CN116240303A - Nucleic acid detection method and kit for distinguishing pathogen DNA/RNA - Google Patents

Abstract

The invention discloses a nucleic acid detection method for distinguishing pathogen DNA/RNA and a kit, wherein an artificial sequence is introduced into the 5 'end of a primer to improve the specificity of the primer, the artificial sequence is not complementary with a template, the artificial sequence is complementary with the 3' end sequence of the primer to form a stem-loop structure, when the template is incompletely complementary with the 3 'end of the primer, the primer exists in the stem-loop structure, and when the template is completely complementary with the 3' end of the primer, the stem-loop structure is melted for reverse transcription; during the amplification of RNA, the synthesized cDNA is provided with the artificial sequence, the primer is completely complementary with the cDNA during the amplification, 7-11 bases are complementary with the template at the 3 'end of the primer, and deoxyuracil is used for replacing deoxythymine bases at the 3' end of the primer. The invention can not amplify DNA under a certain annealing temperature because the Tm value of primer binding is low when amplifying DNA, and can accurately detect RNA because the 5' end of reverse transcription primer has artificial sequence when amplifying RNA, so that the synthesized cDNA has the artificial sequence, and the primer can be completely complementary with cDNA when amplifying.

Description

A nucleic acid detection method and kit for distinguishing pathogen DNA/RNA

Technical Field

The present invention belongs to the technical field of molecular biology, and in particular relates to a nucleic acid detection method and a kit for distinguishing pathogen DNA/RNA.

Background Art

Pathogens refer to infectious microorganisms or biological pathogens that can cause diseases after invading the host. They include bacteria, fungi, viruses, viroids, etc. Food, water sources, soil, etc. can carry pathogens. If people are exposed to pathogen environments for a long time or eat food or water sources contaminated by pathogens, they will develop diseases in the human body. Pathogen infection can cause a variety of diseases in the human respiratory tract, digestive tract, reproductive tract, etc. Timely screening of pathogens is of great significance to the treatment of diseases.

At present, most pathogen diagnosis methods mainly include culture method, immunological diagnosis method and molecular biological diagnosis method. Culture method is a routine laboratory diagnosis method for pathogens. Traditional culture method has high specificity and sensitivity, but the clinical detection time is long (at least 24-48h), the process is cumbersome, and the positive rate is affected by many factors, which is not suitable for large-scale detection. Due to the existence of the window period, the serological method has a lag in diagnosis and cannot accurately reflect whether the current infection is present. Although the specificity is high, the sensitivity is not enough and the detection rate is low. Molecular diagnostic methods include gene sequence sequencing and nucleic acid detection methods, among which gene sequence sequencing has the disadvantages of high cost, complicated and cumbersome steps, and long time consumption, which is not suitable for clinical detection. The pathogen nucleic acid fluorescence PCR detection method has the advantages of rapidity, accuracy and high sensitivity. However, due to the stable structure of DNA, it can remain positive for a long time after the death of bacteria, and it is impossible to judge the specific stage of pathogen infection, resulting in overtreatment. RNA has a short half-life and will degrade quickly after the bacteria die. Testing pathogen RNA alone can distinguish the activity of bacteria. At the same time, since RNA exists in multiple copies in non-viral pathogen microbial cells, it has higher sensitivity and accuracy than technologies such as PCR that target DNA. RNA not only has the advantages of rapid and high sensitivity of nucleic acid detection, but can also screen out dead bacteria, providing more accurate detection, and is suitable for clinical screening and diagnosis.

As for RNA detection technology, one of the main challenges at present is how to eliminate DNA interference in samples, maintain the stability of RNA, and ensure sensitivity and accuracy.

RT-qPCR technology is a mature molecular detection technology that was used earlier in clinical testing and is widely used in clinical practice. However, it has certain limitations when used for some RNA detection. The main reasons are: ① The DNA digestion step inevitably leads to RNA loss, reducing the sensitivity of the detection; ② When the elimination is not thorough, the residual DNA and RNA are amplified together, affecting the accuracy of quantification.

At present, the most commonly used RNA detection method abroad is the transcription mediated amplification (TMA) technology launched by Gen-Probe in 1995. The MMLV reverse transcriptase and T7 RNA polymerase used in this technology can be amplified under constant temperature conditions, and the amplified product RNA is then subjected to endpoint qualitative detection by a matching hybridization protection test. This technology can inspect RNA and has high sensitivity, but the endpoint TMA method is prone to contamination of amplified RNA during actual application, which often results in false positive experimental results, making this technology have great limitations in actual clinical testing.

