HRM (high-resolution melting) label-free probe method, primer and probe for quickly differentiating PRRSV (porcine reproductive and respiratory syndrome virus) classical strains and mutant strains

A technology of PCR-HRM and classic strains, applied in the field of HRM non-labeled probe method and its primers and probes, can solve the problems of PCR product contamination, low sensitivity, expensive price, etc., and achieve high-throughput analysis and repeatability Good, high-accuracy effect

Active Publication Date: 2015-12-16
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above methods can distinguish classic strains of PRRSV from mutant strains, traditional PCR detection techniques such as OneStep RT-PCR require gel electrophoresis analysis of RT-PCR products, which can easily cause contamination of PCR products, resulting in false positives and low sensitivity
Although fluorescent quantitative PCR has high sensitivity, it is expensive, and two or more probes need to be synthesized at the same time

Method used

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  • HRM (high-resolution melting) label-free probe method, primer and probe for quickly differentiating PRRSV (porcine reproductive and respiratory syndrome virus) classical strains and mutant strains
  • HRM (high-resolution melting) label-free probe method, primer and probe for quickly differentiating PRRSV (porcine reproductive and respiratory syndrome virus) classical strains and mutant strains
  • HRM (high-resolution melting) label-free probe method, primer and probe for quickly differentiating PRRSV (porcine reproductive and respiratory syndrome virus) classical strains and mutant strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 A kind of HRM unlabeled probe method for rapidly distinguishing PRRSV classic strains and mutant strains

[0059] Primers and Unlabeled Probes

[0060] 1) PCR pre-amplification primers

[0061] According to the gene sequences of H-PRRSV (PRRSV variant strain) and C-PRRSV (PRRSV classic strain), primer pairs P1 and P2 (PCR pre-amplification primers) for amplifying partial gene fragments of H-PRRSV and C-PRRSV were designed, Its base sequence is shown below.

[0062] P1: 5'-GAYATTCATCATTACACCAGT-3' (SEQ ID NO: 1),

[0063] P2: 5'-AACACYCCGCCAGAGCC-3' (SEQ ID NO: 2).

[0064] 2) PCR-HRM primers:

[0065] After screening a large number of designed primers, it was found that the base sequences of the primers SEQIDNO: 3 and SEQ ID NO: 4 had the best effect on the PCR-HRM method for distinguishing classic and mutant strains of PRRSV, and their base sequences are shown below.

[0066] P3: 5'-GCYACCGCACCAGATGGRACCTACTT-3' (SEQ ID NO: 3),

[0067] P4: 5'-ACGGT...

Embodiment 2

[0071] Example 2 A kind of HRM unlabeled probe method for rapidly distinguishing PRRSV classic strains and mutant strains

[0072] PCR-HRM analysis of standard samples

[0073] 1) Extraction of PRRSV standard nucleic acid:

[0074] Take CH-1R vaccine (C-PRRSV classic strain) and JXA1-R vaccine (H-PRRSV variant strain) respectively and add 3mL of PBS hydrochloric acid buffer solution to dissolve, take 200μL for nucleic acid extraction according to the instructions of TAKARA's MiniBESTViralRNA / DNAExtractionKitVer.4.0 .

[0075] 2) PCR-HRM operation steps for positive standard samples

[0076] In order to verify the ability of the designed primers and non-standard probes to identify actual samples, CH-1R vaccine (C-PRRSV classic strain) and JXA1-R vaccine (H-PRRSV variant strain) were used as standard samples in this study Perform PCR-HRM analysis. The above-mentioned standard samples were used to extract nucleic acids as nucleic acid templates, and the PCR-HRM pre-amplif...

Embodiment 3

[0089] Example 3 Clinical Sample PCR-HRM Analysis

[0090] 1) Extract viral nucleic acid from samples: Take 2g of pig lung tissue samples suspected to be infected with C-PRRSV classic strain and H-PRRSV vaccine strain, add 3mL PBS hydrochloric acid buffer solution for grinding, and grind the homogenate 4000× Centrifuge at g for 8 min, and draw the centrifuged supernatant to -80°C for storage. Or serum samples 200 μL tissue sample homogenate and serum samples were subjected to nucleic acid extraction according to the instructions of TAKARA's MiniBESTViralRNA / DNAExtractionKitVer.4.0.

[0091] 2) Using the extracted viral nucleic acid as a template, perform RT-PCR pre-amplification (refer to the PrimeScript? OneStepRT-PCRKit kit manual), the pre-amplification reaction system is:

[0092]

[0093] The pre-amplification reaction program was reverse transcription at 50°C for 30 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20 s, annealing at 55°C for 20 s, ...

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Abstract

The invention discloses an HRM (high-resolution melting) label-free probe method, a primer and a probe for quickly differentiating PRRSV (porcine reproductive and respiratory syndrome virus) classical strains and mutant strains. The HRM label-free probe method, the primer and the probe have the advantages that the primer and the probe are easy to operate; only the UN-Probe and fluorescent saturable dye need to be added into the strains before PCR (polymerase chain reaction) is carried out; the HRM label-free probe method, the primer and the probe are high in detection speed and throughput; complete operation procedures only take 3 hours, and accordingly required typing time can be greatly shortened; the HRM label-free method, the primer and the probe are low in cost, specific fluorescent label probes can be omitted, and the saturable dye cost of each sample is 1.6RMB; the HRM label-free probe method, the primer and the probe are high in accuracy and good in specificity and repeatability, the PRRSV classical strains and mutant strains can be accurately and quickly analyzed under a high-throughput condition, and accordingly the HRM label-free probe method, the primer and the probe are favorable to popularization and application in clinical practice.

Description

technical field [0001] The invention relates to a method for distinguishing classic virus strains and variant strains, in particular to an HRM non-labeled probe method for quickly distinguishing classic strains and variant strains of PRRSV, as well as primers and probes thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcinereproductiveandrespiratorysyndrome, PRRS) is caused by porcine reproductive and respiratory syndrome virus (Porcinereproductiveandrespiratorysyndromevirus, PRRSV). Respiratory diseases. According to the difference of virus genome, PRRSV can be divided into two genotypes, namely European type and North American type. Although the prevailing strains of PRRSV include American type and European type, so far, the genotypes of PRRSV prevalent in my country are all North American type, including the classic strain (C-PRRSV) represented by the North American type PRRSV CH-1a strain. And the mutant strains (H-PRRSV) represent...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/70C12Q1/701C12Q2527/107C12Q2563/107
Inventor 张建峰刘志成嘎利兵嘎陈琴苓徐志宏张春红康桦华郭鹏举
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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