Detection primers, probe and detection method of highly pathogenic porcine reproductive and respiratory syndrome virus

A porcine blue ear disease virus, highly pathogenic technology, applied in the biological field, can solve the problems of increased detection workload, time and cost, RNA degradation and cross-contamination, and difficulty in realizing high-throughput detection, so as to reduce operation steps and facilitate Degradation, cost-saving effect

Active Publication Date: 2015-06-17
苟德明 +2
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Problems solved by technology

[0004] However, the current methods for detecting PRRS based on RT-PCR all need to extract viral RNA, and then use RNA as a template for detection; this RNA extraction step will undoubtedly greatly increase the detection workload, time and...

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  • Detection primers, probe and detection method of highly pathogenic porcine reproductive and respiratory syndrome virus
  • Detection primers, probe and detection method of highly pathogenic porcine reproductive and respiratory syndrome virus
  • Detection primers, probe and detection method of highly pathogenic porcine reproductive and respiratory syndrome virus

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Embodiment 1

[0039] The detection method (direct amplification RT-PCR method) of embodiment 1 highly pathogenic porcine blue ear disease virus

[0040] 1. Design of specific primers and probes

[0041]The highly pathogenic PRRS virus HP-PRRSV has a discontinuous deletion of 30 amino acids in the non-structural protein 2 (NSP2) coding region, based on which specific primers and probes were designed: respectively designed at both ends of the deletion region Upstream and downstream primers; and a Taqman probe was designed to span the flanking sequences at both ends of the amino acid deletion region.

[0042] The sequence of the upstream primer (dHP-PRRSV-F) is:

[0043] 5'-GGTCGGCACCAGTTCCTG-3' (SEQ ID No: 1),

[0044] The sequence of the downstream primer (dHP-PRRSV-R) is:

[0045] 5'-AAATCCAGAGGCTCATCCTGG-3' (SEQ ID No: 2),

[0046] The sequence of the Taqman probe (dHP-PRRSV-pro) is:

[0047] 5'-FAM-CGCGTAGAACTGTGACAACAACGCTGACG-BHQ1-3' (SEQ ID No: 3).

[0048] 2. Exploration of dire...

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Abstract

The invention discloses a pair of detection primers, a probe and a detection method of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). According to the invention, a serum sample is directly added into a fluorescent quantitation RT-PCR reaction system containing the detection primers and the probe so as to carry out RT-PCR, and whether the sample contains HP-PRRSV is detected after the RT-PCR. By the method provided by the invention, continuous and automatic virus RNA release, reverse transcription and fluorescent quantitative PCR detection are realized in a PCR tube; it only takes 1.5 hours from obtaining the virus serum sample to obtaining detection results; sensitivity and accuracy of the method are comparable with those of traditional RT-PCR; the defect that traditional RT-PCR needs the tedious step of RNA extraction is overcome, operation steps are minimized, detection speed is improved, costs are saved and cross contamination is effectively avoided; and high throughput detection of HP-PRRSV can be carried out, and the method is of great significance for rapid detecting epidemic situation of HP-PRRSV and timely formulating prevention and control measures.

Description

technical field [0001] The invention belongs to the field of biological technology, and more specifically, the invention relates to a detection primer, a probe and a detection method of highly pathogenic porcine blue ear disease virus (HP-PRRSV). Background technique [0002] Porcine blue-ear disease virus (PRRSV) is a single-stranded RNA virus with a genome length of 15 kb, belonging to the Arteriviridae genus Arterivirus. PRRS, also known as "Porcine Reproductive and Respiratory Syndrome", is a highly contagious disease that can cause reproductive disorders, abortion and stillbirth in adult pigs, as well as abnormal breathing in piglets. Pig PRRS was first discovered in North America in 1987 and has now swept the world, becoming one of the most harmful porcine infectious diseases. Porcine PRRS virus can be divided into two genotypes, the American strain represented by VR-2332 strain and the European strain represented by Lelystad virus (LV). Highly pathogenic porcine blu...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2521/107C12Q2527/125C12Q2531/113C12Q2561/101
Inventor 苟德明张利康康
Owner 苟德明
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