The Simultaneous Amplification and Testing (SAT) technology launched by Shanghai Rendu has an amplification principle similar to TMA, but SAT introduces molecular beacons during the detection process, and does not require opening the lid after amplification for endpoint testing, which reduces the possibility of contamination to a certain extent. However, this technology is unstable and is easily affected by a variety of factors, causing amplification failure. During use, it is also necessary to open the lid after reverse transcription and add T7 RNA polymerase, which is cumbersome to operate. Limited by the number of detection fluorescence channels, there are currently no multiple detection products on the market.

The patent of Shanghai Kehua Bioengineering Co., Ltd. (application publication number CN 101948908 A) proposes to modify the primers based on RT-PCR, introduce an auxiliary oligonucleotide sequence, the 3' end of the sequence is complementary to RNA, and introduce an artificial sequence at the 5' end. During the reverse transcription process of the RNA sample, the artificial sequence in the primer is introduced into the cDNA. During amplification, the artificial sequence is completely complementary to the cDNA and can be amplified normally. However, this method has certain limitations. It requires the auxiliary oligonucleotide sequence to be reverse transcribed first, and then the artificial sequence is used for amplification, which is a relatively cumbersome process.

Therefore, the art urgently needs an accurate, sensitive and convenient method for detecting pathogen nucleic acid, which can specifically detect RNA and has high sensitivity. In view of the above situation, the present invention establishes a PCR method and kit for specifically detecting RNA through unique primer design.

Summary of the invention

In order to overcome the problems of poor sensitivity of existing DNA amplification and inability to distinguish between live and dead bacteria, low efficiency, easy contamination, poor stability, cumbersome operation, and high price of RNA amplification technology, the present invention establishes a nucleic acid detection method and kit for distinguishing pathogen DNA/RNA with high sensitivity, strong specificity, and convenient operation on the basis of good stability and high amplification efficiency.

In order to achieve the above object, the present invention adopts the following technical solutions:

The present invention first provides a nucleic acid detection method for distinguishing pathogen DNA/RNA, which comprises: introducing an artificial sequence at the 5' end of a primer to improve the primer specificity, wherein the artificial sequence is not complementary to a template, and the artificial sequence is complementary to a sequence at the 3' end of the primer to form a stem-loop structure, and when the template and the 3' end of the primer are not completely complementary, the primer exists in a stem-loop structure, and when the template and the 3' end of the primer are completely complementary, the stem-loop structure is melted for reverse transcription; when amplifying RNA, the synthesized cDNA is provided with the artificial sequence, and during amplification, the primer and the cDNA are completely complementary.

As a preferred embodiment of the present invention, the 3' end of the primer has 7-11 bases complementary to the template.

As a preferred embodiment of the present invention, deoxyuracil is used at the 3' end of the primer instead of deoxythymine base.

In the present invention, the present invention modifies the primers on the basis of the relatively mature RT-qPCR technology to realize the technology of specifically detecting RNA without the DNA digestion step: the present invention is inspired by the different amplification efficiencies of primers with different binding Tm values at a certain annealing temperature. Through practice, it is found that when the 3' end of the primer is only 7-11 complementary bases to the template, at an annealing temperature of 58-64°C, DNA is basically not amplified. Experiments have found that the fewer the number of binding bases, the lower the DNA amplification efficiency. When the 3' end is only 7-11 complementary bases to the template, there is no significant effect on transcription, but as the number of binding bases decreases, the reverse transcription efficiency will be slightly affected. Studies have found that deoxyuracil can be inserted into oligonucleotides to increase the melting point temperature of the double-stranded chain, thereby increasing the stability of the double-stranded chain and reducing the effect of the small number of bases on the transcription efficiency. Therefore, in order to make DNA unable to amplify and not affect the reverse transcription efficiency, the inventors use deoxyuracil to replace the effect of the small number of deoxythymine bases on the transcription efficiency based on the fact that the 3' end of the primer is only 7-11 complementary bases to the template.

At the same time, an artificial sequence is introduced at the 5' end. In order to minimize the amplification efficiency of DNA and improve the specificity of primers, the artificial sequence must have the following characteristics:

1) The sequence is not complementary to the template;

2) This artificial sequence can complement the 3’ end sequence of the primer to form a simple stem-loop structure. When the template and the 3’ end of the primer are not completely complementary, the primer exists in a stem-loop structure. When the template and the 3’ end of the primer are completely complementary, the stem-loop structure melts and reverse transcription is performed.

