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Primer set, kit and method for multiplex immunofluorescence detection of six viruses in mice

A technology of immunofluorescence detection and primer set, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problems of high difficulty and high cost, and achieve low detection cost, high-throughput detection, Sensitivity of detection results

Active Publication Date: 2018-05-25
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 set, kit and method for multiplex immunofluorescence detection of six viruses in mice
  • Primer set, kit and method for multiplex immunofluorescence detection of six viruses in mice
  • Primer set, kit and method for multiplex immunofluorescence detection of six viruses in mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, the primer group that is used for multiple immunofluorescence detection mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus

[0061] 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 TMEV (GenBank accession number: X56019), the N gene sequence of MHV (GenBank accession number: FJ647223), ORF1-ORF2 binding region gene sequence of MNV (GenBank accession number: AY228235), S1 gene sequence of Reo-3 (GenBank accession number: HM159619), VP2 gene sequence of MVM (GenBank accession number: NC001510), VP2 gene sequence of MPV (GenBank accession number: NC001630) and envelope protein gene sequence of MS2 (GenBank accession number: NC001417, used as internal standard, internal control, IC for short).

[0062] Primers are designed according to the nucleic acid sequence, and the secondary structure of the primers and the a...

Embodiment 2

[0090] Embodiment 2, the kit that is used for multiple immunofluorescence detection mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus

[0091] The kit includes the following components:

[0092] 1) The primer set designed in embodiment 1 for multiple immunofluorescence detection of mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus;

[0093] 2) 7 kinds of fluorescently encoded microspheres containing anti-tag sequences encoding different fluorescent colors. The anti-tag sequences can be complementary to the tag sequences in the primers for multiple fluorescent immunoassays. The 7 kinds of microspheres were purchased from Luminex company, in which TMEV, MHV, MNV, Reo-3, MVM, MPV and MS2 correspond to the fluorescent coded microsphere numbers MTAG-A046, MTAG-A028, MTAG-A015, MTAG-042, MTAG-029, MTAG- 034 and MTAG-036;

[0094] 3) streptavidin-phycoerythrin complex, RT-PCR amplification reagent, internal standard MS2 standard, mixed positive control, negative control; the RT-PCR amplificati...

Embodiment 3

[0095] Embodiment 3, the establishment of a kind of multiple immunofluorescence detection mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus method

[0096] (1) Nucleic acid extraction

[0097] The RNA or DNA of six viruses, TMEV, MHV, MNV, Reo-3, MVM, and MPV, were extracted with an automatic nucleic acid extractor (Tiangen Company) and used as templates for multiplex PCR. 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.

[0098] (2) Preparation of plasmid standard

[0099] Using the RNA or DNA of six viruses, TMEV, MHV, MNV, Reo-3, MVM, and MPV, as templates, use the corresponding original primer pairs in the original primer set designed in Example 1 to carry out RT-PCR amplification, and the amplified The amplified products were detected by agarose gel electrophoresis, the target fragment was recovered with a gel recovery ...

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Abstract

The invention discloses a primer group, a kit and a method for detecting six mouse viruses by multiple immunity fluorescence. The primer group is designed by the aid of multiple RT-PCR (reverse transcription-polymerase chain reaction) technologies and Luminex liquid chip technologies and can simultaneously detect six high-infection viruses such as TMEV, MHV, MNV, Reo-3, MVM and MPV of mice. By the aid of the primer group, target amplification products are acquired through specific PCR, the amplification products, fluorescence encoding microspheres and streptavidin-phycoerythrin are hybridized, and different types of viruses are distinguished when a mean fluorescence intensity value is read by a Luminex detector. The method has the advantages of rapidness, high efficiency, high specificity and sensitivity, good repeatability and the like and can be applied to quality monitoring, epidemiological investigation and early warning of test mice. The primer group is good in flexibility, and varieties detecting the viruses can be increased and decreased as required on the basis.

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 method for multiplex immunofluorescence detection of six kinds of viruses in mice. 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. Viral diseases play an important role in the infectious diseases of experimental animals. In the daily monitoring of animal pathogens in biological experiments, we found that most of the viral infections in experimental mice were recessive, without obvious clinical symptoms, and it was difficult to diagnose the disease; and animals Infection virus often manifests as persistent infection. Once the virus infection exists in the facility, it is difficult to remove it. It nee...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6804C12N15/11
CPCC12Q1/6834C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 袁文张钰郭鹏举黄韧
Owner GUANGDONG LAB ANIMALS MONITORING INST
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