CN113684318A - Fluorescent PCR (polymerase chain reaction) detection kit for M protein gene of porcine reproductive and respiratory syndrome virus - Google Patents
Fluorescent PCR (polymerase chain reaction) detection kit for M protein gene of porcine reproductive and respiratory syndrome virus Download PDFInfo
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Abstract
本发明公开了一种基因2型猪繁殖与呼吸综合征病毒M蛋白基因的荧光PCR检测试剂盒及其制备方法与用途，设计合成了一套特异性的引物及Taqman探针，并利用该引物及探针建立了一种快速简便、特异性强、灵敏度高的荧光PCR检测体系，可在3~4小时内从被检样本中快速、准确、特异、安全、简便地检出基因2型不同谱系PRRSV M蛋白基因，可以在生产标准化试剂盒中应用。The invention discloses a fluorescent PCR detection kit for genotype 2 porcine reproductive and respiratory syndrome virus M protein gene and a preparation method and application thereof. A set of specific primers and Taqman probes are designed and synthesized, and the primers are used. A fast, simple, highly specific, and highly sensitive fluorescent PCR detection system has been established, which can quickly, accurately, specifically, safely and easily detect genotype 2 different genotypes from the tested samples within 3 to 4 hours. Lineage PRRSV M protein genes that can be used in the production of standardized kits.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a fluorescent PCR detection reagent for a gene type 2 porcine reproductive and respiratory syndrome virus M protein gene and application thereof.
Background
PRRSV is an unfragmented single-stranded positive-strand RNA virus with a diameter of about 50-70 nm, a genome length of about 15kb containing 8 Open Reading Frames (ORFs), ORF1 encoding nonstructural proteins, and ORF2-ORF7 encoding structural proteins. The two long polypeptide chains encoded by ORF1a and ORF1b were cleaved to generate 14 non-structural proteins. ORFs 2 to 7 encode 8 structural proteins, including GP2, E, GP3, GP4, GP5a, GP5, M and N. The M protein is the most conserved protein in the arterivirus and has strong immunogenicity, so that the detection of the M protein gene can be used as important evidence that a sample contains PRRSV nucleic acid.
At present, the PRRSV epidemic in china is mainly a genotype 2 strain, which can be specifically divided into 4 lineages: lineage 1, lineage 3, lineage 5 (subline 5.1), and lineage 8 (subline 8.7). Of these, lineage 8 has been predominant in the epidemic strains, including the earliest CH-1a strain and the classical strain before 2006 as well as the HP-PRRSV strain that prevailed after 2006. Lineage 1 began to become prevalent after 2013, with the predominant NADC-like 30 strain, and the clinical detection rate increased year by year with HP-PRRSV of lineage 8 becoming the dominant circulating strain. Pedigree 3 is mainly distributed in southern areas of China, and the clinical detection rate is about 10%. Lineage 5 (BJ-4/VR 2332) was already established as early as 1996, and although no major epidemic was established in China, it was detected clinically. The prevalence trends of PRRS in pig farms in China are diversified due to different pedigrees of PRRSV, so that the seriousness and complexity of PRRS prevention and control in China are increased.
Currently, established methods for detecting PRRSV include virus isolation identification, virus antigen detection, and virus nucleic acid detection. The separation and identification test period of PRRSV is long, and the method is not suitable for rapid detection. The fluorescent antibody experiment is adopted to detect the PRRSV antigen, has the characteristics of rapidness and accuracy, but has insufficient sensitivity to a sample with low virus load and is easy to generate false negative results. The fluorescent PCR technology combines PCR and fluorescent detection, overcomes the defects of time consumption, easy pollution, electrophoresis detection after amplification and the like in the prior art, is applied to PRRSV nucleic acid detection, and primers and labeled probes adopted by the PRRSV fluorescent PCR method reported at present are mainly designed and prepared aiming at Nsp2, GP5 and N protein gene sequences, can detect PRRSV nucleic acids of a certain pedigree, two pedigrees or three pedigrees, but can not realize the detection of PRRSV samples of 4 pedigrees at the same time, namely the PRRSV nucleic acids of pedigrees 1, pedigrees 3, pedigrees 5 and pedigrees 8 realized by 1 pair of primers and 1 probe are not reported yet.
Disclosure of Invention
The invention provides a fluorescent detection reagent for PRRSV M protein gene for solving the technical problem of simultaneous detection of 4 pedigree PRRSV nucleic acids and a preparation method and application thereof, and particularly relates to a detection reagent developed by taking the PRRSV M protein gene as an amplification target sequence.