At this time, at a certain annealing temperature, when amplifying DNA, the DNA cannot be amplified due to the low Tm value of primer binding. When amplifying RNA, since the 5' end of the reverse transcription primer carries an artificial sequence, the synthesized cDNA carries this artificial sequence. During amplification, the primer can be completely complementary to the cDNA, and RNA can be accurately detected.

As a preferred embodiment of the present invention, the pathogen is a Gram-positive pathogen, a Gram-negative pathogen or a fungal pathogen.

As a preferred embodiment of the present invention, when the pathogen is a Gram-positive pathogen, the sequence of the downstream primer is shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, the sequence of the upstream primer is shown in SEQ ID NO: 2, and the sequence of the probe is shown in SEQ ID NO: 7.

As a preferred embodiment of the present invention, when the pathogen is a Gram-negative pathogen, the sequence of the downstream primer is shown in SEQ ID NO: 10, the sequence of the upstream primer is shown in SEQ ID NO: 9, and the sequence of the probe is shown in SEQ ID NO: 11.

As a preferred embodiment of the present invention, when the pathogen is a fungal pathogen, the sequence of the downstream primer is shown as SEQ ID NO: 13, the sequence of the upstream primer is shown as SEQ ID NO: 14, and the sequence of the probe is shown as SEQ ID NO: 15.

The present invention also provides a nucleic acid detection kit for distinguishing pathogen DNA/RNA, comprising the above primers.

Compared with the prior art, the present invention has the following beneficial effects:

1) The present invention introduces an artificial sequence at the 5' end of the primer to minimize the amplification efficiency of DNA and improve the primer specificity.

2) The present invention uses deoxyuracil to replace deoxythymine on the basis that the number of complementary bases between the 3' end of the primer and the template is only 7-11, which affects the transcription efficiency.

3) Under a certain annealing temperature, when amplifying DNA, the present invention cannot amplify DNA due to the low Tm value of primer binding. However, when amplifying RNA, since the 5' end of the reverse transcription primer carries an artificial sequence, the synthesized cDNA carries the artificial sequence. During amplification, the primer can be completely complementary to the cDNA, and RNA can be accurately detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the principle of the present invention.

FIG. 2 shows the amplification of different samples of the present invention using modified primers in the RT-qPCR reaction system.

FIG3 is an amplification curve of Example 1 at 10 ⁴ copies/ml GBS.

FIG. 4 is an amplification curve of Example 1 at 10 ³ copies/ml GBS.

FIG. 5 is an amplification curve of Example 2 at 10 ³ copies/ml NG.

FIG. 6 is an amplification curve of Example 2 at 10 ² copies/ml NG.

FIG. 7 is an amplification curve of Example 3 at 10 ⁴ copies/ml of Candida albicans.

FIG. 8 is an amplification curve of Example 3 at 10 ³ copies/ml of Candida albicans.

DETAILED DESCRIPTION

In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

Referring to Figures 1 and 2, the present invention provides a nucleic acid detection method for distinguishing pathogen DNA/RNA, the nucleic acid detection method comprising: introducing an artificial sequence at the 5' end of the primer to improve the primer specificity, the artificial sequence is not complementary to the template, the artificial sequence is complementary to the 3' end sequence of the primer to form a stem-loop structure, when the template and the 3' end of the primer are not completely complementary, the primer exists in a stem-loop structure, when the template and the 3' end of the primer are completely complementary, the stem-loop structure is melted for reverse transcription; when amplifying RNA, the synthesized cDNA is made to carry the artificial sequence, and the primer and the cDNA are completely complementary during amplification.

The 3’ end of the primer has 7-11 bases that are complementary to the template.

Deoxyuracil was used instead of deoxythymine base at the 3’ end of the primer.

The present invention is inspired by the fact that different amplification efficiencies are caused by different binding Tm values of primers at a certain annealing temperature. It is found through practice that when the 3' end of the primer has only 7-11 complementary bases to the template, at an annealing temperature of 58-64°C, there is basically no amplification of DNA. Experiments have found that the fewer the number of binding bases, the lower the DNA amplification efficiency. When the 3' end has only 7-11 complementary bases to the template, there is no significant effect on transcription, but as the number of binding bases decreases, the reverse transcription efficiency will be slightly affected. Studies have found that deoxyuracil can be inserted into oligonucleotides to increase the melting point temperature of the double-stranded chain, thereby increasing the stability of the double-stranded chain and reducing the effect of a small number of bases on the transcription efficiency. Therefore, in order to prevent DNA from being amplified and not affecting the reverse transcription efficiency, the inventors use deoxyuracil to replace the effect of a small number of deoxythymine bases on the transcription efficiency when the 3' end of the primer has only 7-11 complementary bases to the template.

