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Primers and probe for detecting mouse Sendai virus and method thereof

A Sendai virus and probe technology, applied in the field of biological detection, can solve the problems of short detection time and achieve the effects of simple and fast operation, improved sensitivity, and good linear relationship

Active Publication Date: 2012-02-01
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In order to solve the deficiencies of the existing mouse Sendai virus detection method, the invention provides a primer, probe and method for detecting mouse Sendai virus by real-time fluorescence quantitative PCR. The method is simple and convenient, has high sensitivity and short detection time

Method used

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  • Primers and probe for detecting mouse Sendai virus and method thereof
  • Primers and probe for detecting mouse Sendai virus and method thereof
  • Primers and probe for detecting mouse Sendai virus and method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The design of embodiment 1 mouse Sendai virus special primer and probe

[0040] Sendai virus RNA is composed of 15383 nucleotides, encoding 6 viral structural proteins and one non-structural protein. The 6 structural proteins are L (large), P (RNA polymerase), NP (nucleocapsid), HN (hemagglutinin-neuraminidase ), F (Fusion) and M (membrane). A nonstructural protein, C, is unique to viruses. The gene sequence is 3'-Leader-NP-P+C-M-F-HN-L-Leader-5', with a leader sequence at the 3' end and 5' end.

[0041] By comparing the genomes of different strains of SEV in GenBank, the conserved homologous sequence in the non-structural protein of the virus is selected: the nucleotide sequence shown in SEQ ID NO:4.

[0042] For the found conserved homologous sequence region, special primers and probe sequences for real-time fluorescent quantitative PCR detection were designed, and the amplified target fragment was 100 bases in size. Wherein the preferred primer and probe sequences...

Embodiment 2

[0049] The preparation of embodiment 2 positive RNA standard items

[0050] First prepare the pMD18-T vector containing the nucleotide sequence shown in the homologous sequence SEQ ID NO: 4, use it as the vector to amplify a 100bp DNA fragment, recover it for in vitro transcription reaction, digest the DNA template and purify the RNA after the reaction , quantified as a positive RNA standard.

[0051] 1. Preparation of templates for in vitro transcription

[0052] The nucleotide sequence shown in SEQ ID NO: 4 was synthesized and inserted into the pMD18-T vector (synthesized by Invitrogen), and named pMD18T-SEV. Using pMD18T-SEV as a template, use the upstream primer SEV-100bp-F: 5'-TAATACGACTCACTATAGGGCATCTATG-3' and the downstream primer SEV-100bp-R: 5'-TTTGCAAACACAATAC-3' to amplify a 100bp DNA fragment , recovered by agarose gel electrophoresis, used as a template for in vitro transcription.

[0053] 2. In vitro transcription to prepare a large amount of RNA

[0054] Pr...

Embodiment 3

[0066] Embodiment 3 Establishment of real-time fluorescent quantitative PCR amplification method

[0067] 1. Real-time fluorescence quantitative PCR template preparation - total RNA extraction:

[0068] According to the instructions for use, the sample RNA was extracted with Trizol / Trizol LS kit (provided by Invitrogen), the specific method is as follows:

[0069] 1) Add an appropriate amount of TRIzol / / Trizol LS to the sample (if it is a tissue that needs to be ground and homogenized), mix well, and let stand at room temperature for 5 minutes;

[0070] 2) Add 200 μl of chloroform: isoamyl alcohol (24:1) solution, mix vigorously with vortex for 10 seconds, leave at room temperature for 5 minutes, and centrifuge at 15,000 rpm at 4°C for 15 minutes;

[0071] 3) Take the supernatant, add an equal volume of isopropanol to precipitate, and centrifuge at 15,000 rpm at 4°C for 15 min;

[0072] 4) Remove the supernatant, add pre-cooled 75% ethanol (volume ratio 1:1) to wash the RNA ...

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Abstract

The invention discloses primers and a probe for detecting mouse Sendai virus and a method thereof. The primers have a pair of oligonucleotides constituted by an upstream primer with the SEQ ID No. 1 (sequence identity number 1) in a sequence table and a downstream primer with the SEQ ID No. 2 in the sequence table, and the probe matched with the primers has a nucleotide sequence of the SEQ ID No. 3 in the sequence table. By using the method and a kit for detecting the mouse Sendai virus, the advantages of rapidness, simplicity, high sensitivity, strong specificity and high recovery rate are realized, the defects of being complex and time-consuming in the existing detection means, being long in period and having certain requirements for an operation laboratory are overcome, and the application prospects are great.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a primer, a probe and a method for detecting mouse Sendai virus. Background technique [0002] There are many viruses that naturally infect experimental animals. According to their hazards to humans, they can be divided into three categories; one category is zoonotic viruses, which can infect humans and primates; the second category has no signs of infecting humans. , but can replicate in human, ape and monkey-derived cells cultured in vitro, which is potentially dangerous to humans; the three types of viruses only infect animals themselves under natural conditions, and there is no sign that they can infect humans, so they are harmful to humans. Not much of a threat. [0003] Nowadays, as a major source of biological products such as monoclonal antibodies and protein drugs, mice have potential virus contamination. In the third part of the "Pharmacopoeia of the People's Repub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 谭淑萍李萃王刚蒋立新周志文
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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