In order to solve the technical problems, the technical scheme adopted by the invention is realized by the following steps;
a specific primer for fluorescent PCR detection of M protein gene of porcine reproductive and respiratory syndrome virus genotype 2 is characterized in that:
an upstream primer: M-F: 5'-CTAGGCCGCAAGTACATTCTG-3' SEQ ID NO.1
A downstream primer: M-R: 5'-GACGACAAATGCGTGGTTATC-3' SEQ ID NO.2
The invention further discloses a specific probe for detecting the fluorescent PCR of the M protein gene of the gene type 2 porcine reproductive and respiratory syndrome virus, which is characterized by comprising the following components in parts by weight:
M-P：5'-FAM-TACATTCTGGCCCCTGCCCACCAC-TAMRA-3' SEQ ID NO.3。
the invention also discloses a fluorescent PCR detection kit for the gene 2 type porcine reproductive and respiratory syndrome virus M protein gene, which is characterized by comprising a pair of specific primers, a specific probe and a positive control, wherein the amplification target length is 96bp, and the primers, the probe and the target sequence are as follows:
an upstream primer: M-F: 5'-CTAGGCCGCAAGTACATTCTG-3' SEQ ID NO.1
A downstream primer: M-R: 5'-GACGACAAATGCGTGGTTATC-3' SEQ ID NO.2
And (3) probe: M-P: 5'-FAM-TACATTCTGGCCCCTGCCCACCAC-TAMRA-3' SEQ ID NO.3
Positive control: TGCTAGGCCGCAAGTACATTCTGGCCCCTGCCCACCACGTY
GAAAGTGCCGCRGGCTTTCATCCGATWRCGGCAARTGATAACCACGCATTTGTCGTCCGGCGTCCCGGCTCCACTACGGTYAACGGCACAYTGGTGCCCGGGTTAAAAAGCCTCGTGTTGGGTGGCAGAA；SEQ ID NO.4。
The invention also discloses the application of the fluorescent PCR detection kit for the gene 2 type porcine reproductive and respiratory syndrome virus M protein gene in the aspect of quickly, accurately and specifically detecting the gene 2 type PRRSV M protein genes with different lineages; the different pedigree PRRSV M protein genes refer to 4 pedigree PRRSV nucleic acids which are simultaneously detected, and mainly refer to pedigree 1, pedigree 3, pedigree 5 (subline 5.1) and pedigree 8 (subline 8.7). The experimental results show that: the invention establishes a rapid, simple, convenient, high-specificity and high-sensitivity fluorescent PCR detection system, can rapidly, accurately, specifically, safely and simply detect the PRRSV protein genes of different genetypes and genetypes in different lineages from a detected sample within 3-4 hours, and can be applied to the production of standardized kits.
The invention mainly aims at solving the complex problems that the popular PRRSV pedigrees in the current farms are various, and a plurality of primers and probes are needed for detecting the PRRSV of different pedigrees, and the like, and mainly inspects the possibility of simultaneously detecting the PRRSV nucleic acids of pedigrees 1, 3, 5 and 8 by adopting 1 pair of primers and 1 probe in order to avoid the PRRSV detection loophole and reduce the detection complexity, and the main difficulty lies in the selection of a target sequence and the design of a primer and a probe, and determines the success and failure of the experiment.
In order to elaborate the preparation method of the fluorescent detection reagent for the PRRSV M protein gene, the specific experiment comprises the following steps:
firstly, experimental steps
1. Extraction of viral RNA and cDNA Synthesis
Viral RNA was extracted according to the kit instructions. Adding 10.5 muL of total RNA into a 20 muL reverse transcription system, wherein the total RNA comprises 4 muL 5 XMLV Buffer, 2 muL dNTPmix (10 mmo 1/L), 1 muL random primer (50 mmo 1/L), 2 muL MLV (5U/muL) and 0.5 muL RNase inhibitor (40U/muL), lightly mixing uniformly, performing water bath at 42 ℃ for 1h, and finally performing ice bath for 2min, and then performing fluorescent quantitative PCR or storing at-20 ℃ for later use.
2. Preparation of Positive Standard
Using CMF/CMR as primer to make routine PCR, amplifying M protein gene of PRRSV, recovering PCR product, purifying by kit, connecting to pMD 18-T carrier, converting to DH5 alpha colibacillus, extracting positive clone plasmid identified by PCR and verified by sequence determination analysis, after determining DNA concentration, calculating copy number of standard product according to formula, copy number (copies) = (mass/molecular weight) × 6.0 × 1023. Plasmid standards were serially diluted 10-fold.
3. Optimization of reaction systems and conditions
And optimizing the optimal concentration of the primer and the probe by adopting a matrix method in the template and the reaction system with the same concentration. Meanwhile, different Tm values (50-65 ℃) are adjusted by combining a gradient PCR instrument in TaqMan fluorescent quantitative RT-PCR reaction conditions, and the positive nucleic acid template with the same concentration is detected by selecting the optimal Tm value.
