyme re-catalyzes the substrate to generate a current release detection signal, which is recognized and amplified by the current detecting device, and the result is detected.
  • the detection principle of the present invention is to rapidly detect viral genotypes by using Electric Field-Induced Release and Measurement (EFIRM) technology, especially for detecting 18 high-risk HPVs.
  • EFIRM Electric Field-Induced Release and Measurement
  • the capture probe is immobilized on the bottom of the reaction well on the detection well plate under the action of the electric field; the capture probe captures the target sequence in the sample to be tested, ie, viral DNA, by the principle of base complementary pairing under the electric field;
  • the labeled detection probe is further bound to the target sequence; the catalytic enzyme is recognized by the labeled streptavidin and biotin, and the substrate of the catalytic enzyme is added to the reaction well to generate a redox reaction, current generation, and instrument detection.
  • the current signal is further determined to have a corresponding target sequence in the sample to be tested. Since both the capture probe and the detection probe are designed according to a conserved region of a specific virus type, the type of the virus can be known.
  • kits for detecting HPV virus based on EFIRM technology provided in the present embodiment is described by taking the HPV genotype as an example.
  • the kit for detecting a virus genotyping includes a probe combination comprising 16 probe pairs for detecting HPV subtypes, and the capture probes and detection probes of each probe pair are The solution forms were independent and the final concentration of the probe was 1 ⁇ M.
  • the 16 probe pairs are: a first probe pair for detecting the HPV 16 subtype, and the base sequence of the capture probe is as shown in SEQ ID NO. 1, and the base sequence of the detection probe is SEQ. ID NO. 2; a second probe pair for detecting the HPV 18 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 3, and the base sequence of the detection probe is SEQ ID NO.
  • a probe combination comprising 16 probe pairs for detecting HPV subtypes, and the capture probes and detection probes of each probe pair are The solution forms were independent and the final concentration of the probe was 1 ⁇ M.
  • the 16 probe pairs are: a first probe pair for detecting the HPV 16 subtype, and the base sequence of the capture probe is as shown in SEQ ID
  • a third probe pair for detecting the HPV 31 subtype the base sequence of the capture probe is shown in SEQ ID NO. 5, and the base sequence of the detection probe is SEQ ID NO.
  • the base sequence of the capture probe is shown in SEQ ID NO. 6, and the base sequence of the detection probe is shown.
  • SEQ ID NO. 15 a 5th probe pair for detecting the HPV 35 subtype, the base sequence of the capture probe is shown in SEQ ID NO. 7, and the base sequence of the detection probe is SEQ. ID NO. 14; a 6th probe pair for detecting the HPV 39 subtype, the base sequence of the capture probe is shown in SEQ ID NO.
  • the base sequence of the detection probe is SEQ ID NO. .16; used to detect HPV 45 a 7th probe pair of the type, the base sequence of the capture probe is shown in SEQ ID NO. 9, and the base sequence of the detection probe is shown in SEQ ID NO. 17; for detecting HPV 51 subtype
  • the eighth probe pair the base sequence of the capture probe is shown in SEQ ID NO. 10, the base sequence of the detection probe is shown in SEQ ID NO. 18, and the ninth for detecting the HPV 56 subtype.
  • the probe pair has a base sequence of the capture probe as shown in SEQ ID NO. 11, a base sequence of the detection probe as shown in SEQ ID NO. 18, and a 10th probe for detecting the HPV 59 subtype.
  • the base sequence of the capture probe is shown in SEQ ID NO. 12, the base sequence of the detection probe is shown in SEQ ID NO. 19, and the 11th probe pair for detecting the HPV 68 subtype,
  • the base sequence of the capture probe is shown in SEQ ID NO. 13, the base sequence of the detection probe is shown in SEQ ID NO. 16, and the 12th probe pair for detecting the HPV 26 subtype is captured.
  • the base sequence of the probe is shown in SEQ ID NO. 20, the base sequence of the detection probe is shown in SEQ ID NO. 26; the 13th probe pair for detecting the HPV 53 subtype, and the capture probe thereof.
  • Base sequence such as SEQ As shown in ID NO. 21, the base sequence of the detection probe is shown in SEQ ID NO.
  • the base sequence of the capture probe is SEQ ID NO. 25; the 14th probe pair for detecting the HPV 66 subtype, the base sequence of the capture probe is SEQ ID NO. As shown in Fig. 22, the base sequence of the detection probe is shown in SEQ ID NO. 25; the 15th probe pair for detecting the HPV 73 subtype, the base sequence of the capture probe is SEQ ID NO. As shown, the base sequence of the detection probe is shown in SEQ ID NO. 27; the 16th probe pair for detecting the HPV 82 subtype, the base sequence of the capture probe is shown in SEQ ID NO. The base sequence of the detection probe is shown in SEQ ID NO. The base sequences of the respective HPV subtypes and their corresponding capture probes and detection probes are shown in Table 1.
