Detection method for identifying American classical PRRSV strain, HP-PRRSV strain and new-type viral NADC30 strain at the same time

A technology of classic strains and mutant strains, applied in the field of detection and molecular biology detection, can solve problems such as low accuracy, PCR product contamination, inability to distinguish PRRSV, etc.

Active Publication Date: 2017-12-15
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, PRRSV detection techniques include virus isolation method, neutralization test, immunoperoxidase monolayer test, immunofluorescence antibody test, immunohistochemical technology, enzyme-linked immunosorbent assay, colloidal gold immunochromatography technology, reverse transcription polymerization Enzyme chain reaction, etc., the above methods are general-purpose and can only identify whether they are infected with PRRSV, but cannot distinguish which type of PR

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  • Detection method for identifying American classical PRRSV strain, HP-PRRSV strain and new-type viral NADC30 strain at the same time
  • Detection method for identifying American classical PRRSV strain, HP-PRRSV strain and new-type viral NADC30 strain at the same time
  • Detection method for identifying American classical PRRSV strain, HP-PRRSV strain and new-type viral NADC30 strain at the same time

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072]1. Design of specific primers and specific probes: Download multiple American PRRSVs from Genbank (classic strains HN1, SD9521, VR2332, SD1100, CH1a; highly pathogenic variant strains SX1, TJ, JXA1, HUN4; NADC30like : HNyc15, HNjz15, JL580, HENXX1 and SDlz1601) NSP2 gene and N gene sequence, use BioEdit software package to carry out Clustalw comparison, utilize Primer 3.0 software to design specific TaqMan probe, primer according to different virus types. According to the conserved region of the N gene sequence of American-type and European-type PRRSV, the universal primers and universal probes of American-type PRRSV were designed.

[0073] 2. Synthesis of primers and probes: Synthesized by Shanghai Jierui Biological Co., Ltd.

[0074] 3. See Table 2 for primers, probe sequences and probe modifications.

[0075] Table 2 Primers and sequence information

[0076]

[0077]

[0078] 4. Sample template preparation

[0079] (1) Extraction of viral RNA: use TRIzol reag...

Embodiment 2

[0084] Example 2 Verification of multiple real-time fluorescence methods

[0085] 1. Specificity verification

[0086] Utilize the kit method of the present invention to respectively use NADC30like LZ, highly pathogenic SX-1, classic SD-1 strain, vaccine virus R98 strain, TJM-F92 strain, JXA1-R, and swine fever virus, porcine parvovirus, porcine Epidemic diarrhea virus, porcine pseudorabies virus and porcine circovirus type 2 virus were used as templates for multiplex real-time fluorescent PCR amplification to verify the specificity of their primers and probes. The results are shown in Table 3, and the results show that the designed primers and probes of the present invention have strong specificity.

[0087] Table 3. Specificity Verification Tests

[0088]

[0089]

[0090] 2. Sensitivity evaluation

[0091] Quantify NADC30like, highly pathogenic, classic strain positive standards to 10 7 copy / μL, serially diluted 10 times to 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×...

Embodiment 3

[0092] Example 3 Clinical Suspect Sample Detection

[0093] The multiple real-time fluorescent RT-PCR detection method established by the present invention for the classic type, highly pathogenic variant strain and NADC30like strain of PRRSV simultaneously detects 30 clinically suspicious samples, and the sample types include pig lung, lymph node, tissue and serum. Detection was performed using both virus isolation methods and sequencing. The results are shown in Table 4, and the results show that the method established by the present invention is completely consistent with the verification of the sequencing results after virus isolation, and the method is accurate and reliable.

[0094] Table 4 Test results of clinical samples

[0095]

[0096]

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Abstract

The invention discloses a detection method for identifying the American classical PRRSV strain, the HP-PRRSV strain and a new-type viral NADC30 strain at the same time. In the method, a multiplex real-time fluorescence RT-PCR for identifying three kinds of strains at the same time is established based on an ABI7500 fluorescent quantitative PCR instrument, wherein the strains include the classical PRRSV strain, the highly-pathogenic HP-PRRSV strain with the deletion of L-amino acid at the 481st locus of the Nsp2 gene and the continuous deletion of 29 amino acids at the 533ird-561st loci of the Nsp2 gene, and NADC30like; specific primers and specific probes for the three kinds of strains are designed respectively based on the Nsp2 gene, universal primers and universal probes for PRRSV are designed according to the relatively conserved N gene, missing detection caused by Nsp2 region mutation can be effectively prevented, and false negative can be avoided.

Description

technical field [0001] The invention relates to a detection method, in particular to a detection method for simultaneously identifying American-type PRRSV classic strains, variant strains and new virus-like NADC30 strains. The invention belongs to the technical field of molecular biology detection. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). The disease can cause reproductive disorders such as fever, miscarriage, premature birth, stillbirth, and mummified fetuses in sows, increased mortality of piglets before and after weaning, and respiratory disorders in pigs of various ages. The disease first occurred in the United States in 1987. At present, the two prevalent PRRSVs in the world are the European type (genotype I) and the American typ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2521/107C12Q2563/107C12Q2561/113
Inventor 吴家强刘艳艳于江王淞杨杰李建达曾昊陈智
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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