4. Establishment of a Standard Curve
Plasmid standard 7.6X 10 diluted 10-fold serially4 copies/µL-7.6×1011And (3) calculating a Ct value and drawing a standard curve after the copies/mu L is used as a template and is detected by a fluorescent quantitative PCR instrument.
5. Specificity test
DNA or cDNA of gene type 2 PRRSV pedigree 1 (NADC 30-like), pedigree 3 (GM 2), pedigree 5 (VR 2332) and pedigree 8 (JXA 1-R) and control viruses CSFV, PRV, PPV and PCV2 are selected and used for specific test by using optimized fluorescent quantitative PCR conditions.
6. Sensitivity test
By 7.6X 100 copies/µL-7.6×1011And (3) performing fluorescent quantitative PCR detection on the copies/mu L positive standard substance, detecting the virus nucleic acid of the dilution, and checking the sensitivity of the method.
7. Repeatability test
Plasmid standards of 4 concentrations were used for the inter-and intra-batch reproducibility tests, respectively.
Second, experimental results
1. Obtaining of Positive Standard
Primers CMF and CMR of partial fragment of M protein gene of amplified PRRSV and cDNA of PRRSV JXA1-R as template are used for routine PCR, and specific fragment of PRRSV M protein gene of 171bp is amplified as a result (FIG. 1). And recovering the PCR product, purifying by using a kit, then continuously inoculating the PCR product into a pMD 18-T vector, transforming the vector into DH5 alpha escherichia coli, and obtaining positive clone by PCR identification and sequence determination analysis verification after culture. After extracting positive cloning plasmid with plasmid extraction kit and determining DNA concentration, calculating the copy number of standard product according to formula, copy number (copies) = (mass/molecular weight) × 6.0 × 1023。
2. Optimization of fluorescent quantitative PCR conditions
The present study optimizes the primer and probe concentrations to improve the amplification efficiency and sensitivity of the reaction. The result shows that when the primer concentration is 10 pmol/muL, the probe concentration is 50 pmol/muL and the Tm temperature is 60 ℃, the lowest Ct value and the highest fluorescence intensity increase value can be obtained, and the optimized 25 muL reaction system: 2 × Premix ExTaq(Probe qPCR) 12.5 muL; the upstream primer and the downstream primer (10 pmol/muL) are 0.5 muL respectively, and the probe (50 pmol/muL) is 1 muL; the template (plasmid or cDNA) is 2 muL, and DEPC water is supplemented to 25 muL. Fluorescence quantitative PCR reaction parameters: denaturation at 95 ℃ for 30s, amplification at 95 ℃ for 5s and 60 ℃ for 30s for 40 cycles, and single-point fluorescence signal detection and collection at 60 ℃.
3. Establishment of a Standard Curve
Serially diluting the RNA standard substance, determining by using the established fluorescent quantitative RT-PCR method, drawing a standard curve by using the Ct value and the logarithmic value of the standard substance with different concentrations to obtain a standard curve equation of y = -4.5204X LOG C X) +44.195 and a correlation coefficient R2= 0.99. The resulting standard curve (see FIG. 2).
4. Results of specificity test
DNA or cDNA of gene type 2 PRRSV lineage 1 (NADC 30-like), lineage 3 (GM 2), lineage 5 (VR 2332) and lineage 8 (JXA 1-R) and control viruses CSFV, PRV, PPV and PCV2 are selected to be used for carrying out fluorescence quantitative PCR detection by using optimized fluorescence quantitative PCR conditions, wherein the detection results of the PRRSV lineage 1 (NADC 30-like), lineage 3 (GM 2), lineage 5 (VR 2332) and lineage 8 (JXA 1-R) are positive, and the detection results of the control samples except the PRRSV are negative (a fluorescence signal does not reach the value) (FIG. 3). The results prove that: the method has good specificity.
5. Results of sensitivity test
The standard (JXA 1-R) was subjected to fluorescent quantitative PCR detection using the established method, and the positive determination standard was Ct >0 in 40 cycles and had a typical S amplification curve (FIG. 4). The result shows that the template with the lowest concentration of 104 copies/mu L can be detected by the method, and the sensitivity is good.
6. Results of the repeatability test
The plasmid standard substances with 4 concentrations are used for carrying out repeatability tests, and the coefficient of variation is less than 2 percent, which shows that the repeatability is good.
