CN114292962A - Fluorescent quantitative detection primer and probe set for identifying American PRRSV (porcine reproductive and respiratory syndrome Virus) and European PRRSV (porcine reproductive and respiratory syndrome Virus) - Google Patents

Abstract

The invention discloses a fluorescent quantitative detection primer and a probe set for identifying American type PRRSV and European type PRRSV, wherein the sequence of the primer and the probe for identifying American type PRRSV is AM-F, AM-R, AM-P, and the sequence of the primer and the probe for identifying European type PRRSV is EU-F, EU-R, EU-P. The invention designs specific primers and probes according to conserved sequences of American type and European type PRRSV ORF6 genes, establishes a dual real-time fluorescent quantitative PCR method capable of simultaneously detecting and detecting European type and American type PRRSV, and the standard substance is 1 × 10⁹copies/μL~1×10¹The primer and the probe have strong specificity and good repeatability, and have important significance for quick and sensitive detection of PRRSV of different genotypesIt has important meaning.

Description

Fluorescent quantitative detection primer and probe set for identifying American PRRSV (porcine reproductive and respiratory syndrome Virus) and European PRRSV (porcine reproductive and respiratory syndrome Virus)

Technical Field

The invention relates to the technical field of biology, in particular to a fluorescent quantitative detection primer and a probe set for identifying American PRRSV (porcine reproductive and respiratory syndrome Virus) and European PRRSV (porcine reproductive and respiratory syndrome Virus).

Background

Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The method is distributed all over the world, and causes great economic loss to the pig industry all over the world. PRRSV can be divided into two genotypes based on differences in genetic evolution, a european type represented by the LV strain and an american type represented by the VR-2332 strain. Since 1996 Guobaoqing and the like, PRRSV is firstly separated from aborted fetuses, the American type is the main epidemic of PRRSV in China, and the situation that various subtypes coexist is presented. In 1997, the european type PRRSV was isolated from domestic pigs by grand mingjie et al, and thereafter, the european type PRRSV was in a sporadic state in China and had a rising trend. At present, European PRRSV separated in China belongs to Subtype I, and has low pathogenicity, but European PRRSV strains with high toxicity have been reported abroad, so that the rapid and reliable detection and identification of two genotypes are very important for monitoring the PRRSV.

Disclosure of Invention

The invention aims to provide a fluorescent quantitative detection primer and a probe set for identifying American PRRSV and European PRRSV.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

fluorescent quantitative detection primer and probe set for identifying American type and European type PRRSV, wherein the sequences of the primer and the probe for identifying American type PRRSV are as follows:

AM-F：CCGCRSGSTTTCATCCGA；

AM-R：ARTGYGCCGTTRACYGTAG

AM-P：FAM-AACCACGCATTTGTCGTCCGGC-BHQ1

the sequences of primers and probes for identifying the european type PRRSV are as follows:

EU-F：TTYYAGATGCAGAYTGTGTTGCC；

EU-R：GTATSCYYGGTTACCAGACGC；

EU-P：VIC-CCGGCGATACATTCTGGCCCCT-BHQ1。

further, the invention also provides a PCR primer for amplifying the fluorescent quantitative fragment, which is used for preparing a standard substance, wherein the American type PRRSV standard substance primer sequence is as follows:

AM-PF：ACGCCARTRATGATATATGC；

AM-PR：CAATCAGTGCCAYTCACCA；

the primer sequences of the European PRRSV standard products are as follows:

EU-PF：CACCYATAATGATATAYGCCC；

EU-PR：GGCARCATRAACTCRACCT。

according to the invention, specific primers and probes are designed according to conserved sequences of American type and European type PRRSV ORF6 genes, the optimal reaction conditions are determined through optimization, and sensitivity, specificity and repeatability experiments and detection of clinical samples are carried out, so that the dual real-time fluorescent quantitative PCR method capable of simultaneously detecting European type and American type PRRSV is established. The standard substance is 1 × 10⁹copies/μL～1×10¹The plasmid has a good linear relation in a range of the copies/mu L concentration, and the standard substance positive plasmid with the copies/mu L can be detected at the lowest level; the method has no cross reaction with CSFV, PCV, PEDV, PRV, TGEV and PPV, and the variation Coefficient (CV) between batches and in batches is less than 1 percent. The coincidence rate with a commercial kit in clinical sample detection can reach 95%. The method has the advantages of high sensitivity, strong specificity, good stability, high accuracy, rapid detection and the like, and can be used for early diagnosis of PRRSV infection and rapid detection and quantitative analysis of samples.