  • the capture probes of the 33, 52, and 58 subtypes have the same base sequence
  • the detection probes of the 33, 52, and 58 subtypes have the same base sequence, that is, the capture probe shown by SEQ ID NO.
  • the probe and the detection probe shown in SEQ ID NO. 15 are capable of detecting HPV 33, 52 and 58 subtype viruses, and the detection results indicate that the sample has at least one of HPV types 33, 52 and 58, but specifically Which of the three subtypes of the virus was not detected.
  • the detection probe sequences of the 39 and 68 subtypes are identical
  • the detection probe sequences of the 51 and 56 subtypes are identical
  • the detection probe sequences of the 53 and 66 subtypes are identical.
  • the 5'-end labeled biotin was used to detect the 16 and 18 subtypes of the detection probe, and the remaining subtypes of the detection probe were labeled with biotin at the 3' end (as shown in Table 1).
  • the specific detection steps for the sample 1 and the sample 2 using the virus genotyping detection kit provided in the present embodiment are as follows.
  • E-plate On a 96-well detection plate (E-plate) (the structure and working principle can be seen in the priority document 201620769829.2), add 30 ⁇ l of the prepared mixture of pyrrole and CP to the reaction well according to the instructions.
  • the tip of the gun is attached to the bottom of the hole, but does not touch the bottom electrode, after tilting or tapping the E-plate, the liquid is evenly covered on the surface of the electrode in the hole, and then immediately go to the EFIRM instrument and operate the electric field according to its operating instructions. .
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles were performed. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
  • the lotion A was a 2 ⁇ SSC buffer containing 0.05% by mass of SDS.
  • the hybrid buffer (purchased from thermo fisher) was treated in a water bath at 90 ° C for 10 min in a water bath and then allowed to cool at room temperature for 20 min.
  • the sample to be tested was taken out from the -20 ° C refrigerator and placed in a refrigerator at 4 ° C to be thawed. After complete dissolution, the sample to be tested is pretreated by boiling or 0.4M NaOH, and then the sample to be tested is mixed with the hybrid buffer by a volume ratio of 1:2, vortexed and centrifuged, and then the sample is loaded for detection.
  • four detection groups are set, which are 16 subtype group, 18 subtype group, 11 high risk type groups, and 5 medium and high risk type groups.
  • the 16 subtype group was used to detect the HPV16 subtype (the corresponding pair of probe pairs was added to each well in the group)
  • the 18 subtype group was used to detect the HPV18 subtype (the corresponding probe was added to each well of the group)
  • 11 high-risk groups were used to detect one of 11 high-risk HPVs (the group included 3 to 11 probe pairs for each well, and 5 medium- and high-risk types for 5 medium- and high-risk HPVs).
  • One of the groups (the pair of holes corresponds to the 12th to 16th probe pairs). It should be noted that the number of detection groups can be designed according to the accuracy of the required test results.
  • the common type HPV, the high-risk type virus, and the medium-high-risk type virus can be set according to the setting method of the detection group in this embodiment. If only the sample is required to detect whether the sample contains HPV, only one detection group can be set, and the specific detection method Refer to Embodiment 2. If it is necessary to detect that the sample specifically contains a certain type of virus, 18 detection groups can be set, and the specific detection method is referred to Embodiment 3.
  • a corresponding 30 ⁇ l of a hybrid buffer containing a positive oligonucleotide having a final concentration of 1 pM (which is capable of complementary pairing with the corresponding capture probe and detection probe) was added as a positive control to the positive control well (remarks that The positive control of 11 high-risk groups only selected 52 subtype nucleotide sequences (line 9 in Table 2), and the positive control of 5 medium-high-risk groups only selected 26 subtype nucleotide sequences (Table 2) Line 13) can serve as a positive control.
  • the base sequence of the positive oligonucleotide corresponding to each HPV subtype is shown in Table 2. 30 ⁇ l of hybridization buffer was added to the negative control well as a negative control.
  • the electric field parameters are set to: voltage A: 300 mV, 1 s; voltage B: 500 mV, 1 s; 150 cycles. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
  • the dilution solution was taken out from the refrigerator at 4 ° C, and a 1.5 mL centrifuge tube was taken. 990 ⁇ l of the dilution solution was added, and 10 ⁇ l of DP (100 ⁇ M) was added to the reaction wells of each test group, vortexed and mixed, centrifuged, and set aside. Among them, the diluent was PBS buffer (pH 7.4) containing 0.1% (mass by volume) casein. The role of casein is to block non-specific sites to increase the sensitivity and accuracy of the assay.
  • the lotion B was a PBS buffer containing 0.1% by mass of Tween20.
  • the substrate is a solution containing TMB (commercially available from Thermo Fisher, Cat. No. 34028 the product, the name 1-Step TM Ultra TMB-ELISA ).
  • TMB commercially available from Thermo Fisher, Cat. No. 34028 the product, the name 1-Step TM Ultra TMB-ELISA ).
