CN116004917A - Fluorescent quantitative PCR primer probe group and kit for detecting avian influenza virus H3, H4 and H5 subtypes - Google Patents

Abstract

The invention discloses a fluorescent quantitative PCR primer probe group and a kit for detecting subtype H3, H4 and H5 of avian influenza virus, and a fluorescent quantitative PCR kit containing the primer probe group, wherein the kit also contains a fluorescent quantitative PCR reagent and ddH ₂ O and positive plasmid standard substance, can detect whether the sample to be detected contains avian influenza virus H3, H4 and H5 subtype at the same time. The kit provided by the invention has good specificity for H3, H4 and H5 subtype avian influenza viruses; compared with the common PCR detection method, the kit provided by the invention has higher sensitivity, and the minimum detection limit of the kit for three subtype avian influenza viruses is 2.1 multiplied by 10 ² cobies/. Mu.L; the book is provided withThe kit provided by the invention also has the advantages of high stability and good repeatability. Therefore, the invention can be used for rapid diagnosis and detection of the subtype H3, H4 and H5 of the avian influenza virus, and has important significance for prevention and control of the subtype H3, H4 and H5 of the avian influenza virus.

Description

Fluorescent quantitative PCR primer probe group and kit for detecting avian influenza virus H3, H4 and H5 subtypes

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 easily subjected to false positive due to interference of autoantibodies, xenophils and the like; ordinary PCR, while simple and rapid, requires a longer time than fluorescent quantitative PCR; the recombinase street detection can be carried out under enzyme mediation at constant temperature for rapid amplification, so that the requirement on equipment is reduced, but the requirement on two ends of a primer is met, and the primer is required to be redesigned; ring-mediated Deng Wenkuo increased aerosol contamination during the experiment due to uncapping, and false positives were easily seen in the detection results. The invention patent of China with the application number of CN104498629A discloses a double real-time fluorescence 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 published AIV sequence on NCBI, three pairs of primers and three probes are respectively designed for selecting H3, H4 and H5 subtype avian influenza virus HA genes in nearly 5 years, and the primers and the probes are applied to detection of related pathogens by a fluorescence quantitative Taqman probe method PCR (polymerase chain reaction) and an H3 standard curve equationIs Y= -3.343lgX+39.94, the correlation coefficient R ² =0.997, amplification efficiency e=99.14%; the H4 standard curve equation is Y= -3.42lgX+40.19, and the correlation coefficient R ² =0.999, amplification efficiency e=96.06%; the H5 standard curve equation is Y= -3.405lgX+39.78, and the correlation coefficient R ² =0.998, amplification efficiency e=96.65%. The minimum detection amount of detection was 2.1X10 ² The copies/. Mu.L, the sensitivity is high, the specificity is good, and the variation coefficient in batch and batch-to-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 5 'end of the H3-Probe is marked with a fluorescent gene FAM, and the 3' end is marked with a quenching gene BHQ1; the 5 'end of the H4-Probe is marked with a fluorescent gene HEX, and the 3' end is marked with a quenching gene BHQ1; 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 a fluorescent quantitative PCR reagent 10 mu L, ddH ₂ O5.5. Mu.L 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 the target fragment: using the upstream primer and the downstream primer, carrying out conventional PCR amplification on target fragments by using H3, H4 and H5 gene templates, and purifying PCR products by gel electrophoresis; the reaction procedure for conventional PCR amplification is: (1) the temperature is 95 ℃ for 3min; (2) 95℃for 15s, (3) 55℃for 20s, (4) 72℃for 2min, (2) - (4) 35 cycles; (5) the temperature was 72℃for 10min.

S2: the target fragment is connected: vector ligation of purified product was performed using the Tarkara company pMD18-T Vector;

s3: introducing the recombinant plasmid into a recipient bacterial cell;

s4: screening and identifying recombinant plasmids: selecting single bacterial colony with good growth, culturing, taking suspected positive bacterial liquid as a template, and carrying out PCR identification;

s5: extraction of positive plasmids: 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 fluorescent quantitative PCR kit for detecting the subtype H3, H4 and H5 of the avian influenza virus provided by the invention comprises the following steps: extracting total RNA in a sample to be detected, taking the obtained total RNA as a template, carrying out fluorescence quantitative PCR detection by using the kit provided by the invention, judging whether the sample to be detected is an avian influenza virus H3, H4 or H5 subtype according to a fluorescence CT value, and if the CT value is more than 35, carrying out negative, and if the CT value is less than or equal to 35, carrying out positive.