At the same time, an artificial sequence is introduced at the 5' end. In order to minimize the amplification efficiency of DNA and improve the specificity of primers, the artificial sequence must have the following characteristics:

1) The sequence is not complementary to the template;

2) This artificial sequence can complement the 3’ end sequence of the primer to form a simple stem-loop structure. When the template and the 3’ end of the primer are not completely complementary, the primer exists in a stem-loop structure. When the template and the 3’ end of the primer are completely complementary, the stem-loop structure melts and reverse transcription is performed.

At this time, at a certain annealing temperature, when amplifying DNA, the DNA cannot be amplified due to the low Tm value of primer binding. When amplifying RNA, since the 5' end of the reverse transcription primer carries an artificial sequence, the synthesized cDNA carries this artificial sequence. During amplification, the primer can be completely complementary to the cDNA, and RNA can be accurately detected.

Example 1

Gram-positive pathogen RNA detection

This example takes Group B Streptococcus RNA detection as an example:

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a facultative anaerobic Gram-positive coccus. According to the different capsular polysaccharides of the bacteria, GBS can be divided into Ia, Ib, Ic, II, III, IV, V, VI, VII, VIII and IX. GBS can intermittently, transiently or continuously colonize the digestive tract and reproductive tract. GBS colonization in pregnant women refers to the positive GBS culture in the vagina, rectum or perianal sampling during pregnancy. GBS is a conditional pathogen that can change from a colonized state to a pathogen under certain conditions, causing invasive GBS disease in pregnant women or newborns. Invasive GBS disease refers to the positive GBS culture in the sampling of sterile sites under normal circumstances, accompanied by relevant clinical manifestations.

The GBS determination rate among pregnant women varies in different countries and regions. A meta-analysis shows that the GBS determination rate was 11.3% among 44,716 pregnant women reported in my country from 2000 to 2018.

If GBS colonization in pregnant women is not intervened, 50% of them will be vertically transmitted to the fetus or newborn, which is an important cause of early-onset GBS disease (GBS-EOD) in newborns, and can cause neonatal sepsis and neonatal meningitis. Therefore, establishing an effective and rapid screening method for GBS in pregnant women is of great significance to reduce GBS infection in newborns.

The specific RNA detection method is as follows:

1. Extraction of Group B Streptococcus RNA

RNA was extracted from the collected swabs or urine using our company's RNA nucleic acid extraction kit.

2. Design of primers and probes for Group B Streptococcus

The present invention downloads GBS 16S sequence from NCBI database and performs homology analysis. By comparing a large number of target genes with similar sequences, a region with high conservation is selected for primer probe design. When designing primers, an artificial sequence is introduced at the 5' end of the upstream primer (the bold part of the primer is the introduced artificial sequence, which is not complementary to the bold sequence in the template). The template sequence is as follows:

SEQ ID NO: 1:

CGGCAATGGACGGAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGAT

CGTAAAGCTCTGTTGTTAGAGAAGAACGTTGGTAGGAGTGGAAAATCTACCAAGTGAC

GGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTTGTCCGGATTTATTG.

Primer and probe sequences are shown in Table 1.

Table 1. Primer probe sequences

The above primers were used to test RNA positive samples, DNA positive samples, and negative controls, and qRT-PCR enzyme and DNA amplification enzyme were used for testing. RNA positive samples were RNA dilutions of GBS 16s transcribed using T7 promoter; DNA positive samples were synthetic GBS 16s plasmid dilutions; and negative controls were normal saline.

Experimental results show

DNA amplification enzyme amplification results of the control group:

1) After the artificial sequence was introduced into the downstream primer, the amplification efficiency of the positive sample was significantly reduced, while the common primer could amplify the positive sample normally (comparison between R1 and Ra11); 2) As the number of complementary bases decreased, the downstream primers that introduced the artificial sequence had basically no amplification of the positive sample (comparison between Ra11 and Ra9); 3) Deoxyuracil was used instead of deoxythymine on the basis of Ra9, and the positive sample was also not amplified (comparison between Ra9 and Ra9u).

qRT-PCR enzyme amplification results of the experimental group (see Table 2).

