CN116004917B - Fluorescence quantitative PCR primer probe set and kit for detecting avian influenza virus H3, H4 and H5 subtypes - Google Patents
Fluorescence quantitative PCR primer probe set and kit for detecting avian influenza virus H3, H4 and H5 subtypes Download PDFInfo
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Abstract
本发明公开了一种检测禽流感病毒H3、H4和H5亚型的荧光定量PCR引物探针组及试剂盒，以及含有上述引物探针组的荧光定量PCR试剂盒，该试剂盒还含有荧光定量PCR试剂、ddH2O以及阳性质粒标准品，能够同时检测出待测样本中是否含有禽流感病毒H3、H4和H5亚型。本发明所提供的试剂盒对H3、H4和H5亚型禽流感病毒具有良好的特异性；相比普通的PCR检测方法，本发明提供的试剂盒具有更高的灵敏度，对三种亚型的禽流感病毒的最低检测限均为2.1×102copies/μL；本发明提供的试剂盒还具有稳定性高、重复性良好的优点。因此，本发明可用于对禽流感病毒H3、H4和H5亚型的快速诊断和检测，对禽流感病毒H3、H4和H5亚型的防控有重要意义。
The present invention discloses a fluorescent quantitative PCR primer probe group and a kit for detecting H3, H4 and H5 subtypes of avian influenza virus, and a fluorescent quantitative PCR kit containing the primer probe group. The kit also contains fluorescent quantitative PCR reagent, ddH2O and positive plasmid standard products, and can simultaneously detect whether the sample to be tested contains H3, H4 and H5 subtypes of avian influenza virus. The kit provided by the present invention has good specificity for H3, H4 and H5 subtypes of avian influenza virus; compared with the common PCR detection method, the kit provided by the present invention has higher sensitivity, and the minimum detection limit for the three subtypes of avian influenza virus is 2.1× 102 copies/μL; the kit provided by the present invention also has the advantages of high stability and good repeatability. Therefore, the present invention can be used for rapid diagnosis and detection of H3, H4 and H5 subtypes of avian influenza virus, which is of great significance for the prevention and control of H3, H4 and H5 subtypes of avian influenza virus.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a fluorescent quantitative PCR primer probe set and a kit for detecting avian influenza virus H3, H4 and H5 subtypes.
Background
Avian influenza virus is a respiratory disease in poultry, which not only causes great economic loss to the poultry industry, but also constantly poses a threat to human health. The gene bank of influenza a viruses in waterfowl provides all genetic diversity required for the appearance of pandemic influenza viruses in humans, lower animals and birds, and in recent years, H3, H4 and H5 subtype avian influenza viruses have spread widely in waterfowl, and infections with H3 and H5 subtype avian influenza viruses have been observed in humans.
Common detection methods for avian influenza viruses today comprise ELISA antibody detection, PCR, recombinase-mediated detection (RAA), loop-mediated isothermal amplification (LAMP) and other detection technologies. ELISA antibody detection repeatability is poor and is interfered by autoantibodies, xenophilic antibodies and the like, false positives are easy to occur, common PCR is simple and rapid, but longer time is required compared with fluorescent quantitative PCR, recombinase street detection can be carried out at constant temperature and rapidly under enzyme mediation, requirements on equipment are reduced, but the two ends of a primer are required, the primer is required to be redesigned, ring mediation Deng Wenkuo is added in the experimental process, aerosol pollution caused by uncapping is caused, and false positives are easy to occur in detection results. The invention patent of China with the application number of CN104498629A discloses a double real-time fluorescent quantitative PCR detection kit for H3N2 subtype avian influenza virus, which has the defect that only H3N2 subtype avian influenza virus can be detected, and a plurality of avian influenza subtype viruses possibly occurring in waterfowl cannot be detected.
