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Detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV

A technology for detection primers and detection methods, applied in the field of molecular biology, can solve the problems of complex operation procedures of WB method, poor specificity of detection methods, and high false positive rate, and achieve shortened detection time and sample volume, high-throughput detection, high specific effect

Active Publication Date: 2017-05-10
GUANGDONG LAB ANIMALS MONITORING INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the existing experimental animal quality testing standards, the detection methods for the above three pathogens still use ELISA or IFA detection methods, which have poor specificity, low sensitivity, and high false positives, and must also be combined with WB detection specificity Antibodies can finally confirm the diagnosis
The false positive rate of ELISA method and IFA method is high and the operation procedure of WB method is complicated, which limits its application scope to a certain extent.

Method used

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  • Detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV
  • Detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV
  • Detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 can simultaneously detect and distinguish the design of the detection primer set of SIV, SRV, STLV

[0043] The main core protein gag gene partial sequence of simian retrovirus (SIV) (GenBank accession number is M74931.1), part of the conserved gene sequence in the p27 protein of simian retrovirus (SRV1, SRV2, SRV3) (GenBank accession number Numbers are M11841.1, AF126467.1, M12349), and the env gene partial sequence of monkey T lymphotropic virus type Ⅰ is mainly the target gene. Primer Premier 6 is used to design specific primers, and the designed upstream primers are respectively A Tag sequence was added to the 5' end, and Spacer C18 was used to separate the Tag sequence from the primer sequence, and a biotin label was added to the 5' end of the downstream primer. The sequences of the detection primers used for PCR amplification are shown in Table 1.

[0044] Table 1. SIV, SRV, STLV detection primer set

[0045]

Embodiment 2

[0046] Example 2 Establishment of a method for simultaneously detecting and distinguishing SIV, SRV, and STLV.

[0047] (1) Extraction of total viral RNA: The total viral RNA in the SIV and SRV virus culture medium was extracted by using the total viral RNA extraction kit of Meiji Biotechnology Co., Ltd., and reverse-transcribed into cDNA by using the reverse transcription kit.

[0048] (2) PCR amplification reaction:

[0049] The 20 μl reaction system contains: SIVfd 0.2 μM, SIVbk 0.2 μM, SRVfd 0.2 μM, SRVbk 0.2 μM, STLV1fd 0.2 μM, STLVbk 0.2 μM, multiplex PCR Assay Kit reaction solution 10.0 μl, PCR EnzymeMix 0.1 μl, cDNA to be tested 2.0 μl, Make up to 20 μl with DEPC water; set positive control and negative control; mix the prepared PCR tube and centrifuge, and perform PCR reaction according to the following procedure: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30s, 58°C for 30s, 72°C Extend at ℃ for 30s, cycle 33 times; extend at 72℃ for 5min.

[0050] (...

Embodiment 3

[0058] Embodiment 3 specificity experiment

[0059] The mixed cDNA templates of SIV, SRV, and STLV were simultaneously detected by the method in Example 2 to verify the specificity.

[0060] The test results showed that the three viral primers had no cross-reaction with each other (see figure 1 ).

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Abstract

The invention discloses a detection primer set and a method capable of simultaneously detecting and distinguishing SIV, SRV and STLV. The detection primer set comprises specific sequence primers of three kinds of different viruses, SIV, SRV and STLV, with specific Tag sequences. The detection method comprises the steps of, extracting DNA of the viruses in a blood sample, simultaneously amplifying target segments of the three viruses by multiplex PCR, hybridizing an amplification product with fluorescence coded microspheres, streptavidin-phycoerythrin solution, reading an MFI value by a detection instrument, and accordingly determining whether the blood sample contains the three kinds of viruses. According to the detection primer set and the method provided by the invention, the three kinds of different viruses, SIV, SRV and STLV, can be simultaneously detected and distinguished. The detection primer set and the method have the advantages of high specificity and sensitivity, rapidness and high efficiency, low cost and accurate and legible result, and are particularly applicable to popularization to use in detection units at various levels.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a detection method for pathogens related to experimental animals, in particular to a detection primer set and method capable of simultaneously detecting and distinguishing SIV, SRV and STLV. Background technique [0002] Non-human primates are closely related to humans in evolution, and have the characteristics of wide natural distribution, large numbers, and easy breeding. They are widely used in the research of reproductive physiology, drug toxicology, biological products and human disease models. my country is rich in non-human primate resources, which are mainly concentrated in southern regions such as Guangdong, Guangxi, Yunnan, Sichuan, and Hainan. There are currently more than 50 breeding companies with a population of more than 300,000 monkeys, which are used for scientific research every year. There are more than 30,000 pieces, and more than 20,000 pieces are expor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6816C12Q1/701C12Q1/702C12Q1/703C12Q2600/16C12Q2537/143C12Q2563/149C12Q2563/107
Inventor 刘助红朱余军王静练月晓黄碧洪徐凤姣郭鹏举张钰黄韧
Owner GUANGDONG LAB ANIMALS MONITORING INST
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