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Multiplex fluorescence analysis method for simultaneous detection of four rat parvoviruses and kit

A parvovirus and multiple fluorescence technology, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial measurement/inspection, can solve the detection of four strains of rat parvovirus and multiple fluorescence analysis methods that have not been seen. Rat parvovirus and other problems, to achieve the effect of ensuring renaturation temperature and hybridization efficiency, less sample amount, and avoiding cross-hybridization

Active Publication Date: 2016-11-30
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no method in China that can detect and distinguish four strains of rat parvovirus at the same time, and there is no report on the application of multiple fluorescence analysis methods to detect rat parvovirus

Method used

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  • Multiplex fluorescence analysis method for simultaneous detection of four rat parvoviruses and kit
  • Multiplex fluorescence analysis method for simultaneous detection of four rat parvoviruses and kit
  • Multiplex fluorescence analysis method for simultaneous detection of four rat parvoviruses and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Primer Design

[0092] After screening a large number of designed primers, it was found that the primer pairs M1 and M2, K1 and K2, H1 and H2, P1 and P2 paired multiple fluorescence to simultaneously detect 4 strains of rat parvovirus RMV, KRV, H1 and RPV -1a strain has the best effect, and its base sequence is shown below.

[0093] Primer M1: ACACCATGCCAACTGCAGATG (SEQ ID NO: 1),

[0094] Primer M2: ATTGTTCACTCCCTGTGTTTGTGTT (SEQ ID NO: 2);

[0095] Primer K1: AACCAGACGCTGGAATCGCTAA (SEQ ID NO: 3),

[0096] Primer K2: TGTAGCAGTCTAGATGCATGA (SEQ ID NO: 4);

[0097] Primer H1: CTCTAGCAACTCTGCTGAAG (SEQ ID NO: 5),

[0098] Primer H2: CAGTTATTCCTTGGAGGCAT (SEQ ID NO: 6);

[0099] Primer P1: GATGATAAGCGGTTCAGGG (SEQ ID NO: 7),

[0100] Primer P2: AAGAGCTCCGGTATCTCTGTC (SEQ ID NO: 8).

[0101] The present invention adopts the method of multiple fluorescence analysis to distinguish rat parvovirus RMV strain, KRV strain, H1 strain and RPV-1a strain, so the abo...

Embodiment 2

[0107] Example 2: Multiplex fluorescence assay kit for simultaneous detection of 4 strains of rat parvovirus

[0108] The kit includes the following components:

[0109] (1) The primers designed in Example 1 for multiplex fluorescence analysis;

[0110] (2) 4 kinds of fluorescently encoded microspheres containing anti-tag sequences that encode different fluorescent colors, and the anti-tag sequences can be complementary paired with the tag sequences in the primers for multiple fluorescence analysis; 4 kinds of microspheres were purchased from luminex company, wherein RMV, KRV, H-1 and PRV-1a respectively correspond to the fluorescent coded microsphere numbers MTAG-A015, MTAG-A036, MTAG-A062 and MTAG-077.

[0111] (3) Streptavidin-phycoerythrin complex.

Embodiment 3

[0112] Example 3 Establishment of multiple fluorescence analysis method for simultaneous detection of 4 strains of rat parvovirus

[0113] (1) Construction of plasmids

[0114] Using Tiangen’s automatic nucleic acid extractor to extract the DNA of 4 parvovirus strains RMV, KRV, H1 and RPV-1a respectively, and perform PCR with primer pairs M1 and M2, K1 and K2, H1 and H2, P1 and P2 respectively For amplification, the amplified products were detected by agarose gel electrophoresis and purified by gel cutting. Then use Tiangen’s kit to connect the purified cDNA to the pMD-19T vector, transform the ligated product into DH5a competent cells, select a single clone, carry out bacterial liquid PCR identification, and carry out plasmid extraction on the colonies identified as positive bacteria. Mention, send for sequencing.

[0115] (2) Plasmid PCR amplification

[0116] The RMV, KRV, H-1 and PRV-1a primers were used for single-fold and quadruple PCR amplification, respectively, usi...

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Abstract

The invention discloses a multiplex fluorescence analysis method for simultaneous detection of four rat parvoviruses and a kit. The method is simple to operate. By PCR, a target amplified fragment is obtained; then, the amplified product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized; and through a detector, MFI value is read to distinguish pathogenies of different types. The method can simultaneously detect and identify rat parvoviruses RMV, KRV, H-1 and PRV-1a, has advantages of strong specificity, high sensitivity, good repeatability and the like, and can realize simultaneous detection of various different target molecules in a same sample. The method has good flexibility. Types of detected pathogenies can be increased or reduced according to needs.

Description

technical field [0001] The invention belongs to the field of pathogen detection of experimental animals, and in particular relates to a multiple fluorescence analysis method for simultaneously detecting four strains of rat parvoviruses (RMV, KRV, H1 and RPV-1a). Background technique [0002] Rat parvovirus (Rat parvovirus) is one of the most harmful viruses to experimental rats. It can cause a decrease in the reproductive rate of the breeding group of rats. Reinfection of susceptible animals results in the persistence of rat parvovirus in the population. Rat parvovirus can also contaminate tumor grafts and cell lines, which can seriously interfere with experimental studies. [0003] Up to now, a total of 4 strains of rat parvovirus RMV, KRV, H-1 and RPV-1a have been isolated, among which the previously identified KRV and H-1 strains are SPF-grade large strains in the national standard for experimental animals (GB 14922.2-2011). One of the must-check items for rat virus. S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6816C12Q1/701C12Q2600/16C12Q2563/107C12Q2537/143C12Q2563/149
Inventor 徐凤姣郭鹏举黄韧张钰练月晓饶丹
Owner GUANGDONG LAB ANIMALS MONITORING INST
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