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Primer group for simultaneously detecting five pathogens of rats, kit and multiple immunofluorescence analysis method

A technology of pathogens and primer sets, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as high cost and difficulty, and achieve low environmental interference, low detection cost, and strong detection specificity Effect

Active Publication Date: 2017-05-31
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself, and can only detect up to 5 targets, and the success of the experiment is extremely difficult and the cost is relatively high

Method used

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  • Primer group for simultaneously detecting five pathogens of rats, kit and multiple immunofluorescence analysis method
  • Primer group for simultaneously detecting five pathogens of rats, kit and multiple immunofluorescence analysis method
  • Primer group for simultaneously detecting five pathogens of rats, kit and multiple immunofluorescence analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, simultaneously detect the primer group of five kinds of pathogens of rat RTV, TMEV, RCV, PVM, M.pulmonis

[0058] According to the purpose of the invention, a large number of sequences are analyzed and compared, and the specific conserved sequences of the following viruses are screened out: the 5'UTR gene sequence of RTV (GenBank accession number: EU542581), the 5'UTR gene sequence of TMEV (GenBank accession number: X56019 ), the N gene sequence of RCV (GenBank accession number: AF088984), the NS1 gene sequence of PVM (GenBank accession number: AY743910), the 16sRNA gene sequence of M.pulmonis (GenBank accession number: AF125582) and the envelope protein gene sequence of MS2 (GenBank accession number: NC001417, used as internal standard, internal control, referred to as IC).

[0059] Primers are designed according to the nucleic acid sequence, and the secondary structure of the primers and the ability to form primer-dimers with each other are evaluated. T...

Embodiment 2

[0084] Embodiment 2, the kit that detects five kinds of pathogens of rat RTV, TMEV, RCV, PVM, M.pulmonis simultaneously

[0085] The kit includes the following components:

[0086] 1) The primer set designed in embodiment 1 to simultaneously detect rat RTV, TMEV, RCV, PVM, M.pulmonis pathogen;

[0087] 2) 6 kinds of fluorescently encoded microspheres containing anti-tag sequences that encode different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in multiple fluorescent immunoassay primers; 6 kinds of microspheres were purchased from luminex company, where RTV, TMEV, RCV, PVM, M.pulmonis, and IC correspond to fluorescently coded microsphere numbers MTAG-A034, MTAG-A046, MTAG-A028, MTAG-019, MTAG-029, and MTAG-036;

[0088] 3) streptavidin-phycoerythrin complex, RT-PCR amplification reagent, internal standard MS2 standard, mixed positive control, negative control; the RT-PCR amplification reagent contains 5×buffer, dNTP, Enzyme mix; th...

Embodiment 3

[0089] Example 3, Establishment of Multiple Fluorescence Immunoassay Method for Simultaneously Detecting Five Pathogens of RTV, TMEV, RCV, PVM and M.pulmonis in Rats

[0090] (1) Nucleic acid extraction

[0091] The RNA or DNA of five pathogens, RTV, TMEV, RCV, PVM, and M.pulmonis, were extracted with an automatic nucleic acid extractor (Tiangen Company) and used as templates for multiple fluorescent immunoassay detection methods. The internal standard MS2 standard is added to each sample, and simultaneously participates in the whole process of sample nucleic acid extraction, sample addition, RT-PCR amplification and signal detection.

[0092] (2) Preparation of plasmid standard

[0093] With RTV, TMEV, RCV, PVM, the RN or DNA of five kinds of pathogens of M.pulmonis as a template, use the corresponding original primer pair in the original primer set designed in Example 1 to carry out RT-PCR amplification respectively, and the amplified product Carry out agarose gel electrop...

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Abstract

The invention discloses a primer group for simultaneously detecting five pathogens of rats, a kit and a multiple immunofluorescence analysis method. A multiple PCR (polymerase chain reaction) technology and a Luminex liquid phase chip technology are combined; the primer group capable of simultaneously detecting the five pathogens including RTV (rat theilovirus), TMEV (Theiler's murine encephalomyelitis virus), RCV (rat coronavirus), PVM (pneumonia virus of mice) and M. pulmonis of rats is designed. The primer group uses the specific PCR to obtain a target amplification product; then, the amplification product, fluorescence coded microspheres and streptavidin-phycoerythrin are hybridized; different types of pathogens are distinguished by reading an MFI value through an Luminex detection instrument. The method has the beneficial effects of high speed, high efficiency, high specificity, high sensitivity, good repeatability and the like, and can be applied to the experiment rat quality monitoring, epidemiology survey and early-stage early warning. The primer group has the advantages that the flexibility is good; the pathogen detection types can be increased or decreased on the basis according to requirements.

Description

technical field [0001] The invention belongs to the field of biological detection, and more specifically relates to a primer set, a kit and a multiple immunofluorescence analysis method for simultaneously detecting five pathogens in rats. Background technique [0002] Microbiological quality testing of experimental animals is an important indicator for evaluating the quality of experimental animals. Microbial infection not only causes harm to experimental animals, but also potentially interferes with scientific research. Most of the pathogenic microorganisms in the experimental rats were recessively infected, without obvious clinical symptoms, and the diagnosis of the disease was difficult. Moreover, pathogens infected with animals are often manifested as persistent infections. Once pathogenic infections exist in facilities, it is difficult to remove them. It requires a test-and-removal method to completely eradicate pathogens from contaminated facilities. Therefore, it is n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11C12R1/35C12R1/93
CPCC12Q1/686C12Q1/689C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/131C12Q2563/107C12Q2563/149
Inventor 黄韧袁文郭鹏举张钰
Owner GUANGDONG LAB ANIMALS MONITORING INST
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