Primer pair, probe, kit and method for fluorescent quantitative RT-PCR detection of suspected theiler virus in rat

A fluorescent quantitative and primer pair technology, which is applied in the field of fluorescent quantitative RT-PCR to detect suspected Theileria virus in rats, can solve the problems of time-consuming, low sensitivity, and tediousness, and achieve broad application prospects, high sensitivity, and specificity strong effect

Inactive Publication Date: 2017-06-23
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used TLV detection method in the world is serological detection, in which the blood collection and serum separation of rats are particularly cumbersome and time-consuming, and there are ELISA kits for the detection of suspected Theileria virus infection in rats on the market today. Some are imported kits, the price is around 2000-3000 yuan, and they can only detect 96 times 2 samples
Therefore, the traditional methods have disadvantages such as cumbersomeness, limitations, low sensitivity, and high price, and there is an urgent need to develop a simple, fast, and accurate method and kit for detecting TLV.

Method used

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  • Primer pair, probe, kit and method for fluorescent quantitative RT-PCR detection of suspected theiler virus in rat
  • Primer pair, probe, kit and method for fluorescent quantitative RT-PCR detection of suspected theiler virus in rat
  • Primer pair, probe, kit and method for fluorescent quantitative RT-PCR detection of suspected theiler virus in rat

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Establishment of TaqMan real-time fluorescent quantitative PCR detection method for suspected Theileria virus in rats

[0027] 1. Design of primers and probes

[0028] 根据大鼠疑似泰勒氏病毒TLV NGS910毒株的全基因组序列(Genbank登录号为:AB090161.1),选取3622~3729nt这一段特异区域序列:ggctttggatacaagggaacacttcagtggctgtgagagttaggtataagaaaatgaaagttttctgtccccgtcccactctcttccttccttggccctcgaccaccactaccagaatccatgcagataacccagtgt(SEQ ID NO:4)。

[0029] Connect with pUC57 vector to construct the plasmid standard of TLV.

[0030] Design primers and TaqMan-specific probes at 3622-3729nt:

[0031] qTLV-F: 5'-CTTCAGTGGCTGTGAGAGTTAG-3' (SEQ ID NO: 1);

[0032] qTLV-R: 5'-ATCTGCATGGATTCTGGTAGTG-3' (SEQ ID NO: 2);

[0033] FAM-TLV: 5'-FAM-CCGTCCCACTCTCTTCCTCCTTG-3'-BHQ1 (SEQ ID NO: 3).

[0034] 2. pUC-TLV108 plasmid synthesis and common PCR results

[0035] Send the pUC-TLV108 plasmid synthesized by GENEWIZ Company, and dilute it by 10 times, and the final concentration is 3×10 6 ~3×10 0 Copy number / μL, using...

Embodiment 2

[0042] Embodiment 2 Sensitivity and repeatability detection

[0043] Using the pUC-TLV108 plasmid standard as a template, press 3×10 7 ~3×10 0 The copy number / μL ratio was diluted, and the fluorescent quantitative PCR amplification was carried out respectively. The Ct value greater than 35 was considered negative. The amplification curves of the three groups of samples under different concentrations of templates were plotted. The results show that the method has high sensitivity, and the minimum detectable template concentration is 30 copies / μL ( image 3 ), about 100fgDNA, which is 100 times higher than the common PCR method. image 3 Among them, the copy numbers of templates corresponding to Nos. 1-8 are respectively: 3×10 7 , 3×10 6 , 3×10 5 , 3×10 4 , 3×10 3 , 3×10 2 , 3×10 1 , 3×10 0 9. Negative control.

[0044] Take 3×10 of the pUC-TLV108 plasmid standard 6 and 3×10 4 Copy number / μL Two dilutions of DNA were used as templates, and the two dilutions were me...

Embodiment 3

[0047] Embodiment 3 Specificity experiment of rat suspected Theileria virus real-time fluorescent quantitative PCR method

[0048] The concentrations of all template cDNAs used for specific detection were uniformly diluted to 50 ng / μL, and the reaction conditions and reaction system were carried out referring to the method in Example 1 above. see results Figure 4 : Rat parvovirus KRV strain, rat parvovirus H1 strain, Sendai virus and mousepox virus were detected by the established TLV TaqMan fluorescent quantitative PCR detection method. There is no amplification curve. This shows that the method has no cross-reaction with other rat virus pathogens to be tested ( Figure 4 ).

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Abstract

The invention discloses a primer pair, probe, kit and method for fluorescent quantitative RT-PCR detection of a suspected theiler virus in a rat. According to the detection method, by employing cDNA of a brain tissue of the rat as a template, an SEQ ID No:1 and an SEQ ID No:2 as primers and an SEQ ID No:3 as a probe, fluorescent quantitative PCR amplification is carried out to collect a fluorescent signal. According to the fluorescent quantitative RT-PCR detection method, fast, accurate, convenient, fast and specific detection of suspected theiler virus nucleic acid in the rat can be achieved; and the method is high in sensitivity, high in specificity and good in repeatability, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a primer pair, a probe, a kit and a method for detecting suspected Theileria virus in rats by fluorescent quantitative RT-PCR. Background technique [0002] Rat Theiler's-like virus (TLV) is suspected in rats. TLV is a member of the Picornaviridae Cardiovirus genus. The representative strain is NGS910 with a genome size of 8.021kb. The open reading frame (ORF) of its L gene has 72% homology with Theiler's murine encephalomyelitis virus (TMEV), and 55% homology with encephalomyocarditis virus (EMCV) 56%. TLV mainly affects the central nervous system of rats, most of which are recessive infections, and sometimes manifest symptoms such as encephalomyelitis, meningitis, and hindlimb paralysis. Once TLV is contaminated, it will cause huge economic losses to experimental rats and their biotechnology products. [0003] The positive rate of TLV antibody in the sampl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107C12Q2521/107
Inventor 魏晓锋蔡骁垚熊炜胡建华张泉其他发明人请求不公开姓名
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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