Disclosure of Invention
Based on the detection, the invention provides a fluorescent quantitative PCR primer probe group and a kit for detecting H3, H4 and H5 subtype avian influenza viruses, so as to realize the simultaneous detection of the H3, H4 and H5 subtype avian influenza viruses.
In a first aspect of the present invention, there is provided a fluorescent quantitative PCR primer probe set for detecting subtypes H3, H4 and H5 of avian influenza virus, characterized by comprising the following primers and probes:
an H3 subtype upstream primer H3-F, which has a nucleotide sequence shown as SEQ ID NO. 1;
A H3 subtype downstream primer H3-R having a nucleotide sequence as shown in SEQ ID No. 2;
An H4 subtype upstream primer H4-F having a nucleotide sequence as shown in SEQ ID NO. 3;
A H4 subtype downstream primer H4-R having a nucleotide sequence as shown in SEQ ID NO. 4;
an H5 subtype upstream primer H5-F having a nucleotide sequence as shown in SEQ ID NO. 5;
a H5 subtype downstream primer H5-R having a nucleotide sequence as shown in SEQ ID NO. 6;
an H3 subtype Probe H3-Probe having a nucleotide sequence as shown in SEQ ID NO. 7;
An H4 subtype Probe H4-Probe having a nucleotide sequence as shown in SEQ ID NO. 8;
H5 subtype Probe H5-Probe, which has the nucleotide sequence shown as SEQ ID NO. 9.
According to the method, three pairs of primers and three probes are respectively designed according to the published AIV sequences on NCBI, H3, H4 and H5 subtype avian influenza virus HA genes in nearly 5 years, the primers and the probes are applied to detection of related pathogens by a fluorescence quantitative Taqman probe method PCR, an H3 standard curve equation is Y= -3.343lgX+39.94, a correlation coefficient R 2 =0.997, an amplification efficiency E=99.14, an H4 standard curve equation is Y= -3.42lgX+40.19, a correlation coefficient R 2 =0.999, an amplification efficiency E=96.06, an H5 standard curve equation is Y= -3.405lgX+39.78, a correlation coefficient R 2 =0.998, and an amplification efficiency E=96.65%. The minimum detection amount of the detection is 2.1 multiplied by 10 2 copies/. Mu.L, the sensitivity is high, the specificity is good, and the variation coefficient in batch and batch is less than 1.5%. Therefore, the invention can be used as a rapid, sensitive and accurate detection means to be applied to actual production and epidemic situation monitoring, and provides reference for the identification and detection of H3, H4 and H5 subtype avian influenza viruses in the future.
Further, the 5 'ends of the H3-Probe, the H4-Probe and the H5-Probe are respectively marked with fluorescent genes, and the 3' ends are respectively marked with quenching genes.
Further, the H3-Probe is marked with a fluorescent gene FAM at the 5 'end and a quenching gene BHQ1 at the 3' end, the H4-Probe is marked with a fluorescent gene HEX at the 5 'end and a quenching gene BHQ1 at the 3' end, and the H5-Probe is marked with a fluorescent gene CY5 at the 5 'end and a quenching gene BHQ2 at the 3' end.
In a second aspect of the present invention, the use of the primer probe set described above for preparing a fluorescent quantitative PCR kit for detecting avian influenza virus H3, H4 and H5 subtypes is also claimed.
In a third aspect of the invention, there is provided a fluorescent quantitative PCR kit for detecting avian influenza virus H3, H4 and H5 subtypes, in particular, the kit comprising a primer probe set as described in any one of the above.
Further, the kit contains H3-F0.4. Mu. L, H3-R0.4. Mu. L, H3-Probe 0.2. Mu. L, H4-F0.4. Mu. L, H4-R0.4. Mu. L, H4-Probe 0.2. Mu. L, H5-F0.2. Mu. L, H5-R0.2. Mu.L and H5-Probe 0.1. Mu.L, and the concentration of each of the primers and probes is 10. Mu.M.