  • the substrate of the enzyme is added, a redox reaction occurs, a current is generated, and the current value in each well is detected to complete the entire detection process.
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: -200 mV, 60 s; voltage B: 0 mV, 0 s; one cycle was performed. After the electric field treatment is completed, remove it immediately and clean the E-plate plate.
  • the instrument will automatically complete the test and the test data will be automatically uploaded to the cloud computing platform.
  • the histogram is drawn according to the detected data, the abscissa is the category of the detection group, and the ordinate is the current value (Current) of each detection hole in each detection group, and the unit is nanoamperes (-nA, negative sign indicates direction).
  • the detection results of this embodiment are shown in FIG.
  • the test result of the sample 1 was positive, and it contained the HPV16 subtype virus (the negative control current value was 28.67 nA, the standard deviation was 5.62, and the positive control current value was 130.22 nA, the sample The current value of 1 is 192.05 nA, and the current value of sample 2 is 30.53 nA).
  • the test result of sample 2 is positive (the negative control current value is 24.94 nA, and the standard deviation is 5.91, positive control)
  • the current value is 132.94nA
  • the current value of sample 1 is 29.57nA
  • the current value of sample 2 is 151.30nA), indicating that it contains high-risk HPV31, 33, 52, 58, 35, 39, 45, 51, 56, 59, At least one of the 68 subtypes.
  • the kit for detecting HPV virus based on EFIRM technology includes a probe combination (same as in Example 1), a fixative, streptavidin-labeled horseradish peroxidase (present in solution), and a bottom thereof.
  • the immobilizer is a conductive polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride.
  • the substrate is a solution containing TMB. The rest are the same as in the first embodiment.
  • the detection group is set to one, and the 18 subtypes are simultaneously detected, and the detection group includes one positive control hole, one negative control hole (repeated 4 times), one sample 1 detection hole and one sample. 2 detection holes.
  • the 16 probe pairs described in Example 1 were added to each well and 18 HPV subtypes were simultaneously detected). The rest are the same as in the first embodiment. If there is a positive detection result, the detected result can indicate that the sample to be tested contains HPV 16, 18, 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68, 26, 53, 66, At least one of the 73 and 82 subtypes (as shown in Figure 2).
  • the detection result of this embodiment is as shown in FIG. 2.
  • test result of sample 1 is positive (the negative control current value is 26.59 nA, the standard deviation is 7.91, the positive control current value is 110.46 nA, and the sample 1 current value is 189.66 nA), indicating that it contains HPV16, At least one of 18, 31, 33, 52, 58, 35, 39, 45, 51, 56, 59, 68, 26, 53, 66, 73, 82 subtypes; sample 2 is positive (negative)
  • the control current value was 26.59 nA, the standard deviation was 7.91, the positive control current value was 110.46 nA, and the sample 2 current value was 80.60 nA), indicating that sample 2 contained HPV 16, 18, 31, 33, 52, 58, 35, 39. At least one of the 45, 51, 56, 59, 68, 26, 53, 66, 73, 82 subtypes.
  • the kit for detecting HPV virus based on EFIRM technology includes a probe combination (same as in Example 1), a fixative, streptavidin-labeled horseradish peroxidase (present in solution), and a bottom thereof. Matter, cleaning solution.
  • the anchor is a conductive polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride.
  • the substrate is a solution containing TMB.
  • the cleaning solution includes a lotion A and a lotion B, the lot A is an SDS buffer containing SDS, and the lotion B is a PBS buffer containing Tween 20. The rest are the same as in the first embodiment.
  • the detection group is set to 16 groups, which are 16 subtype group, 18 subtype group, 26 subtype group, 31 subtype group, 35 subtype group, 39 subtype group, and 45 subtype group. , 51 subtype group, 52 subtype group, 53 subtype group, 56 subtype group, 59 subtype group, 66 subtype group, 68 subtype group, 73 subtype group and 82 subtype group.
  • One positive control well was set for each test group (each test group was added with the corresponding positive oligonucleotide shown in Table 2), and one negative control well (heavy Repeat 4 times), one sample 1 detection hole and one sample 2 detection hole, and each corresponding detection hole is added to its corresponding probe pair. The rest are the same as in the first embodiment. If each test group has a positive detection result, the detected result can indicate the corresponding HPV subtype virus contained in the sample to be tested.
  • the positive result detected by the 52 subtype indicates that the sample to be tested contains one or more of the HPV 33, 52, and 58 subtypes.
  • the detection result of this embodiment is shown in FIG.
  • the test result of the sample 1 was positive (the negative control current value was 28.67 nA, the standard deviation was 5.62, the positive control current value was 130.22 nA, and the sample 1 current value was 192.05 nA.