Compared with the prior art, the invention has the following advantages:

the invention has good specificity to H3, H4 and H5 subtype avian influenza viruses, and can detect H3, H4 and H5 subtype avian influenza viruses widely spread in waterfowl simultaneously; the kit provided by the invention contains positive plasmid standard substances, the standard substances are subjected to gradient dilution and amplification, and the results show that the standard substances with different concentrations have good linear relation and meet the expected results; dilution of the standard to 2.1X10 ⁹ copies/μL-2.1×10 ⁰ cobies/. Mu.L, andthe reaction system provided by the invention is used for detecting the avian influenza virus, the minimum detection limit is determined, and the minimum detection limit of the avian influenza virus of the H3, H4 and H5 subtypes is 2.1 multiplied by 10 ² The sensitivity of the copies/. Mu.L is about 1 order of magnitude higher than that of the common PCR detection method; the standard substance is subjected to real-time fluorescence quantitative PCR amplification in batches and between batches, and the variation coefficient in batches is found to be smaller than 1 percent, and the variation coefficient between batches is found to be smaller than 1.5 percent, so that the primer probe set and the kit provided by the invention have high stability and good repeatability.

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 the table 1, wherein the 5 'end of the H3-Probe is marked with a fluorescent gene FAM, and the 3' end is marked with a quenching gene BHQ1; the 5 'end of the H4-Probe is marked with a fluorescent gene HEX, and the 3' end is marked with a quenching gene BHQ1; the H5-Probe 5 'end marks the fluorescent gene CY5, and the 3' end marks the 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 with H3, H4 and H5 gene templates using the upstream and downstream primers (10. Mu.M), the reaction procedure was: (1) the temperature is 95 ℃ for 3min; (2) 95℃for 15s, (3) 55℃for 20s, (4) 72℃for 2min, (2) - (4) 35 cycles; (5) the temperature was 72℃for 10min.

TABLE 1 conventional PCR reaction System

After the PCR procedure is finished, adding all PCR products into 2% agar gel containing Goldview, electrophoresis for 30min, cutting off target strips under the irradiation of an ultraviolet lamp, and using a Simer flying 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 a 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 the GeneJET purification column. 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 the GeneJET purification column into a clean 1.5mL microcentrifuge tube. 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 column was discarded and its concentration was examined, 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 from Tarkara, the reaction system is shown in Table 3, and after mixing the reactants shown in Table 3, the mixture was placed in a PCR apparatus at 16℃for 4 hours and at 4℃overnight 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 10 competent cells, respectively.

(3-2) after gently mixing the ligation product and competent cells, immediately ice-bath for 30min, then heat-shock for 45s at 42℃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 an incubator at 37℃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

PCR reaction procedure: (1) the temperature is 95 ℃ for 3min; (2) 95℃for 15s, (3) 55℃for 20s, (4) 72℃for 2min, (2) - (4) 35 cycles; (5) the temperature was 72℃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 P3 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. The mixture was allowed to stand at room temperature for 2min, and the DNA was eluted by centrifugation at 13,000Xg for 1min.

(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 was determined according to gene copy number (copies/. Mu.L) =6.02X10 ²³ Xplasmid concentration (ng/. Mu.L). Times.10 ^-9 Plasmid size (bp) ×660]The gene copy number was calculated.

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)

(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 800. Mu.L Buffer W2 was added thereto, and the mixture was centrifuged at 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 transcription

III 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 of a PCR instrument at 65 ℃ for 5min, and rapidly placing the RNA solution on ice for standing for 2min.

(2-2) genome removal

The following solutions were carefully mixed and incubated for 2min at 42℃in a transient-free post-PCR apparatus.

10. Mu.L of the solution obtained in the step (1-9) 5 XgDNA wind Mix 5.5. Mu.L

(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:

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the reaction procedure is: (1) lasting for 2min at 37 ℃; (2) 95℃for 5min, (3) 95℃for 10s, (4) 60℃for 30s, steps (3) - (4) were repeated for 40 cycles.

(4) Result determination

Negative: and if the Ct value of the detection sample is greater than 35, the detection sample is negative.

Positive: and if the Ct value of the detection sample is less than or equal to 35, the detection sample is positive.

Example 4 construction of fluorescent quantitative PCR System and Standard Curve

The 18T-H3, 18T-H4 and 18T-H5 plasmids were combined in a 1:1:1 and carrying out gradient dilution to obtain 2.1X10 ⁹ copies/μL-2,1×10 ⁰ copies/. Mu.L of 10 dilutions of standard, 2.1X10 ⁸ copies/μL-2.1×10 ² The copies/. Mu.L was used as a reaction template, 3 replicates per dilution and negative controls were established. The reaction system is shown in Table 5, and the result is shown in FIG. 1, wherein the H3 standard curve equation is Y= -3.343lgX+39.94; correlation coefficient R ² =0.997; amplification efficiency e=99.14%, H4 standard curve equation y= -3.42lgx+40.19; correlation coefficient R ² =0.999; amplification efficiency e=96.06%, H5 standard curve equation is y= -3.405lgx+39.78; correlation coefficient R ² =0.998; amplification efficiency e=96.65%, and the results show 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, and the expected results are met.