Further, the kit also contains 10 mu L, ddH 2 O5.5 mu L of fluorescent quantitative PCR reagent and 2 mu L of positive plasmid standard.
Further, the fluorescent quantitative PCR reagent is AceQ Universal U + Probe Master Mix V2.
In one embodiment, the positive plasmid standard is prepared by the steps of:
S1, PCR amplification and purification of target fragments, namely, conventional PCR amplification is carried out on the target fragments by using an upstream primer and a downstream primer and H3, H4 and H5 gene templates, and PCR products are purified by gel electrophoresis, wherein the conventional PCR amplification reaction procedures are that the temperature of the target fragments is ① ℃ for 3min, the temperature of the target fragments is ② ℃ for 15S, the temperature of the target fragments is ③ ℃ for 20S, the temperature of the target fragments is ④ ℃ for 2min, the temperature of the target fragments is ②-④ for 35 cycles, and the temperature of the target fragments is ⑤ ℃ for 10min.
S2, target fragment ligation, namely carrying out carrier ligation on the purified product by using a Tarkara company pMD18-T Vector;
s3, introducing the recombinant plasmid into a recipient bacterial cell;
S4, screening and identifying recombinant plasmids, namely selecting single bacterial colonies with good growth, culturing, and taking suspected positive bacterial liquid as a template for PCR identification;
S5, extracting positive plasmids, namely performing amplification culture on the bacterial liquid with the correct sequencing result in the step 4, and extracting plasmids by using a plasmid extraction kit.
In one embodiment, the use of the fluorescent quantitative PCR kit for detecting the subtype H3, H4 and H5 of the avian influenza virus provided by the invention comprises the steps of extracting total RNA in a sample to be detected, taking the obtained total RNA as a template, carrying out fluorescent quantitative PCR detection by using the kit provided by the invention, judging whether the sample to be detected is the subtype H3, H4 or H5 of the avian influenza virus according to a fluorescent CT value, and judging that the sample to be detected is negative if the CT value is more than 35, and judging that the sample to be detected is positive if the CT value is less than or equal to 35.
Compared with the prior art, the invention has the following advantages:
The kit provided by the invention contains positive plasmid standard substances, the standard substances are subjected to gradient dilution and amplification, the results show that the standard substances with different concentrations have good linear relation and accord with expected results, the standard substances are diluted to be 2.1 multiplied by 10 9copies/μL-2.1×100 copies/mu L, the reaction system provided by the invention is used for detecting the subtype H3, H4 and H5 avian influenza viruses, the minimum detection limit is determined to be 2.1 multiplied by 10 2 copies/mu L, compared with the common PCR detection method, the sensitivity is about 1 order of magnitude higher, and the real-time fluorescent quantitative PCR amplification of the standard substances is carried out, so that the intra-batch variation coefficient is less than 1% and the inter-batch variation coefficient is less than 1.5%, therefore, the stability of the primer probe group and the kit provided by the invention is high, and the repeatability is good.
Drawings
FIG. 1 is a standard chart of fluorescence quantitative PCR for H3 subtype avian influenza virus of example 4 of the present invention;
FIG. 2 is a standard chart of fluorescence quantitative PCR for H4 subtype avian influenza virus of example 4 of the present invention;
FIG. 3 is a standard chart of fluorescence quantitative PCR for H5 subtype avian influenza virus of example 4 of the present invention;
FIG. 4 is a fluorescent quantitative PCR sensitivity detection graph of subtype H3 avian influenza virus of example 5 of the present invention;
FIG. 5 is a fluorescent quantitative PCR sensitivity detection graph of subtype H4 avian influenza virus of example 5 of the present invention;
FIG. 6 is a fluorescent quantitative PCR sensitivity detection graph of subtype H5 avian influenza virus of example 5 of the present invention;
FIG. 7 is a diagram showing the general PCR electrophoresis of example 5 of the present invention;
FIG. 8 is a fluorescent quantitative PCR-specific assay graph according to example 6 of the present invention.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present invention, "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of technical features indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics. In the present invention, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The percentage content referred to in the present invention refers to mass percentage for both solid-liquid mixing and solid-solid mixing and volume percentage for liquid-liquid mixing unless otherwise specified. The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system after the component is added. The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
Example 1, synthesis and design of primers:
according to published AIV sequences on NCBI, three pairs of primers and three probes are respectively designed for the HA genes of H3, H4 and H5 subtype avian influenza viruses in nearly 5 years, the HA gene sequences of the three avian influenza subtype viruses and the designed primer pairs and Probe sequences are shown in a table 1, wherein the H3-Probe 5 'end marks fluorescent gene FAM, the 3' end marks quenching gene BHQ1, the H4-Probe 5 'end marks fluorescent gene HEX, the 3' end marks quenching gene BHQ1, the H5-Probe 5 'end marks fluorescent gene CY5 and the 3' end marks quenching gene BHQ2.
