Taqman real-time fluorescent PCR kit for detecting pseudorabies virus and detection method thereof

A pseudorabies virus, real-time fluorescence technology, applied in microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve problems such as low sensitivity and low efficiency, and achieve good repeatability and precision. High-grade, easy-to-control effect

Inactive Publication Date: 2018-12-18
SHANGHAI SHENYA ANIMAL HEALTH PROD FUYANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a Taqman real-time fluorescent PCR kit and detection method for detecting pseudorabies virus in order to overcome the low efficiency and low sensitivity defects in the prior art

Method used

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  • Taqman real-time fluorescent PCR kit for detecting pseudorabies virus and detection method thereof
  • Taqman real-time fluorescent PCR kit for detecting pseudorabies virus and detection method thereof
  • Taqman real-time fluorescent PCR kit for detecting pseudorabies virus and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0054] The invention detects porcine blood pseudorabies virus.

[0055] Specifically include the following steps: (1) sample collection; (2) RNA extraction; (3) preparation of PCR reaction system; (4) sample addition; (5) amplification; (6) judgment result;

[0056] (1), sample collection and processing

[0057] Live samples: Tonsil samples from 7 pigs were taken with a sampling gun, numbered: No. 001, No. 002, No. 003, No. 004, No. 005, No. 006 and No. 007. Blood was collected with a sterile syringe and injected with 1 / 10 4% EDTA solution in a sterile container, mix well, number for later use;

[0058] (2) Extraction of sample RNA (sample processing room)

[0059] Use the QIAampMinElute Virus Spin Kit (Catalog No.: 57704) from QIAGEN. For specific extraction steps, please refer to the instructions of the corresponding extraction kit.

[0060] Sample extraction quality control results such as figure 1

[0061]

[0062] (3), preparation of PCR reaction system

[0063] ...

Embodiment 2

[0077] Embodiment 2 Sensitivity detection

[0078] The positive control clone plasmid was used to evaluate the sensitivity of the kit of the present invention, and the positive control clone plasmid was diluted 10 times, and the detection range was 107-101copiea / ul. The results show that 107-101copiea / ul can be detected, and the pseudorabies virus can be detected within this range, that is, the kit of the present invention can detect samples with 10 copies of the pseudorabies virus content. See the test results figure 2 .

Embodiment 3

[0079] Embodiment 3 specific detection

[0080] Utilize the kit of the present invention to detect 8 kinds of blue ear classic strain, porcine parvovirus, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine circovirus, swine fever virus, Japanese encephalitis and porcine epidemic diarrhea Virus.

[0081] The test results show that the kit can specifically amplify the pseudorabies virus without cross-reaction with other nucleic acids. See the test results image 3 .

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Abstract

The invention discloses a Taqman real-time fluorescent PCR kit for detecting pseudorabies virus and a detection method thereof. The kit comprises a PCR reaction buffer solution A, a PRV primer probe mixture B, an enzyme system C, a negative control, and positive control; and the method comprises the following steps: 1) sample collection and processing; 2) sample RNA extraction, wherein sample RNAextraction is carried out in a sample processing chamber; 3) preparation of a PCR reaction system; 4) sample adding, 5) amplification; and 6) negative result determination, wherein a FAM channel amplification curve is not in S shape and a Ct value is UNDET; the positive result determination is characterized in that when the sample is in S shape in the FAM channel amplification curve and the Ct value is less than or equal to 36, the result is positive; an experimental gray area is characterized in that if the sample is in S shape in the FAM channel amplification curve and the Ct value is greater than 36 and less than 38, the result is in the experimental gray area, and the sample should be re-examined; if the amplification curve is in S shape, the result of the re-examination is positive, and if the amplification curve is not in S shape, the result is negative. The method has the advantages of high sensitivity, good use effect and the like.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to a Taqman real-time fluorescent PCR kit for detecting pseudorabies virus and a detection method thereof. Background technique [0002] Pseudorabies virus (PRV), also known as Aujieszky'sdisease virus (ADV), belongs to the α-herpesvirus subfamily (Alpheresvae) of the Herpesuiridae family. The complete virion is round, with a diameter of 150-180nm, and the nucleocapsid is icosahedral, with a diameter of 105-110nm, and has a capsule and a fiber. The full length of the PRV genome is 140-150kb, and its GC content is as high as 73%. It is a conjoined circular linear double-stranded DNA with high molecular weight. Research and analysis found that the entire PRV gene encodes about 100 viral proteins, among which gC, gE, gG, gI, and gM are non-essential glycoproteins for virus replication. The gE gene is an important virulence gene of PRV, which promotes the fusion between in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/705C12Q2561/101C12Q2561/113
Inventor 徐邦伟王勇葛好林
Owner SHANGHAI SHENYA ANIMAL HEALTH PROD FUYANG CO LTD
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