  • the current value of sample 2 is 30.53nA), indicating that sample 1 contains HPV 16 subtype virus; in group 52, sample 2 is positive (negative control current value is 25.58nA, and its standard deviation is 5.33, The positive control current value was 75.70 nA, the sample 1 current value was 23.90 nA, and the sample 2 current value was 166.10 nA).
  • sample 2 test result was positive (the negative control current value was 25.92).
  • nA its standard deviation is 6.20
  • positive control current value is 115.75nA
  • sample 1 current value is 34.47nA
  • sample 2 current value is 72.72nA)
  • the detection group of 18 by setting the detection group of 18 to detect the detection result of the sample to be tested, the specific virus subtype contained therein can be detected, and the detection result is accurate and reliable.
  • the kit for detecting HPV virus based on EFIRM technology includes a probe combination, a detection plate, a fluorescein isothiocyanate antibody-labeled horseradish peroxidase (present in solution) and a substrate thereof (ABTS) , in the form of a solution), cleaning solution.
  • the probe combination comprises 9 probe pairs, respectively: a first probe pair for detecting the HPV 16 subtype, a second probe pair for detecting the HPV 18 subtype, and For detecting the third probe pair of the HPV 31 subtype, the fourth probe pair for detecting the HPV 33 subtype or the 52 subtype or the 58 subtype, and the eighth probe pair for detecting the HPV 51 subtype, 9th probe pair for detecting HPV 56 subtype, 12th probe pair for detecting HPV 26 subtype, 13th probe pair for detecting HPV53 subtype, 15th for detecting HPV 73 subtype Probe pair.
  • Each test probe was provided with fluorescein isothiocyanate at the 3' end.
  • the base sequences of the respective probe pairs and the corresponding capture probes and detection probes are shown in Table 1 in Example 1.
  • the capture probe of the above probe pair is fixed in the reaction well of the detection well plate, and the method of fixing the capture probe to the detection well plate is the same as the "1 capture probe fixation" step in the first embodiment, of course, in other implementations.
  • the detection of the capture probe of the present invention by using other methods to the detection well plate is also within the scope of the present invention.
  • the cleaning solution includes a lotion A and a lotion B, the lotion A is an SSS buffer containing SDS, and the lotion B is a PBS buffer containing Tween20.
  • the rest are the same as in the first embodiment.
  • the sample 1 and the sample 2 are simultaneously detected by using the embodiment, and the detecting step is basically the same as that of the first embodiment, and the detection result is as shown in FIG. 4 .
  • sample 1 contains HPV16 subtype virus
  • sample 2 is positive (negative control current value is 24.25nA, standard deviation is 5.27, positive control current value is 115.92nA, sample)
  • the current value of 2 is 170.42 nA), indicating that it contains at least one of the high-risk types HPV 31, 33, 51, 52, 58 and 56 subtypes.
  • the kit for detecting HPV virus based on EFIRM technology includes a probe combination, a fixer, a digoxin antibody-labeled alkaline phosphatase enzyme (present in solution) and a substrate thereof (including BCIP and NBT) A solution of the composition is present), a cleaning solution.
  • the probe combination includes four probe pairs, which are: a first probe pair for detecting the HPV 16 subtype, a second probe pair for detecting the HPV 18 subtype, and A 4th probe pair for detecting HPV 33 subtype or 52 subtype or 58 subtype, and a 12th probe pair for detecting HPV 26 subtype.
  • Each detection probe has a digoxin at the 5' end.
  • the base sequences of the respective probe pairs and the corresponding capture probes and detection probes are shown in Table 1 in Example 1.
  • the anchor is a conductive polymer and an ionic compound, wherein the conductive polymer is thiophene, and of course, in other embodiments, aniline.
  • the ionic compound is sodium chloride.
  • the cleaning solution includes a lotion A and a lotion B, the lot A is an SDS buffer containing SDS, and the lotion B is a PBS buffer containing Tween 20. The rest are the same as in the first embodiment.
  • the sample 1 and the sample 2 are simultaneously detected by using the embodiment, and the detection step is basically the same as that of the embodiment 1, and the detection result is shown in FIG. 5.
  • the test result of the sample 1 was positive (the negative control current value was 26.31 nA, the standard deviation was 5.29, the positive control current value was 118.74 nA, and the sample 1 current value was 185.47 nA. ), indicating that sample 1 contains HPV16 subtype virus; in the three high-risk groups, sample 2 was positive (negative control current value was 27.45 nA, standard deviation was 5.15, positive control current value was 122.62 nA, sample) The current value of 2 is 150.07 nA), indicating that sample 2 contains at least one of the high-risk types HPV 33, 51, 52, and 58 subtypes.
  • the kit for detecting HPV virus based on EFIRM technology includes a probe combination, a immobilizer, streptavidin-labeled horseradish peroxidase (present in solution) and a substrate thereof, and the immobilizer is conductive A polymer and an ionic compound, wherein the conductive polymer is pyrrole and the ionic compound is potassium chloride.