TABLE 1 primer pairs and probes designed and primer pairs for HA genes of H3, H4 and H5 subtype avian influenza viruses
Example 2 preparation of plasmid standards:
(1) PCR amplification and purification of fragments of interest
The target fragment was amplified using the H3, H4 and H5 gene templates using the upstream and downstream primers (10. Mu.M) with a reaction procedure of ① ℃for 3min, 3495℃for 15s, ③ ℃for 20s, ④ ℃for 2min, 3835 cycles, ⑤ ℃for 10min.
TABLE 2 conventional PCR reaction System
After the PCR procedure is finished, adding all PCR products into Goldview% agar gel, electrophoresis for 30min, cutting off a target strip under the irradiation of an ultraviolet lamp, and using a Simer-fly-glue recovery kit to carry out glue recovery, wherein the Binding Buffer and the Wash Buffer in the step (1) are both from the kit, and the specific steps are as follows:
(1-1) cutting gel sections containing DNA fragments with a clean scalpel or blade as close as possible to the DNA to minimize gel volume, weighing the gel sections in a pre-weighed 1.5mL tube, and recording the weight of the gel sheets.
(1-2) 1:1 Volume of Binding Buffer (volume: weight, e.g., 100. Mu.L of Binding Buffer per 100mg of agarose gel) was added to the gel sheet.
(1-3) Incubation of the gel mixture at 55℃for 10 minutes or until the gel sections are completely dissolved. The mixing tubes were inverted every few minutes to facilitate the melting process. Ensure complete dissolution of the gel. The gel mixture was vortexed briefly before loading. The color of the solution was checked. Yellow indicates the optimal pH for DNA binding.
(1-4) Transfer up to 800. Mu.L of the dissolved gel solution from the above step to GeneJET purification column. Centrifuge 12000rpm for 1min. The filtrate was discarded and the column was then returned to the same collection tube.
(1-5) 100. Mu.L of Binding Buffer was added, centrifuged for 1min, the filtrate was discarded, and the column was returned to the same collection tube. (1-6) 700. Mu.L Wash Buffer was added to GeneJET purification columns. Centrifuge 12000rpm for 1min. The filtrate was discarded and the column was then returned to the same collection tube.
(1-7) The empty GeneJET purification column was centrifuged at 12000rpm for 1min to completely remove the residual wash buffer. (1-8) transfer GeneJET purification columns to clean 1.5mL microcentrifuge tubes. 40. Mu.L of DEPC water was added to the center of the purification column membrane. Centrifuge at 12000rpm for 1min.
(1-9) The GeneJET purification columns were discarded and their concentrations were checked and the purified DNA was stored at-20 ℃.
(2) Ligation of fragments of interest
The PCR purified products obtained in the steps (1-9) were subjected to carrier ligation with reference to the instructions for use of pMD18-T Vector (Tarkara Co., ltd.) in the reaction system shown in Table 3, and after the reactants shown in Table 3 were mixed uniformly, they were subjected to 16℃for 4 hours and 4℃overnight in a PCR apparatus to obtain ligation products.
