Pig atypical pestivirus RT-PCR detection specific primer, kit and detection method

A type of atypical pestivirus and detection kit technology, applied in the field of animal virology and molecular biology, can solve the problems of poor applicability, rapid gene sequence variation, large gene sequence differences, etc., to achieve fast detection, simple operation, The effect of high specificity and sensitivity

Active Publication Date: 2017-06-06
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] These findings may help to understand the cause of congenital tremor in piglets. The PCR method is often used to detect the virus in foreign countries, but the virus is an RNA virus with rapid gene sequence variation and large gene sequence differences between different strains. NCBI currently The homology of the landing sequence is only 88.1%-90.9%. The PCR method established abroad is not very applicable in China, and there is no detection method for this virus in China.

Method used

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  • Pig atypical pestivirus RT-PCR detection specific primer, kit and detection method
  • Pig atypical pestivirus RT-PCR detection specific primer, kit and detection method
  • Pig atypical pestivirus RT-PCR detection specific primer, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 A kind of porcine SARS virus PCR detection primer, detection kit and detection method

[0035] 1, a kind of porcine SARS virus PCR detection primer

[0036] According to the strain sequence of porcine SARS virus registered by NCBI, accession numbers: KU194229, KX929062, LT594521, KU041638, KU041639, KU041637, KX929063, KX929069 and other information provided, using DNAMAN6.0 for comparative analysis, the following PCR was designed The primers:

[0037] Upstream primer F: ctcacyagtgatgggtggga (SEQ ID NO: 1), wherein, y represents: c or t;

[0038] Downstream primer R: cctatyttcttcatgaayaccatggc (SEQ ID NO: 2), wherein, y represents: c or t;

[0039] Using the above primers for PCR amplification can identify porcine atypical fever virus quickly and accurately.

[0040] 2. A porcine SARS virus PCR detection kit

[0041] A porcine atypical fever virus detection kit, comprising the above-mentioned porcine atypical fever virus PCR primers SEQID NO: 1 and ...

Embodiment 2

[0060] Example 2 specificity test

[0061] Serum sample RNA with positive detection is used as positive control, and sterile distilled water is used as negative negative control, according to embodiment 1 step (2), (3) amplification respectively, do electrophoresis identification by step (4) method, the result is as follows figure 1 shown.

[0062] Serum samples tested positive were used as positive controls, culture medium of porcine transmissible gastroenteritis virus, porcine rotavirus, porcine epidemic diarrhea virus, bovine viral diarrhea virus, swine fever virus, porcine reproductive and respiratory syndrome virus Carry out specificity detection, use the method of embodiment 1 and primer, the result does not all amplify except positive control, and positive disease material has band respectively at 439bp place through PCR amplification (see figure 2 ).

Embodiment 3

[0063] Embodiment 3 Sensitivity test

[0064] RNase Free H is used for the RNA extracted from the serum sample in Example 1 2 O made 10-fold serial dilution, the RNA content was 1.22μg / μL, 0.122μg / μL, 0.0122μg / μL, 1.22ng / μL, 0.122ng / μL, 0.0122ng / μL as a template, each dilution was taken 8 μL was used as a template, detected according to the method in Example 1, and the positive band was observed, and the sensitivity was calculated based on the highest dilution of the template amount used for the positive expected band, and the result showed that the minimum detection amount was 0.976ng, that is, 0.122ng / μL gradient. (See image 3 ).

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Abstract

The invention belongs to the technical fields of animal virology and molecular biology, and particularly relates to a pig atypical pestivirus RT-PCR detection specific primer, a kit and a detection method. The nucleotide sequences of the specific primer are as shown in SEQ ID NO:1 and SEQ ID NO:2; the kit comprises the primer, a Prime Script 1 step enzyme mixture, a 2*1 step buffer solution and nuclease-free water. The invention also discloses a method for detecting pig atypical pestivirus by using the specific primer or the kit. The primer provided by the invention has high specificity and sensitivity and can be used for detecting and identifying the pig atypical pestivirus from various clinical samples, so that prevention and control measures can be taken quickly and timely, and economic loss is reduced.

Description

technical field [0001] The invention belongs to the technical field of animal virology and molecular biology, and specifically relates to a specific primer, a kit and a detection method for porcine atypical fever virus RT-PCR detection. Background technique [0002] Pestivirus belongs to the Flaviviridae family and is a highly mutated RNA virus with an envelope. The length of its genome is about 12.3kb. Four species have been identified: bovine viral diarrhea virus type 1 (BVDV-1), bovine viral diarrhea virus type 2 (BVDV-2), swine fever virus (CSFV) and border virus (BDV). There are also several unidentified atypical pestiviruses, including giraffe pestivirus, antelope pestivirus, HoBi-like virus, and Bungowannah virus. [0003] In 2015, when American scholars performed metagenomics analysis on a PRRSV positive sample, they found that there were fragments with pestiviruses CSFV, BVDV and chrysanthemum bat pestilence virus (RaPV) expected value (E) from 8e-6-1e-88 , and ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 王东东欧阳海平叶敏慧潘永飞宋延华
Owner WENS FOOD GRP CO LTD
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