TABLE 1 PRRSV real-time fluorescent quantitation RT-PCR in-and between-batch repeatability tests
Drawings
FIG. 1 is a specific fragment of the PRRSV M protein gene;
FIG. 2 is a standard curve for fluorescent quantitative RT-PCR detection of PRRSV;
FIG. 3 shows the result of the fluorescent quantitative RT-PCR specificity test;
FIG. 4 is a kinetic curve of fluorescent quantitative RT-PCR for detecting PRRSV.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
Example 1
The preparation method of the fluorescent PCR detection kit for the gene 2 type porcine reproductive and respiratory syndrome virus M protein gene comprises the following steps:
(1) selecting a gene sequence conserved fragment of gene 2 type PRRSV 4 lineage virus M protein as a target sequence prepared and amplified by a positive control, wherein the nucleotide sequence is as follows; TGCTAGGCCGCAAGTACATTCTGGCCCCTGCCCACCACGTYGAAAGTGCCGCRGGCTTTCATCCGATWRCGGCAARTGATAACCACGCATTTGTCGTCCGGCGTCCCGGCTCCACTACGGTYAACGGCACAYTGGTGCCCGGGTTAAAAAGCCTCGTGTTGGGTGGCAGAA as shown in SEQ ID NO. 4;
(2) designing a plurality of primers according to gene sequences of M protein genes of gene 2 type PRRSV 4 lineage viruses, determining the most suitable 1 pair of primers through a large number of comparison screening experiments, amplifying and preparing M protein gene positive control, wherein the primer sequences are as follows:
an upstream primer: CMF: 5'-TGCTAGGCCGCAAGTACATTC-3' SEQ ID NO.5
A downstream primer: CMR: 5'-TTCTGCCACCCAACACGAG-3' SEQ ID NO.6
(3) Designing a plurality of pairs of primers and a plurality of probes according to the gene positive control sequence of the M protein gene of the gene type 2 PRRSV 4 lineage viruses, and determining the most suitable combination of the primers and the labeled probes through a large number of comparison screening tests:
an upstream primer: M-F: 5'-CTAGGCCGCAAGTACATTCTG-3' SEQ ID NO.1
A downstream primer: M-R: 5'-GACGACAAATGCGTGGTTATC-3' SEQ ID NO.2
And (3) probe: M-P: 5'-FAM-TACATTCTGGCCCCTGCCCACCAC-TAMRA-3' SEQ ID NO.3
Example 2
The real-time fluorescent quantitative RT-PCR detection and the commercial fluorescent RCR kit (aiming at the Nsp2 of PRRSV pedigree 3, pedigree 5 and pedigree 8 and the GP5 protein gene aiming at the PRRSV pedigree 1) detection are respectively carried out on the collected 20 suspected PRRSV clinical samples, and the results show that the positive detection rate (40%) of the real-time fluorescent quantitative RT-PCR method is higher than that of the commercial fluorescent quantitative PCR detection kit (the positive rates of the two kits are 35% in total), thereby realizing the simultaneous detection of the PRRSV nucleic acids of pedigree 1, pedigree 3, pedigree 5 and pedigree 8, and indicating that the sensitivity and the accuracy of the detection (aiming at the PRRSV M protein gene) are higher, the detection time is short, and the kit can be applied to the production of standardized kits.
SEQUENCE LISTING
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Fluorescent PCR (polymerase chain reaction) detection kit for <120> porcine reproductive and respiratory syndrome virus M protein gene
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064961A (en) * | 2022-11-14 | 2023-05-05 | 盐城师范学院 | Products and methods for fluorescent RT-RAA detection of genotype 2 porcine reproductive and respiratory syndrome virus |
| CN116121454A (en) * | 2022-12-09 | 2023-05-16 | 山东信达基因科技有限公司 | Combined detection reagent for swine fever, porcine reproductive and respiratory syndrome and porcine pseudorabies |
| CN117965808A (en) * | 2024-02-21 | 2024-05-03 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | PRRSV-1 and PRRSV-2 dual fluorescence one-step RT-PCR primer probe combination, kit and application |
| CN121046580A (en) * | 2025-10-31 | 2025-12-02 | 泰州蕾灵百奥生物科技有限公司 | A nucleic acid composition, product, and application for detecting porcine reproductive and respiratory syndrome virus (PRRSV) of the Americas type. |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064961A (en) * | 2022-11-14 | 2023-05-05 | 盐城师范学院 | Products and methods for fluorescent RT-RAA detection of genotype 2 porcine reproductive and respiratory syndrome virus |
| CN116121454A (en) * | 2022-12-09 | 2023-05-16 | 山东信达基因科技有限公司 | Combined detection reagent for swine fever, porcine reproductive and respiratory syndrome and porcine pseudorabies |
| CN117965808A (en) * | 2024-02-21 | 2024-05-03 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | PRRSV-1 and PRRSV-2 dual fluorescence one-step RT-PCR primer probe combination, kit and application |
| CN121046580A (en) * | 2025-10-31 | 2025-12-02 | 泰州蕾灵百奥生物科技有限公司 | A nucleic acid composition, product, and application for detecting porcine reproductive and respiratory syndrome virus (PRRSV) of the Americas type. |
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