TABLE 3 Carrier ligation reaction System
(3) Introduction of recombinant plasmid into recipient bacterial cells (3-1) the ligation products obtained in step (2) were added to 50. Mu. Ltop.sup.10 competent cells, respectively.
(3-2) After gently mixing the ligation product and competent cells, immediately ice-bath for 30min, followed by 42℃heat shock for 45s, and then immediately ice-bath for 2min.
(3-3) 400. Mu.L of fresh LB liquid medium was added to each tube, and the culture was slowly shaken in a 37℃incubator for 1 hour.
(3-4) 100. Mu.L of the bacterial liquid was spread on 1.5% (W/V) LB agar plates containing Amp (100. Mu.g/mL), and cultured upside down at 37℃for 12-16 hours.
(4) And (5) screening and identifying recombinant plasmids.
Single colonies with good growth are selected and placed in 500 mu L of LB liquid medium containing Ampicillin (AMP), and placed in a shaking table at a constant temperature of 37 ℃ for shaking culture for 4-6 hours, and a suspected positive bacterial liquid is taken as a template for PCR identification, wherein a PCR identification reaction system is shown in Table 4.
TABLE 4 PCR identification reaction System
The PCR reaction procedure was ① ℃for 3min, ② ℃for 15s, ③ ℃for 20s, ④ ℃for 2min, 3835 cycles, ⑤ ℃for 10min. And (5) sending the screened positive bacterial liquid to a biological engineering limited company for sequencing.
(5) Extraction of positive plasmid
The bacterial liquids with correct sequencing results in the step (4) are named as 18T-H3, 18T-H4 and 18T-H5, and are subjected to expansion culture, and plasmid extraction is carried out by using a Norvezan plasmid extraction kit, wherein the specific steps are as follows:
(5-1) cultivation of E.coli Single colonies were selected from the plate medium and inoculated into 5ml of LB liquid medium containing Ampicillin (AMP) and cultured overnight at 37℃for 12-16 hours.
(5-2) 5Ml of the overnight culture broth was centrifuged at 12000rpm for 5 minutes, and the supernatant was discarded.
(5-3) To the centrifuge tube with the bacterial pellet left, 250. Mu.L of Buffer P1 was added and mixed by pipetting or vortexing.
(5-4) 250. Mu.L Buffer P2 was added to step (5-3), and the mixture was gently mixed upside down for 8-10 times to allow the cells to be completely lysed.
(5-5) 350 Mu LBuffer P of 3 was added to step (5-4), and the solution was gently turned upside down 8-10 times immediately to thoroughly neutralize Buffer P2. At this point a white flocculent precipitate should appear. Centrifuge at 13,000Xg for 10min.
(5-6) The FastPure DNA Mini Columns adsorption column was placed in a 2ml collection tube. The supernatant from step (5-5) was carefully transferred to an adsorption column using a pipette, taking care not to aspirate the pellet, and centrifuged at 13,000Xg for 1min. The waste liquid in the collecting pipe is poured out, and the adsorption column is put back into the collecting pipe.
(5-7) Into the adsorption column, 500. Mu.l Buffer PW 1. Centrifuge at 12,000rpm (13,400Xg) for 30-60sec. Discarding the waste liquid, and putting the adsorption column back into the collecting pipe.
(5-8) Into the adsorption column, 600 u L Buffer PW 2. Centrifuge at 13,000Xg for 1min. Discarding the waste liquid, and putting the adsorption column back into the collection pipe.
(5-9) Repeating the step (5-8).
(5-10) The column was placed in a fresh sterilized 1.5ml centrifuge tube. Add 30-100. Mu.L of the Elution Buffer to the center of the membrane of the column. Standing at room temperature for 2min,13,000× and g, eluting DNA by centrifugation for 1 min.