  • the substrate is a solution containing TMB. The rest are the same as in the first embodiment.
  • the probe combination includes only the first probe pair, the second probe pair, the twelfth probe pair, the thirteenth probe pair, the fourteen probe pair, the fifteenth probe pair, and the sixteenth probe pair (each The sequence of the capture probe and the detection probe of the probe pair is the same as in Example 1).
  • the probe combination includes a first probe pair, a second probe pair, and at least one selected from the group consisting of 12th to 16th probe pairs, for example, two or three types. .
  • the sample 2 was only tested by the kit of the present embodiment, and the detection step was basically the same as that of the first embodiment, and the detection result is shown in FIG. 6.
  • the test result of sample 2 is positive (the negative control current value is 28.36nA, the standard deviation is 7.15, the positive control current value is 147.47nA, and the current value of sample 2 is 66.91nA), indicating that sample 2 contains one or more of five medium-high-risk viruses; in the 16-subtype group (negative control current value is 26.36nA, standard deviation is 5.10, positive control current value is 142.15nA) , sample 2 current value is 28.54nA) and 18 subtype group (negative control current value is 23.51nA, its standard deviation is 5.90, positive control current value is 116.36nA, sample 2 current value is 31.21nA), sample The test result of 2 was negative, indicating that sample 2 did not contain HPV16 subtype and HPV18 subtype.
  • the capture probe is fixed at the bottom of the detection orifice plate.
  • the capture solution contained the following components: 5% by weight of thiophene, 2 mol/L of NaCl, and 1.5 ⁇ mol/L of capture probe.
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 1500 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
  • the hybrid buffer (using the hybrid buffer 3 in the Summary of the Invention) was treated in a water bath at 95 ° C for 5 min in a water bath and then allowed to cool at room temperature.
  • the samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:2.5, vortexed and centrifuged, and the sample can be tested.
  • a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the sample volume was 80 ⁇ l.
  • WT negative control
  • MT positive control
  • the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched.
  • the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 10 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
  • the detection solution was PBS as a solvent, wherein the weight percentage of casein was 5%, the concentration of the detection probe was 1.5 ⁇ mol/L, vortexed and mixed, centrifuged, and set aside.
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: 500 mV, 1 s; voltage B: 800 mV, 1 s; 8 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
  • TMB/H2O2 solution (purchased from thermo fisher, product no. 34022, named as
  • the electric field parameter is set to: voltage A: -300mV, 100s, and the current reading is obtained.
  • test results were in agreement with the results of Examples 1-6 obtained based on the procedure described in Example 1.
  • test sample the specific steps are as follows:
  • the capture probe is fixed at the bottom of the detection orifice plate.
  • the capture solution contained the following components: the weight percentage of aniline was 0.1%, the concentration of NaCl was 0.01 mol/L, and the concentration of the capture probe was 0.5 ⁇ mol/L.
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 800 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
  • the hybrid buffer (using the hybrid buffer 6 in the Summary of the Invention) was treated in a water bath at 85 ° C for 15 min in a water bath and then left to cool at room temperature.
  • the samples were taken out from the -20 ° C refrigerator and thawed in a 4 ° C refrigerator. After complete dissolution, the sample and the hybrid buffer are mixed at a volume ratio of 1:1.5, vortexed and centrifuged, and the sample can be tested.
  • a blank control buffer, a corresponding concentration of the negative control (WT) and a positive control (MT) were added to the corresponding wells, and the amount of the sample was 20 ⁇ l.
  • WT negative control
  • MT positive control
  • the tip of the gun is attached to the bottom of the hole, but the bottom electrode is not touched.
  • the tilting or tapping of the detecting plate allows the liquid to uniformly cover the surface of the electrode in the hole, and then immediately goes to the EFIMR for electric field operation.
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
  • the detection solution was PBS as a solvent, wherein the weight percentage of casein was 0.1%, the concentration of the detection probe was 0.5 ⁇ mol/L, vortexed and mixed, centrifuged, and set aside.
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the electric field parameters were set to: voltage A: 200 mV, 5 s; voltage B: 300 mV, 5 s; 3 cycles were performed. After the electric field is processed, remove it immediately and clean the detection orifice.
  • the electric field parameter is set to: voltage A: -100mV, 40s, and the current reading is obtained.
  • the detecting orifice plate used in the method of the present invention has the common feature that the inner bottom of the reaction hole is provided with an electrode for applying an electric field to the solution in the reaction hole after the EFIRM detector is turned on, and the commercially available product can be used.
  • the capture probe is immobilized in the reaction well during use, or prefabricated for use or for sale.
  • the detection orifice plate is shown in FIG. 7 and includes: a reaction well bottom plate 101, the reaction well bottom plate 101 includes at least one detection region 102, a working electrode 103, and the working electrode 103 is disposed on the reaction well bottom plate 101. And configured to apply a voltage to form an electric field; and the opposite electrode 104, the opposite electrode 104 is disposed on the reaction aperture substrate 101 and configured to acquire a detection signal and output the detection signal.