1) After the artificial sequence was introduced into the downstream primers, the amplification efficiency of DNA-positive samples was significantly reduced, while there was no significant change in RNA-positive samples. Ordinary primers could amplify RNA and DNA-positive samples normally (comparison between R1 and Ra11); 2) As the number of complementary bases decreased, the amplification efficiency of DNA-positive samples was further reduced when the artificial sequence of the downstream primers was introduced, and the amplification efficiency of RNA-positive samples was also slightly reduced (comparison between Ra11 and Ra9); 3) Deoxyuracil was used instead of deoxythymine on the basis of Ra9, and there was no amplification of DNA-positive samples, while the efficiency of RNA-positive samples was slightly increased (comparison between Ra9 and Ra9u).

Table 2. Amplification results

3. Preparation of PCR reaction system for Group B Streptococcus RNA detection kit

(1) Take the kit out of the refrigerator, equilibrate to room temperature, fully dissolve all components, and centrifuge quickly for 5 seconds.

(2) Calculate the number of reactions required for the experiment (n = number of samples + 2T (control), and prepare the reaction system according to the following table: Prepare the reaction solution according to the system in Table 3.

Table 3. GBS RNA detection reaction system configuration

3. Procedure for using the Group B Streptococcus RNA Detection Kit and interpretation of test results

On-line procedures

Set up the temperature cycle and signal acquisition program according to the steps in Table 4.

Table 4. Temperature cycle and signal acquisition program

Fluorescence channel selection during detection: Select FAM and ROX fluorescence detection channels.

Test result interpretation

Instrument Setup

1. After the reaction is completed, according to the amplification curve, the appropriate baseline (generally the start is set to 3 and the end is set to 15) and the fluorescence threshold (generally the threshold is set in the middle of the exponential growth period in the logarithmic form of the amplification curve) are defined to obtain the CT values of different channels.

2. When analyzing the results, add background signal correction and set Passive reference to None.

Quality Control

The following conditions must be met simultaneously for a successful PCR reaction:

Negative control: no amplification in the FAM detection channel or Ct(FAM)>38; Ct(ROX)≤35.

Positive quality control: FAM detection channel has an amplification curve and Ct(FAM)≤38; ROX detection channel has or does not have an amplification curve.

When any of the above conditions is not met, the PCR reaction is deemed to have failed, the sample test results are invalid and need to be repeated.

Interpretation of results

If the FAM detection channel has an amplification curve and Ct(FAM)≤38 and the ROX detection channel has or does not have an amplification curve, the sample can be judged to be GBS positive.

If there is no amplification in the FAM detection channel or Ct(FAM)>38; if Ct(ROX)≤35 in the ROX detection channel, the sample can be judged as GBS negative.

If there is no amplification in the FAM detection channel or Ct(FAM)>38; there is no amplification in the ROX detection channel or Ct(ROX)>35, the PCR reaction failed. There may be inhibitors in the RNA sample preparation process. It is recommended to dilute the RNA 10 times or 100 times and repeat the measurement. If there is a fluorescent signal, judge the result according to the above. Otherwise, it is recommended to repeat the measurement.

5. Experimental results

Results of low concentration samples tested with the Group B Streptococcus RNA Detection Kit

The low copy samples were tested according to the above method. As shown in Figures 3 and 4, the kit can accurately detect samples with 10 ³ copies/ml (Note: HBB is an internal reference).

Group B Streptococcus RNA Detection Kit Specificity Detection

RNA detection for other pathogens

In the above manner, samples of bacteria closely related to group B streptococci, microorganisms that are likely to cause the same or similar clinical symptoms, and bacteria colonizing the infection site are tested: Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, herpes simplex virus, Candida albicans, Mycoplasma hominis, etc.

The test showed that except for the strain of group B streptococcus, the rest of the strains were negative.

Group B Streptococcus RNA Detection Kit for GBS DNA Detection

According to the above method, 10 ⁶ copies/ml GBS bacterial solution and 10 ⁶ copies/ml GBS bacterial solution inactivated by high concentration antibiotics were tested respectively. At the same time, a competitive DNA kit was used for comparative testing (see Table 5). The results showed that the GBS bacterial solution of the present invention can be amplified normally, while the GBS bacterial solution inactivated by antibiotics has no amplification signal, and the competitive DNA kit has signals before and after GBS inactivation.

Table 5. Comparative test results

Group B Streptococcus RNA Detection Kit Clinical Sample Testing

150 clinical samples were examined using the kit, culture method and DNA detection method respectively. The results are shown in Tables 6 and 7.