(5-11) Discarding the adsorption column, and preserving the DNA product in-20' C to prevent DNA degradation.
Plasmid concentration was measured using an ultramicro-fluorescence spectrophotometer, and gene copy number was calculated from gene copy number (copies/. Mu.L) =6.02X10 23 ×plasmid concentration (ng/. Mu.L) ×10 -9/[ plasmid size (bp) ×660 ].
Example 3 detection of H3, H4 and H5 influenza Virus by the primers and probes of the invention
(1) RNA extraction (AxyPrepTM Body Fluid Viral DNA/RNA MINIPREP KIT kit)
(1-1) First 12000 Xg centrifugal 5min, collect 200 u L sample, transfer into 1.5ml centrifugal tube.
(1-2) Adding 200 mu L Buffer V-L, mixing uniformly by vortex oscillation, and standing at room temperature for 5min.
(1-3) Adding 75. Mu.L Buffer V-N, mixing well by vortex, centrifuging at 12000 Xg for 5min.
(1-4) The supernatant was transferred to a new 2ml centrifuge tube (provided in the kit), 300. Mu.L of isopropyl alcohol (1% glacial acetic acid) was added, and the mixture was inverted 6-8 times upside down, and gently mixed.
(1-5) The preparation tube was placed in a 2ml centrifuge tube (provided in a kit, at most 800. Mu.L), the mixture in step 4 was transferred into the preparation tube in two batches, and the first addition of 800. Mu.L, 6000 Xg was centrifuged for 1min, and the filtrate was discarded. The remaining 750ul was transferred and centrifuged at 6000 Xg for 1min.
(1-6) The filtrate was discarded, and the preparation tube was returned to a 2ml centrifuge tube, 500. Mu.L Buffer W1A was added thereto, and the mixture was allowed to stand at room temperature for 1min. Centrifuge 12000 Xg for 1min.
(1-7) The filtrate was discarded, and the preparation tube was returned to a 2ml centrifuge tube, and centrifuged at 800. Mu.L Buffer W2,12000 Xg for 1min. (1-8) the preparation tube was placed back into a 2ml centrifuge tube and centrifuged at 12000 Xg for 1min.
(1-9) The preparation tube was placed in a clean 1.5ml centrifuge tube (provided in the kit), 30. Mu.L of DEPC water was added to the center of the preparation tube film, and the tube was allowed to stand at room temperature for 1min. RNA was eluted by centrifugation at 12000 Xg for 1min.
(2) Reverse transcriptionIII 1stStrand cDNA synthesis Kit kit
(2-1) RNA template denaturation:
And (3) placing the RNA solution obtained in the step (1-9) in a water bath at 65 ℃ for 5min, and rapidly standing on ice for 2min.
(2-2) Genome removal
The following solutions were carefully mixed and incubated for 2min at 42℃in a transient-off post-PCR apparatus.
10. Mu.L of the solution obtained in step (1-9)
5×gDNA wiper Mix 5.5μL
(2-3) The following solutions were gently mixed by blowing with a pipette.
Placing the mixed solution in a PCR instrument for PCR amplification, wherein the reaction procedure is as follows:
The amplified product was stored in a-80 ℃ refrigerator.
(3) H3, H4 and H5 subtype avian influenza virus fluorescent real-time PCR
The reaction system is as follows:
The reaction procedure was ① for 2min at 37 ℃, ② for 5min at 95 ℃, ③ for 10s at 95 ℃ and ④ for 30s at ④, and step ③-④ was repeated for 40 cycles.
(4) Result determination
Negative: detecting sample Ct value A value greater than 35 is negative.
Positive: the Ct value of the detection sample is smaller than A positive result was found to be 35.