  • the working electrode 103 and the opposite electrode 104 are both disposed on the same surface of the reaction cell bottom plate, and therefore, the working electrode 103 and the opposite electrode 104 may be in the same plane. As shown in FIG.
  • the working electrode 103 includes at least one first linear portion 1031 having a uniform width; the opposite electrode 104 includes at least one second linear portion 1041 of uniform width; the first linear portion 1031 and the second linear portion 1041 They are disposed within the detection zone 102 and are alternately spaced apart from one another.
  • the electrode 104 located on the right side of FIG. 1 can be configured as a working electrode to apply a voltage to form an electric field; the electrode 103 located on the left side of FIG.
  • the counter electrode is configured to acquire a detection signal and output the detection signal, and the disclosure is not limited herein.
  • the working electrode 103 can apply a voltage to generate an electric field to move and concentrate the target substance.
  • the working electrode 103 can apply a square wave alternating voltage to first include the target substance in the liquid to be detected.
  • the charged substance moves to the working electrode 103 to be enriched, so that the target substance can be combined with the probe on the working electrode 103, and then the polarity of the voltage is changed, so that other substances in the charged substance that are not combined with the probe are away from the working electrode 103 (
  • the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe; then, the opposite electrode 104 can acquire a detection signal about the target substance and output the detection signal, for example, a target substance bound to the probe.
  • the current reacts with a specific reagent to generate a current, so the opposite electrode 104 can acquire a detection signal about the target substance by detecting the current and output the detection signal; and then, by analyzing the output detection signal, the target substance can be obtained.
  • Information (such as the concentration of the target substance) so that it can be detected quickly and accurately. Since the first linear portion 1031 and the second linear portion 1041 are linear structures having a uniform width, and within the detection region 102, the first linear portion 1031 and the second linear portion 1041 are alternately equidistantly spaced within the detection region 102.
  • the first linear portion 1031 of the working electrode 103 can generate a uniform electric field in the detection region 102, and the second linear portion 1041 of the opposite electrode 104 can detect a minute current in the detection region 102, thus Can improve the accuracy of detection.
  • the density and uniformity of the probe formed on the working electrode 103 can be controlled, and the probe is not overly dense, thereby giving The binding of the target substance to the probe provides space to increase the efficiency of binding of the target substance to the probe, thereby improving the reaction speed of the liquid biopsy and further improving the accuracy of the detection.
  • the detecting electrode structure in the detecting orifice plate has a circular shape
  • the working electrode 103 may include an arc-shaped first body portion 1030 and a plurality of extending from the first body portion 1030.
  • the first linear portion 1031 that is parallel to each other.
  • the counter electrode 104 includes an arc-shaped second body portion 1040 and a plurality of second linear portions 1041 that are parallel to each other and extend from the second body portion 1040.
  • the first body portion 1030 is disposed opposite to the second body portion 1040, and the plurality of first linear portions 1031 and the plurality of second linear portions 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed.
  • the working electrode 103 and the opposite electrode 104 have a comb-like structure, and the working electrode 103 and the opposite electrode 104 cross each other to form an interdigitated structure.
  • the range of the detection area may include a plurality of first linear portions and a plurality of second linear portions, and may further include a first main body portion and a second main body portion, which are not limited herein.
  • the working electrode may not be provided with a fixing portion that is in direct contact with the probe, so the first linear portion may be formed into a line having a uniform width, thereby providing a more uniform electric field, so that the arrangement of the probe is more regular, thereby improving the efficiency and accuracy of detection. .
  • the detecting area 102 has a circular shape
  • the working electrode 103 includes a first linear portion 1031 which is spirally arranged
  • the opposite electrode 104 includes a second line which is spirally arranged.
  • the portion 1041, the first linear portion 1031 and the second linear portion 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed.
  • the width of the first linear portion is the same as the width of the second linear portion, so that the accuracy of the detection can be improved; in addition, the width of the first linear portion and the second linear portion
  • the range can be 3-20 mils (thousandths of an inch).
  • the pitch of the first linear portion and the second linear portion may range from 3 to 20 mils (thousandths of an inch).
  • the width of the first linear portion and the width of the second linear portion are equal to the spacing between the first linear portion and the second linear portion.
  • FIG. 9 is a plan view showing another structure of the detecting electrode
  • FIG. 10 is a plan view showing another structure of the detecting electrode.
  • the detecting electrode structure provided in an example of the embodiment further includes setting.
  • the reference electrode At the reference electrode at the edge of the detection zone, since the reference electrode is disposed at the edge of the detection zone, the outer side of the first linear portion and the second linear portion, the reference electrode can provide a contrast in the process of acquiring the detection signal about the target substance. The polarity error of the working electrode is eliminated, thereby further improving the accuracy of the detection.