Table 6. Comparison of DNA amplification and culture methods

Culture positive Culture negative total DNA amplification positive 30 6 36 DNA amplification negative 3 111 114 total 33 117 150

Table 7. Comparison of Group B Streptococcus RNA Detection Kit and Culture Method

Culture positive Culture negative total RNA amplification positive 31 3 34 RNA amplification negative 2 114 116 total 33 117 150

Using the gold standard of culture method, the sensitivity of DNA detection method was 90.9% (30/33), and the specificity was 94.9% (111/117); using the gold standard of culture method, the sensitivity of the group B streptococcus RNA detection kit was 93.9% (31/33), and the specificity was 97.4% (114/117). The results showed that the sensitivity and specificity of RNA detection method were higher than those of DNA detection method.

Embodiment 2:

Gram-negative pathogen RNA detection

This example takes gonococcal RNA detection as an example:

Gonorrhea is one of the main sexually transmitted diseases in my country. It is caused by infection with Neisseria gonorrhoeae (NG). It is characterized by a short incubation period and strong infectiousness. If it is not cured in time, serious complications and sequelae may occur. According to clinical manifestations, it is divided into symptomatic urogenital infection (commonly manifested as suppurative inflammation), asymptomatic urogenital infection, infection of the eyes, pharynx, skin, rectum, pelvic cavity and other parts, and hematogenous disseminated infection. The common manifestation in men is urethritis, and complications include epididymitis, prostatitis, seminal vesiculitis, etc.; the common manifestation in women is cervicitis, urethritis, vestibular glanditis, perianal inflammation, and complications include pelvic inflammatory disease; it can also induce decreased fertility and neonatal conjunctivitis.

Establishing an effective and rapid method for detecting gonococcal RNA is of great significance for the treatment of gonorrhea. The specific RNA detection method is as follows:

1. Extraction of Neisseria gonorrhoeae RNA

RNA was extracted from the collected swabs or urine using our company's RNA nucleic acid extraction kit.

2. Design of Neisseria gonorrhoeae Primer Probe

The present invention downloads the gonococcal 16S sequence from the NCBI database and performs homology analysis. By comparing a large number of target genes with similar sequences, a region with high conservation is selected for primer probe design. When designing the primer, an artificial sequence is introduced at the 5' end of the upstream primer (the bold part of the primer is the introduced artificial sequence, which is not complementary to the bold sequence in the template), and the template sequence is as follows:

SEQ ID NO: 8:

CCTGATCCAGCCATGCCGCGTGTCTGAAGAAGGCCTTCGGGTGTAAAGGACTTTTGTCAGGGAAGAAAAGGCTGTTGCCAATATCGGCGGCCGATGACGGT.

The primer and probe sequences are shown in Table 8.

Table 8. Primer probe sequences

name Sequence (5'-3') NG-F (SEQ ID NO: 9) TGATCCAGCCATGCCGCGTGTC NG-Ra (SEQ ID NO: 10) ccaaCGTGATTGGTAACA/ideoxyU/AT/ideoxyU/GG NG-P (SEQ ID NO: 11) CGGGTTGTAAAGGACTTTT

3. Preparation of PCR reaction system for Neisseria gonorrhoeae RNA detection kit

(1) Take the kit out of the refrigerator, equilibrate to room temperature, fully dissolve all components, and centrifuge quickly for 5 seconds.

(2) Calculate the number of reactions required for the experiment (n = number of samples + 2T (control), and prepare the reaction system according to the following table: Prepare the reaction solution according to the system in Table 9.

Table 9. Neisseria gonorrhoeae RNA detection reaction system

4. Procedures for using the Neisseria gonorrhoeae RNA test kit and interpretation of test results

On-line procedures

Set up the temperature cycle and signal acquisition program according to the steps in Table 10.

Table 10. Temperature cycle and signal acquisition program

Fluorescence channel selection during detection: Select FAM and ROX fluorescence detection channels.

Test result interpretation

Instrument Setup

1. After the reaction is completed, according to the amplification curve, the appropriate baseline (generally the start is set to 3 and the end is set to 15) and the fluorescence threshold (generally the threshold is set in the middle of the exponential growth period in the logarithmic form of the amplification curve) are defined to obtain the CT values of different channels.

2. When analyzing the results, add background signal correction and set Passive reference to None.

Quality Control

The following conditions must be met simultaneously for a successful PCR reaction:

Negative control: No amplification in FAM detection channel or Ct(FAM)>38; Ct(ROX)≤35

Positive quality control: FAM detection channel has an amplification curve and Ct(FAM)≤38; ROX detection channel has or does not have an amplification curve.

When any of the above conditions is not met, the PCR reaction is deemed to have failed, the sample test results are invalid and need to be repeated.

Interpretation of results

If the FAM detection channel has an amplification curve and Ct(FAM)≤38 and the ROX detection channel has or does not have an amplification curve, the sample can be judged to be positive for gonococci.