Example 4 construction of fluorescent quantitative PCR System and Standard Curve
The 18T-H3, 18T-H4 and 18T-H5 plasmids were mixed at 1:1:1 and subjected to gradient dilution to give 2.1X10 9copies/μL-2,1×100 copies/. Mu.L of 10-dilution standard, 2.1X10 8copies/μL-2.1×102 copies/. Mu.L of which were used as reaction templates, 3 replicates per dilution and negative controls were established. The reaction system is shown in Table 5, the result is shown in FIG. 1, the H3 standard curve equation is Y= -3.343lgX+39.94, the correlation coefficient R 2 =0.997, the amplification efficiency E=99.14%, the H4 standard curve equation is Y= -3.42lgX+40.19, the correlation coefficient R 2 =0.999, the amplification efficiency E=96.06%, the H5 standard curve equation is Y= -3.405lgX+39.78, the correlation coefficient R 2 =0.998, the amplification efficiency E=96.65%, and the result shows that the standards with different concentrations have good linear relation, wherein the X axis is the copy number of the plasmid standard, and the Y axis is the circulation threshold value, and the expected result is met.
TABLE 5 fluorescent quantitative PCR reaction System
The reaction procedure was ① for 2min at 37 ℃, ② for 5min at ③ for 10s at 95 and ④ for 30s at 35 60, and step ③-④ was repeated for 40 cycles.
Example 5 sensitivity experiment
The 2.1X 7copies/μL-2.1×100 copies/. Mu.L 8 dilutions of plasmid standard was used as a template, amplified according to the fluorescent quantitative PCR reaction system provided in example 3, and the same template was subjected to ordinary PCR amplification, and the lowest detection limit of the two methods of real-time fluorescent quantitative PCR and ordinary PCR was determined and the sensitivity of the two methods was compared. As shown in FIG. 2, the minimum detection amount of the positive standard substance detected by the fluorescent quantitative PCR reaction system provided in example 3 is 2.1X10 2 copies/. Mu.L. As shown in FIG. 3, lanes M are DL500 DNA MARKER, lanes 2-10 are samples of 2.1X10. 10 9copies/μL-2.1×100 copies/. Mu.L, respectively, and lane 1 is a negative control. The minimum detection amount of the ordinary PCR was found to be 2.1X10 3 copies/. Mu.L. In contrast, the real-time fluorescent quantitative PCR detection method established in example 3 is about 1 order of magnitude higher in sensitivity than the conventional PCR detection method.
Example 6 specificity experiments
The established real-time fluorescent quantitative PCR method is adopted to amplify pathogens of H1N1, H3N8, H4N6, H5N8, H6N6, H7N9 and H9N2 subtype avian influenza virus, MDRV (Muscovy duck reovirus), NDRV (novel duck reovirus), MDV (chicken Marek's disease virus), AILT (chicken infectious laryngotracheitis), CIAV (chicken infectious anemia virus), IBV (chicken infectious bronchitis virus) and NDV (chicken Newcastle disease virus), and the specificity of the fluorescent quantitative PCR reaction system provided in the example 3 is verified. As shown in FIG. 4, the results show that the fluorescent quantitative PCR reaction system provided in example 3 specifically amplifies H3, H4 and H5 subtype avian influenza viruses, and has no fluorescent signal to H1N1, H6N6, H7N9, H9N2 subtype AIV, AILT, CIAV, IBV and NDV, but the Ct value of MDRV, NDRV, MDV is 35 later, so that the Ct value of the method is less than or equal to 35 and is positive, and is negative when the Ct value is greater than 35.
Example 7 repeatability test
And respectively carrying out real-time fluorescence quantitative PCR amplification on 3 batches of plasmids, namely 3 in-batch repetition and 3 in-batch repetition, comparing the variation condition of Ct values, verifying the stability of the method, and evaluating the stability of the method by using the in-batch and in-batch variation coefficients. As shown in Table 6, the intra-batch variation coefficient was less than 1%, the inter-batch variation coefficient was less than 1.5%, and the reproducibility was good.
TABLE 6 fluorescent quantitative PCR repeatability test results
The foregoing examples have shown only the preferred embodiments of the invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
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