  • the material of the working electrode and the opposing electrode includes gold. Since the chemical nature of the gold element is stable and does not react with the liquid to be detected and has a lower impedance, the accuracy of the detection can be further improved.
  • other conductive materials such as platinum or indium tin oxide may also be used.
  • Figures 11a and 11b illustrate a detection orifice plate used in the method of the present invention, the detection orifice plate comprising: a cartridge body 200 and a detection electrode structure 100, the cartridge body 200 comprising a plurality of reaction wells 211, the size of the reaction wells Reference may be made to the design of a conventional 96-well plate. Of course, the present disclosure includes but is not limited thereto, and the size of the reaction well may be designed according to the concentration and kind of the liquid to be detected. As shown in FIGS.
  • the detecting electrode structure 100 is disposed at the bottom of the casing 200
  • the detecting electrode structure 100 may be the detecting electrode structure of any of the above-described first embodiment
  • the detecting portion 102 is disposed at the bottom of the reaction hole 211.
  • the bottom of the reaction well 211 is sealed.
  • the reaction cell bottom plate 101 of the detecting electrode structure 100 and the casing 200 can be made of the same material. Thereby, the liquid to be detected can be contained in the accommodating space composed of the detection zone 102 and the reaction well 211, thereby detecting the liquid to be detected.
  • the working electrode 103 may apply a square wave alternating voltage to form a vertical electric field perpendicular to the bottom surface of the through hole 211, first causing the charged substance including the target substance in the liquid to be detected from the respective positions of the reaction hole 211 to the reaction hole 211.
  • the bottom moves and moves to the working electrode 103 to enrich, so that the target substance can be combined with the probe on the working electrode 103 (a substance that can bind to the target substance, such as a DNA polymer molecule), and then the polarity of the voltage is converted to make it perpendicular to the pass.
  • the direction of the vertical electric field on the bottom surface of the hole 211 is reversed, so that other substances not charged with the probe in the charged substance in the liquid to be detected move from the bottom of the reaction hole 211 to the upper portion of the reaction hole 211, thereby causing other substances in the charged substance.
  • the substance not bound to the probe is away from the working electrode 103 (the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe); then, the opposite electrode 104 can acquire the detection signal about the target substance and detect
  • the signal output for example, the target substance bound to the probe reacts with a specific reagent to generate a current, so the opposite electrode 104 can pass the inspection.
  • the current is measured to obtain a detection signal about the target substance and the detection signal is output; then, the information about the target substance (for example, the concentration of the target substance) can be obtained by analyzing the output detection signal.
  • a plurality of reaction wells 211 are arranged in a matrix in the cartridge body 200.
  • the plurality of reaction holes 211 are cylindrical through holes.
  • the shape of the reaction well includes, but is not limited to, the shape of the plurality of reaction holes 211 may also be a square cylinder, a triangular cylinder or other cylinders.
  • the number of the plurality of reaction holes 211 is a multiple of four, as shown in FIG. 8, four adjacent reaction holes 211 correspond to the four working electrodes 103 in the detection electrode structure. Electrically connected.
  • the reaction hole bottom plate 101 is formed with a wire 111 and a wire 112.
  • the wire 111 electrically connects the four working electrodes 103.
  • the wires 112 are electrically connected to the four opposite electrodes 104 to extract the electrical signals of the opposite electrode 104.
  • the four adjacent through holes can be used as a detection group.
  • the voltages applied to the four working electrodes are uniform, and four adjacent through holes are The control experiment can be performed better, so the detection accuracy can be further improved.
  • any number of working electrodes in the detection electrode structure corresponding to any number of through holes may be electrically connected to provide a uniform voltage.
  • the detection aperture board used in the present invention further includes: a circuit board 110 electrically connected to the detection pole structure.
  • An amplification circuit may be disposed on the circuit board 110 to amplify the electrical signal outputted by the opposite electrode or the reference electrode to improve detection accuracy;
  • a voltage stabilization circuit may also be disposed on the circuit board 110 to provide a stable voltage to the working electrode to improve detection accuracy.
  • the present disclosure includes but is not limited thereto, and an overcurrent, overvoltage protection circuit, or the like may be disposed on the circuit board 110.
  • the circuit board 110 can be disposed under the reaction hole bottom plate 101, so that the space can be utilized more reasonably.
  • the circuit board 110 can also be disposed at other positions, and the disclosure is not limited herein.
  • virus genotyping detection kit provided by the present invention has the following advantages:
  • the conventional probe fixing method is to fix one end of the probe on the planar support. This method reduces the hybridization efficiency between the probe and the target DNA to be detected due to the hydrophobicity of the surface of the support, etc.
  • the invention fixes the capture probe in the polypyrrole hole by charge adsorption to ensure the ultra-high activity of the capture probe; the traditional nucleic acid hybridization process improves the hybridization efficiency by controlling the hybridization temperature, the salt ion, the reaction time, etc., and the invention increases the electric field.