If there is no amplification in the FAM detection channel or Ct(FAM)>38; if Ct(ROX)≤35 in the ROX detection channel, the sample can be judged as negative for gonococci.

If there is no amplification in the FAM detection channel or Ct(FAM)>38; there is no amplification in the ROX detection channel or Ct(ROX)>35, the PCR reaction failed. There may be inhibitors in the RNA sample preparation process. It is recommended to dilute the RNA 10 times or 100 times and repeat the measurement. If there is a fluorescent signal, judge the result according to the above. Otherwise, it is recommended to repeat the measurement.

Experimental Results

Gonococcal RNA detection kit test results of low concentration samples

The low copy samples were tested according to the above method. As shown in FIG5 and FIG6 , the kit can accurately detect samples with 10 ² copies/ml (Note: HBB is an internal reference).

Gonococcal RNA detection kit specific detection

RNA detection for other pathogens

In the above manner, samples of bacteria closely related to gonococci, microorganisms that are likely to cause the same or similar clinical symptoms, and bacteria colonizing the infection site are tested: Streptococci, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Chlamydia trachomatis, Ureaplasma urealyticum, herpes simplex virus, Candida albicans, and Mycoplasma hominis.

The test showed that except for the gonococcal strain which was positive, the other strains were negative.

Gonorrhea RNA detection kit for gonococcal DNA detection

According to the above method, 10 ⁶ copies/ml gonococci and 10 ⁶ copies/ml gonococci inactivated by high concentration antibiotics were tested respectively. At the same time, a competitive DNA kit was used for comparative testing (see Table 11). The results showed that the gonococci detected by the present invention can be amplified normally, while the gonococci inactivated by antibiotics have no amplification signal, and the competitive DNA kit has signals before and after the gonococci are inactivated.

Table 11. Comparative test results

Example 3

Fungal Pathogen RNA Detection

This example takes Candida albicans RNA detection as an example:

Candidiasis is an acute, subacute or chronic infection caused by Candida albicans, and is the most common fungal disease. It often invades the skin and mucous membranes, and can also cause visceral or systemic infection. The clinical symptoms are complex and vary in severity. Children are mostly acute secondary infections. In recent years, with the use of large doses of antibiotics, hormones, and immunosuppressants, as well as the development of organ transplantation, its incidence has gradually increased, and it can be life-threatening and cause serious consequences.

The currently used microscopic examination, culture method and serological method for detecting fungal antigens and antibodies or metabolites lack sufficient sensitivity and specificity. Early detection and treatment can reduce the mortality rate. Therefore, it is very important to establish a rapid and accurate early detection method for fungal infection. The specific RNA detection method is as follows:

1. Extraction of RNA from Candida albicans

RNA was extracted from the collected swabs or urine using our company's RNA nucleic acid extraction kit.

2. Design of Primer Probes for Candida albicans

The present invention downloads the Candida albicans 18S sequence from the NCBI database and performs homology analysis. By comparing a large number of target genes with similar sequences, a region with high conservation is selected for primer probe design. When designing the primer, an artificial sequence is introduced at the 5' end of the upstream primer (the bold part of the primer is the introduced artificial sequence, which is not complementary to the bold sequence in the template), and the template sequence is as follows:

SEQ ID NO: 12:

CAAGAACGAAAGTTAGGGGATCGAAGATGATCAGATACCGTCGTAGTCTTAACCAT AAACTATGCCGACTAGGGATCGGTTGTTGTTCTTTTATTGACGCAATCGGCACCTTACGA GAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTT.

The primer and probe sequences are shown in Table 12.

Table 12. Primer probe sequences

3. Preparation of PCR reaction system for Candida albicans RNA detection kit

(1) Take the kit out of the refrigerator, equilibrate to room temperature, fully dissolve all components, and centrifuge quickly for 5 seconds.

(2) Calculate the number of reactions required for the experiment (n = number of samples + 2T (control), and prepare the reaction system according to the following table: Prepare the reaction solution according to the system in Table 13.

Table 13. Candida albicans RNA detection reaction system

Candida albicans RNA detection kit on-line procedure and test result interpretation

On-line procedures

Set up the temperature cycle and signal acquisition program according to the steps in Table 14.

Table 14. Temperature Cycle and Signal Acquisition Procedure

Fluorescence channel selection during detection: Select FAM and ROX fluorescence detection channels.

Test result interpretation

Instrument Setup

1. After the reaction is completed, according to the amplification curve, the appropriate baseline (generally the start is set to 3 and the end is set to 15) and the fluorescence threshold (generally the threshold is set in the middle of the exponential growth period in the logarithmic form of the amplification curve) are defined to obtain the CT values of different channels.