Drawings

FIG. 1 is an electrophoretogram of PCR identification of a standard; wherein, M: DNA molecular mass standard; 1: american type standards; 2: a European-type standard; 3: and (5) negative control.

FIG. 2 is a standard curve of a dual fluorescent quantitative RT-PCR detection method for American and European PRRSV; wherein, 1-10: 1X 10⁹copies/μL～1×10¹copies/. mu.L; 11: and (5) negative control.

FIG. 3 shows the specific test results of the dual fluorescent quantitative RT-PCR detection method for American and European PRRSV; wherein, 1-7: HuN4-F112 strain, JXA1-R strain, CH-1R strain, R98 strain,

PRRS MLV strain, PC strain, FJZ 03; 8-12: swine fever live vaccine, HB-98 strain, attenuated virus + attenuated CV777 strain + NX strain, PCV, PPV; 13: negative control; 14: amervac PRRSV strain.

Detailed Description

Strains and vaccines:

the highly pathogenic porcine reproductive and respiratory syndrome live vaccine (HnN4-F112 strain) is purchased from Harbin group biological vaccine GmbH; the highly pathogenic porcine respiratory and reproductive syndrome live vaccine (JXA1-R) is purchased from Wicke bioengineering, Inc. of Yangzhou, a national drug group; porcine reproductive and respiratory syndrome vaccine (CH-1R strain) was purchased from Haerbin Vitaceae Biotechnology development Inc.; a porcine reproductive and respiratory syndrome virus live vaccine (R98 strain) is purchased from south agriculture high technology, Inc. of Jiangsu; porcine reproductive and respiratory syndrome live vaccine (

PRRS MLV) was purchased from Jiangsu Bolinger Invighan biologicals, Inc.; porcine reproductive and respiratory syndrome mosaic virus live vaccine (PC strain) was purchased from wuhan zhongbo bio incorporated; european attenuated vaccine strain Amervac PRRSV; the swine fever live vaccine (cell source) is purchased from Hayao group biological vaccine GmbH; the porcine pseudorabies live vaccine (HB-98 strain) is purchased from Wuhan department of Probiotics GmbH; porcine transmissible gastroenteritis, porcine epidemic diarrhea and porcine rotavirus (G5 type) triple live vaccines (attenuated virus Hua virus + attenuated CV777 strain + NX strain) are purchased from Jilin Zhengye biological products GmbH; FJZ03, Porcine Circovirus (PCV), Porcine Parvovirus (PPV) nucleic acids were stored by the laboratory.

The main reagents are as follows:

the total RNA extraction kit is purchased from Tiangen Biotechnology (Beijing) Co., Ltd; DNA purification and recovery kit (

Gel Extraction Kit) and plasmid Extraction Kit (

Plasmid Mini Kit) from OMEGA; reverse Transcriptase M-MLV (RNase H-), Reverse RNase Inhibitor, oligo (dT)18Primer, Random Primer (pd (N)6), dNTP mix, pMD^TM19-T Vector Cloning Kit was purchased from Takara, Inc. (Beijing); 2 × Taq Plus Master Mix (Dve Plus), DL2000 Plus DNA Marker, DH5 α chemically competent cells, HiScript II One Step qRT-PCR Probe Kit purchased from Biotech GmbH of Nanjing Novowed.

1. Design of primers and probes

The representative strains of PRRSV and the whole genome sequence of strains circulating in china were downloaded in NCBI GenBank for a total of 100, of which european strain 31 and american strain 69. The sequences were aligned and analyzed by MEGA7 biological software to find the conserved ORF6 of PRRSV encoding gene of American and European strains, and specific primers and probes were designed by using Oligo7 software, and the sequences are shown in Table 1 (AM-F, AM-R, AM-P, EU-F, EU-R, EU-P). And PCR primers for amplifying the fluorescent quantitative fragment-containing PCR primers were designed for the preparation of standards, and the sequences are shown in Table 2 (AM-PF, AM-PR, EU-PF, EU-PR). Both primers and probes were synthesized by Biotechnology engineering (Shanghai) Inc.