  • the capture efficiency of the capture probe to the DNA of the target sequence is improved by the electric field; in this method, the electronic signal generated by the HRP-catalyzed oxidation of TMB is determined as a detection result, since the catalytic efficiency of the enzyme is very high.
  • High, indirect amplification of the results of the hybridization reaction increases the sensitivity of the assay.
  • the three core technologies of instantaneous target molecular capture, ultra-high activity molecular probe immobilization, and capture molecular signal specific amplification ensure that the method has ultra-high sensitivity, far higher than Pap smear, TCT technology and HC2 technology, and Polymerase chain reaction and gene chip and other technologies are equivalent.
  • each subtype of HPV detection process comprises a capture probe and a detection probe, and the probe length is between 25-40 bp, and the hybridization efficiency is affected by the mismatched base. Obviously, only the target sequence DNA and the two probes can be accurately paired at the same time to have a detection signal, which greatly improves the specificity of the detection.
  • the sample does not need to be pre-processed such as purification and PCR amplification, thereby effectively avoiding false positives caused by environmental pollution, and the test operation does not need to be specialized.
  • the operator does not need to obtain the permission for clinical gene amplification, and can be operated by general technicians, and the requirements for the experimental environment and the quality of the operators are low.
  • EFIRM technology is based on the principle of nucleic acid hybridization, using a uniquely designed electrochemical technique.
  • the nucleic acid probe used in the present invention has a length of 25-40 bp, and is selected from the E7 region or the L1 region with a large difference between the HPV subtypes, and artificial synthetic oligonucleotide probes are used, wherein the CP does not need to be modified, DP
  • Biotin modification method the preparation of the probe is completed by a commercial DNA chemical synthesis company, which has low technical difficulty and good stability, and some subtypes share CP or DP, which reduces the total number of probes and reduces the cost. .
  • PCR-based fluorescence quantification requires both ends of the probe to be modified, and one end is a fluorescent group, and the synthesis cost is high; reverse dot blot hybridization and gene chip technology mostly need to immobilize the hybridization probe to the solid phase carrier, and the treatment process Complex, increasing the cost of testing, and the probe passes The immobilization activity is reduced to a certain extent; the probe used for HC2 is a full-length RNA probe with a length of 7000-8000 bases.
  • the preparation process is complicated, time-consuming, costly, and because the probe is very long, hybridization Efficiency is less affected by mismatched bases, and subtypes may cross.
  • the EFIRM technology saves the steps of DNA extraction and purification during the detection process, so the cost of the detection reagent is greatly reduced compared to other techniques.
  • the EFIRM-based HPV probe detection method has the characteristics of high sensitivity, high specificity, short detection time, low experimental site requirement, low cost, and the like, and is suitable for a large number of clinical tests and large-scale epidemics. Pathological screening.

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Abstract

L'invention concerne une combinaison de sondes permettant la détection de génotypage du papillomavirus humain (HPV) au moyen d'un kit, d'un procédé et d'une technique EFIRM, se rapportant au domaine de la technologie de détection de gènes. La combinaison de sondes comprend une pluralité de paires de sondes, et chaque paire de sondes est dirigée vers un génotype du HPV, peut détecter le génotype du HPV dans un échantillon à détecter, et présente une spécificité relativement forte, une sensibilité relativement élevée et un taux de faux positifs relativement faible. Les résultats de la détection sont précis et fiables.
PCT/CN2017/104829 2016-09-30 2017-09-30 Sonde permettant la détection de génotypage du papillomavirus humain au moyen d'un kit, utilisation et technique efirm Ceased WO2018059581A1 (fr)

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CN201610877670.0 2016-09-30
CN201610877670.0A CN106282413B (zh) 2016-09-30 2016-09-30 Hpv高危毒株基因分型检测的探针组合、试剂盒以及方法
CN201610877702.7 2016-09-30
CN201610877702.7A CN106399589B (zh) 2016-09-30 2016-09-30 一种用于病毒基因分型检测的探针组合、试剂盒以及方法
CN201610881088.1 2016-09-30
CN201610881088.1A CN106367536A (zh) 2016-09-30 2016-09-30 一种基于efirm技术hpv病毒检测的探针组合、试剂盒以及方法

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CN116884550A (zh) * 2023-06-28 2023-10-13 广州凯普医学检验所有限公司 多平台hpv分型检测结果智能交互校验审核及处置指引系统

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CN111218520A (zh) * 2019-10-26 2020-06-02 大连海关技术中心 转基因大豆gts-40-3-2品系efirm检测探针及其应用
CN116884550A (zh) * 2023-06-28 2023-10-13 广州凯普医学检验所有限公司 多平台hpv分型检测结果智能交互校验审核及处置指引系统
CN116884550B (zh) * 2023-06-28 2024-03-22 广州凯普医学检验所有限公司 多平台hpv分型检测结果智能交互校验审核及处置指引系统

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