2. When analyzing the results, add background signal correction and set Passive reference to None.

Quality Control

The following conditions must be met simultaneously for a successful PCR reaction:

Negative control: no amplification in the FAM detection channel or Ct(FAM)>38; Ct(ROX)≤35.

Positive quality control: FAM detection channel has an amplification curve and Ct(FAM)≤38; ROX detection channel has or does not have an amplification curve.

When any of the above conditions is not met, the PCR reaction is deemed to have failed, the sample test results are invalid and need to be repeated.

Interpretation of results

If the FAM detection channel has an amplification curve and Ct(FAM)≤38 and the ROX detection channel has or does not have an amplification curve, the sample can be judged to be positive for Candida albicans.

If there is no amplification in the FAM detection channel or Ct(FAM)>38; if Ct(ROX)≤35 in the ROX detection channel, the sample can be judged as negative for Candida albicans.

If there is no amplification in the FAM detection channel or Ct(FAM)>38; there is no amplification in the ROX detection channel or Ct(ROX)>35, the PCR reaction failed. It may be that there are inhibitors in the RNA sample preparation process. It is recommended to dilute the RNA 10 times or 100 times and repeat the measurement. If there is a fluorescent signal, follow the above judgment result. Otherwise, it is recommended to repeat the measurement.

Experimental Results

Candida albicans RNA detection kit test results of low concentration samples

The low copy samples were tested according to the above method. As shown in FIG7 and FIG8 , the kit can accurately detect samples with 10 ³ copies/ml (Note: HBB is an internal reference).

Candida albicans RNA detection kit specific detection

RNA detection for other pathogens

The samples of closely related bacteria of Candida albicans, microorganisms that are likely to cause the same or similar clinical symptoms, and colonizing bacteria at the infection site were tested in the above manner: Trichomonas vaginalis, Streptococcus, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, herpes simplex virus, Mycoplasma hominis, etc.

The test showed that except for the strain of Candida albicans which was positive, the other strains were negative.

Candida albicans RNA detection kit for DNA detection

According to the above method, 10 ⁶ copies/ml of Candida albicans liquid and 10 ⁶ copies/ml of Candida albicans liquid inactivated by high concentration antibiotics were tested respectively. At the same time, a competitive DNA kit was used for comparative testing (see Table 15). The results showed that the Candida albicans liquid of the present invention can be amplified normally, while the Candida albicans liquid inactivated by antibiotics has no amplification signal, and the competitive DNA kit has signals before and after Candida albicans inactivation.

Table 15. Comparative test results

The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims (8)

1. A method of nucleic acid detection for pathogen DNA/RNA discrimination, the method comprising: introducing a section of artificial sequence at the 5 'end of the primer to improve the specificity of the primer, wherein the artificial sequence is not complementary with the template, the artificial sequence is complementary with the 3' end sequence of the primer to form a stem-loop structure, the primer exists in the stem-loop structure when the template is not fully complementary with the 3 'end of the primer, and the stem-loop structure is melted for reverse transcription when the template is fully complementary with the 3' end of the primer; in the amplification of RNA, the synthesized cDNA is carried with the artificial sequence, and the primer is completely complementary to the cDNA in the amplification.

2. The method of claim 1, wherein the primer has 7-11 bases at its 3' end complementary to the template.

3. The method of claim 1, wherein the 3' -end of the primer is a deoxyuracil instead of a deoxythymine base.

4. A method of nucleic acid detection for distinguishing between DNA/RNA of a pathogen according to claim 1 or 2 or 3, wherein the pathogen is a gram positive pathogen, a gram negative pathogen or a fungal pathogen.

5. The method of claim 4, wherein the sequence of the downstream primer is set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ id no:6, the sequence of the upstream primer is shown as SEQ ID NO:2, the sequence of the probe is shown as SEQ ID NO: shown at 7.

6. The method of claim 4, wherein the sequence of the downstream primer is set forth in SEQ ID NO:10, the sequence of the upstream primer is shown as SEQ ID NO:9, the sequence of the probe is shown as SEQ ID NO: 11.

7. The method of claim 4, wherein the sequence of the downstream primer is set forth in SEQ ID NO:13, the sequence of the upstream primer is shown as SEQ ID NO:14, the sequence of the probe is shown as SEQ ID NO: 15.

8. A nucleic acid detection kit for distinguishing between DNA/RNA of a pathogen, comprising the primer of any one of claims 1-3.

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