TABLE 1 Dual fluorescent quantitative RT-PCR primer and Probe sequences for American and European PRRSV

TABLE 2 Standard primer sequences

2. Preparation of fluorescent quantitative PCR standard

Extracting total RNA from positive pathological material with total RNA extraction kit, reverse transcribing to cDNA, performing PCR amplification with the primers in Table 2, and running gel to determine whether the size of the band is correct or notAnd (3) recovering the gel from the strip by using a DNA purification recovery kit, connecting a recovered product with a pMD-19T vector, transferring the product into DH5 alpha competent cells, selecting bacteria, carrying out bacteria liquid identification by using primers in the table 2, carrying out PCR identification, and using the bacteria liquid with correct sequencing for preparing a standard substance. And (4) carrying out plasmid extraction on the bacterial liquid with correct sequencing according to the specification of the plasmid extraction kit. Measuring the concentration of positive plasmid with a microspectrophotometer, converting the concentration into copy number according to the following formula, first diluting the positive plasmid to 1 × 10¹⁰copies/. mu.L, then diluted 10-fold, the diluted plasmid was used as template for standard. Copy number (copies/. mu.L) ═ CxNA/MW, where C is the concentration of positive plasmid assay, in ng/. mu.L, and NA is 6.02X 10²³copies/mol, MW is the average molecular mass in units of Dolton.

The results show that PCR amplification of recombinant plasmids AM and EU with the primers in Table 2 gave fragments of about 875bp and 758bp, and the electrophoretogram is shown in FIG. 1. The sequencing result was correct and no mutation occurred. The AM plasmid concentration is 216.08 ng/muL through the determination of a micro-spectrophotometer; the EU plasmid concentration was 97.5 ng/. mu.L, corresponding to copy numbers of 5.53X 10, respectively¹⁰copies/μL、2.58×10¹⁰copies/. mu.L, diluted 1X 10 with water¹⁰copies/μL～1×10⁰copies/μL。

3. Optimization of fluorescent quantitative PCR reaction conditions

The prepared standard substance is used as a template to optimize the fluorescent quantitative PCR reaction condition, the fluorescent quantitative PCR reaction condition is prepared according to the HiScript II One Step qRT-PCR Probe Kit instruction system, the total system is 20ul, wherein the added amounts of the primers and the probes in the reaction system are optimized by 2 xOne Step QProbe Mix 10 mu L, One Step Q Probe Mix 1 mu L and the template 2 mu L, and the rest system uses RNase free ddH₂And O filling, and simultaneously optimizing a reaction program so as to obtain the lowest CT value and the maximum amplification efficiency by the same template amount.

The optimized optimal reaction system is as follows: 2 Xone Step Q Probe Mixa 10. mu.L, One Step Q Probe Mixb 1. mu.L, template 2. mu.L, 0.5. mu.L each of AM-F, AM-R, EU-F, EU-R, 0.2. mu.L each of AM-P, EU-P, and water 4.6. mu.L, for a total of 20. mu.L. The optimal reaction conditions are as follows: 50 ℃ for 15 min; at 95 ℃ for 30 s; 95 ℃, 10s, 60 ℃, 30s, 40 cycles.

4. Drawing of standard curve and sensitivity test

Selecting 1X 10⁹Taking 10 standard substances with different concentrations of copies/mu L-1 × 100 copies/mu L as templates, carrying out fluorescence quantitative RT-PCR amplification under the optimal reaction condition, and determining the lowest dilution concentration detected by the method; and drawing a standard curve by taking the logarithm of the copy number as a horizontal ordinate and taking the corresponding Ct value as an ordinate.

As shown in FIG. 2, the lowest copy number detectable by this method was 10 copies/. mu.L at 1X 10⁹copies/μL～1×10¹The copies/mu L gradient range has good linear relation, the correlation coefficients R2 of the standard curve are all larger than 0.999, which indicates that the correlation is higher; the amplification efficiency is 92.9 percent and 98.1 percent respectively, and the amplification effect is good.