TABLE 5 fluorescent quantitative PCR reaction System

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The reaction procedure: (1) lasting for 2min at 37 ℃; (2) 95℃for 5min, (3) 95℃for 10s, (4) 60℃for 30s, steps (3) - (4) were repeated for 40 cycles.

Example 5 sensitivity experiment

Will be 2.1X10 ⁷ copies/μL-2.1×10 ⁰ The fluorescence quantitative PCR reaction system provided in example 3 was used to amplify 8 dilutions of the plasmid standard as a template, and the same template was subjected to ordinary PCR amplification, and the lowest detection limit of both real-time fluorescence quantitative PCR and ordinary PCR methods was determined and the sensitivity of both methods was compared. As shown in FIG. 2, the minimum detectable 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, lane M is DL500 DNA Marker; lanes 2-10 are 2.1X10, respectively ⁹ copies/μL-2.1×10 ⁰ Samples of copies/. Mu.L; lane 1 negative control. As seen from the results, the minimum detection amount of the conventional PCR was 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 (infectious laryngotracheitis), CIAV (infectious chicken anemia virus), IBV (infectious bronchitis virus) and NDV (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.

Claims (9)

1. A fluorescent quantitative PCR primer probe set for detecting subtype H3, H4 and H5 of avian influenza virus, which is characterized by comprising the following primer pairs 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 H3 subtype Probe H3-Probe having a nucleotide sequence as shown in SEQ ID NO. 3;

an H4 subtype upstream primer H4-F having a nucleotide sequence as shown in SEQ ID NO. 4;

a H4 subtype downstream primer H4-R having a nucleotide sequence as shown in SEQ ID NO. 5;

an H4 subtype Probe H4-Probe having a nucleotide sequence as shown in SEQ ID NO. 6;

an H5 subtype upstream primer H5-F having a nucleotide sequence as shown in SEQ ID NO. 7;

a H5 subtype downstream primer H5-R 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.

2. The fluorescent quantitative PCR primer Probe set for detecting the subtype H3, H4 and H5 of the avian influenza virus according to claim 2, wherein 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.

3. The fluorescent quantitative PCR primer Probe set for detecting subtype H3, H4 and H5 of avian influenza virus according to claim 3, wherein the H3-Probe is marked with fluorescent gene FAM at the 5 'end and with quenching gene BHQ1 at the 3' end; the 5 'end of the H4-Probe is marked with a fluorescent gene HEX, and the 3' end is marked with a quenching gene BHQ1; the H5-Probe is marked with a fluorescent gene CY5 at the 5 'end and a quenching gene BHQ2 at the 3' end.

4. Use of a primer-probe set according to any one of claims 1 to 3 for the preparation of a fluorescent quantitative PCR kit for detecting the H3, H4 and H5 subtypes of avian influenza virus.

5. A fluorescent quantitative PCR kit for detecting the H3, H4 and H5 subtypes of avian influenza virus, characterized by comprising the primer probe set of any one of claims 1 to 3.

6. The fluorescent quantitative PCR kit according to claim 5, wherein the concentration of each of the primers and the probes is 10. Mu.M, comprising 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.

7. The fluorescent quantitative PCR kit according to claim 6, further comprising a fluorescent quantitative PCR reagent 10. Mu. L, ddH ₂ O5.5. Mu.L and 2. Mu.L of positive plasmid standard.

8. The fluorescent quantitative PCR kit of claim 7 wherein the fluorescent quantitative PCR reagent is AceQ Universal u+ Probe Master Mix V2.

9. The fluorescent quantitative PCR kit of claim 7, wherein the positive plasmid standard is prepared by:

s1: PCR amplification and purification of the target fragment: using the upstream primer and the downstream primer, carrying out conventional PCR amplification on target fragments by using H3, H4 and H5 gene templates, and purifying PCR products by gel electrophoresis;

s2: the target fragment is connected: vector ligation of purified product using pMD18-T Vector;

s3: introducing the recombinant plasmid into a recipient bacterial cell;

s4: screening and identifying recombinant plasmids: selecting single bacterial colony with good growth, culturing, taking suspected positive bacterial liquid as a template, and carrying out PCR identification;

s5: extraction of positive plasmids: and (3) performing amplification culture on the bacterial liquid with the correct sequencing result in the step (S4), and extracting plasmids by using a plasmid extraction kit.

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