5. Experiment of specificity

Respectively extracting HuN4-F112 strain, JXA1-R strain, CH-1R strain, R98 strain, live vaccine of porcine reproductive and respiratory syndrome,

The detection method comprises the following steps of amplifying PRRS MLV strains, PC strains, European attenuated vaccine strain Amervac PRRSV, swine fever live vaccines, porcine pseudorabies live vaccine HB-98 strains, porcine transmissible gastroenteritis, porcine epidemic diarrhea, porcine rotavirus (G5 type) triple live vaccine (attenuated Chinese virus + attenuated CV777 strain + NX strain) nucleic acids, extracted vaccine nucleic acids, laboratory-stored FJZ03, Porcine Circovirus (PCV) and Porcine Parvovirus (PPV) nucleic acids by using the established detection method, and the result is shown in figure 3, wherein the FAM channel has no amplification curve except for the amplification curves of the American porcine reproductive and respiratory syndrome live vaccine and the FJZ03 strain; the VIC channel has no amplification curve except that the European attenuated vaccine strain Amervac PRRSV has an amplification curve. The established detection method is shown to have good specificity.

6. Repeatability test

Get 10⁵copies/μL、10⁴copies/μL、10³3 concentrations of copies/. mu.L standard, three replicates per gradient with the same batch of reagents were calculatedInter-batch coefficient of variation. Three reagents are prepared at different times, and the inter-batch variation coefficient is calculated.

The results are shown in tables 3 and 4, the variation coefficient of the in-batch repeatability test is between 0.08 and 0.28 percent, the variation coefficient of the in-batch repeatability test is between 0.11 and 0.62 percent, and the variation coefficients are all less than 1 percent, which indicates that the established method has good repeatability and high stability.

TABLE 3 results of the dual fluorescent quantitative RT-PCR detection method for American and European PRRSV in the repetitive test group

TABLE 4 results of the reproducibility test between the groups of the dual fluorescent quantitative RT-PCR detection methods for American and European PRRSV

7. Compliance test

The method and the commercialized kit established by the invention are used for simultaneously detecting the pathological material suspected to be infected with PRRSV, and simultaneously, the general RT-PCR detection kit for the porcine reproductive and respiratory syndrome virus of Beijing Shijiheng animal epidemic prevention technology Limited company and the fluorescent RT-PCR detection kit for the porcine reproductive and respiratory syndrome virus of Qingdao instant biological technology Limited company are used for detecting to verify the accuracy.

The results are shown in tables 5 and 6, and the coincidence rate of the kit with the universal real-time fluorescent RT-PCR detection kit for the century-yuan Henry porcine reproductive and respiratory syndrome virus is 95.26%; the coincidence rate of the fluorescent RT-PCR detection kit for porcine reproductive and respiratory syndrome virus found in Qingdao is 95.14%. 1328 samples are detected by the method established by the invention, wherein the PRRSV American type positive sample is 795 parts, and the PRRSV American type negative sample is 533 parts; the coincidence rate of the European positive sample 2 parts and the negative sample 1326 parts with the century Yuan Henry porcine reproductive and respiratory syndrome virus European strain real-time fluorescence RT-PCR detection kit is 100%.

TABLE 5 fluorescent quantitative RT-PCR detection method for American PRRSV meets test results

TABLE 6 fluorescent quantitative RT-PCR detection method for European PRRSV meets test results

Claims (2)

1. A fluorescent quantitative detection primer and a probe group for identifying American PRRSV and European PRRSV are characterized in that,

the primer and probe sequences for identifying the American type PRRSV are as follows:

AM-F：CCGCRSGSTTTCATCCGA；

AM-R：ARTGYGCCGTTRACYGTAG

AM-P：FAM-AACCACGCATTTGTCGTCCGGC-BHQ1

the sequences of primers and probes for identifying the european type PRRSV are as follows:

EU-F：TTYYAGATGCAGAYTGTGTTGCC；

EU-R：GTATSCYYGGTTACCAGACGC；

EU-P：VIC-CCGGCGATACATTCTGGCCCCT-BHQ1。

2. the primer and probe set for quantitative fluorescent detection for identifying PRRSV types america and europe according to claim 1, wherein the PCR primer sequences for amplifying the PCR primer sequences comprising the quantitative fluorescent fragments are as follows:

the American type PRRSV standard primer sequences are as follows:

AM-PF：ACGCCARTRATGATATATGC；

AM-PR：CAATCAGTGCCAYTCACCA；

the primer sequences of the European PRRSV standard products are as follows:

EU-PF：CACCYATAATGATATAYGCCC；

EU-PR：GGCARCATRAACTCRACCT。

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