Hybrid snakehead rhabdovirus fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof

A detection kit, fluorescence quantitative technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., to achieve high sensitivity, accurate qualitative and quantitative, and good specificity

Active Publication Date: 2013-11-20
湖南振业动物检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, traditional viral disease diagnosis requires methods such as virus isolation, cell transfection, electron microscope observation, and ordinary PCR detection. There is a lack of detection and early warning methods for rapid, accurate, and quantitative analysis of viruses.

Method used

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  • Hybrid snakehead rhabdovirus fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof
  • Hybrid snakehead rhabdovirus fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof
  • Hybrid snakehead rhabdovirus fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of primers and probes:

[0036]The length of the amplified product of the first pair of primers (P1, P2) is 1560bp, which is used to construct a recombinant plasmid and provide a template for making a positive control standard curve; the second pair of primers (HSHRV-2F, HSHRV-2R) and probe (HSHRV- 2probe) for fluorescent quantitative PCR amplification, the length of the amplified product is 143bp. Primers and probes were synthesized by Guangzhou Jige Biotechnology Co., Ltd. The primer and probe sequences are as follows:

[0037] P1: 5'-CGCGGATCCATCATGAAATCAATCATTGCACT-3' (SEQ ID NO. 1);

[0038] P2: 5'-CATCTGCAGAATTGATACTGCTGCAAAGGG-3' (SEQ ID NO. 2);

[0039] HSHRV-2F: 5'-CACCAGTCATATCAATCC-3' (SEQ ID NO. 3);

[0040] HSHRV-2R: 5'-CGGACTTAAACCTCATTC-3' (SEQ ID NO. 4);

[0041] HSHRV-2 probe: 5'-ACCTCTCCGCACATTGACATCT-3' (SEQ ID NO.5);

[0042] The 5' end of the HSHRV-2 probe is labeled with the fluorescent dye FAM, and the 3' end is labeled with the ...

Embodiment 2

[0044] The establishment of a fluorescent quantitative PCR detection kit for rhabdovirus of hybrid snakehead and the optimization of detection method:

[0045] 1. Preparation of standard template

[0046] 1) Infect EPC with HSHRV, collect the cytotoxic material after the cells appear lesions, extract the total viral RNA according to the instructions of Trizol Reagent, and measure the absorbance of the RNA samples at 260nm and 280nm wavelengths after being treated with DNase I RNase-free (Fermentas) value, to calculate the concentration and purity of the RNA sample.

[0047] 2) Using PrimeScript TM RT reagent Kit (TaKaRa) kit was used for reverse transcription to obtain cDNA. The obtained cDNA was used as a template, and P1 and P2 were used as primers for PCR amplification. The reaction parameters were pre-denaturation at 94°C for 3 min, 30 cycles at 94°C for 30 s, 30 s at 55°C, and 90 s at 72°C, and a final extension at 72°C for 10 min. At the same time, a negative contro...

Embodiment 3

[0063] Sensitivity, repeatability and specificity tests of the hybrid rhabdovirus fluorescent quantitative PCR detection kit:

[0064] 1. Perform 10-fold serial dilution of the positive control recombinant plasmid so that the concentration is 1×10 6 ~1 copies / μL as standard template, with sterilized ddH 2 O replaces the template as a negative control. The hybrid rhabdovirus fluorescent quantitative PCR detection kit established in Example 2 and the optimized reaction system and conditions are used to carry out the fluorescent quantitative PCR reaction. According to the linear relationship between the sample copy number and the Ct value and The correlation coefficient determines the minimum detection amount. At the same time, common PCR reaction was carried out with primers HSHRV-2F and HSHRV-2R, the minimum template concentration that could be detected by the two methods was calculated, and the sensitivity of the two methods was compared.

[0065] The results show that the t...

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Abstract

The invention discloses a hybrid snakehead rhabdovirus fluorescent quantitative PCR (polymerase chain reaction) detection kit and a detection method thereof. The kit contains specific primers HSHRV-2F and HSHRV-2R and a probe HSHRV-2probe. When the kit is used for detecting hybrid snakehead rhabdovirus, the kit has the characteristics of high sensitivity, high repetitiveness and good specificity, can quickly and accurately realize qualitative and quantitative detection of hybrid snakehead rhabdovirus, and is of far reaching importance in early diagnosis of hybrid snakehead virus diseases, molecular epidemiology research, prevention and control technology research and the like.

Description

technical field [0001] The invention belongs to the detection field of pathogenic microorganisms, in particular to a fluorescent quantitative PCR detection kit and a detection method for rhabdoviruses of hybrid snakeheads. Background technique [0002] hybrid snakehead ( Channa maculata♀×C. argus♂ ) is a generation of offspring obtained by hybridization with the spotted snakehead as the female parent and the black snakehead as the male parent. The hybrid snakehead combines the advantages of the parents. In the actual production process, it has fast growth speed, strong stress resistance, high survival rate, and transportation resistance. , Easy to tame artificial feed and other advantages. Due to the obvious economic benefits of breeding hybrid snakeheads, the scale of breeding has been expanding nationwide in recent years, and has gradually replaced snakeheads such as black snakeheads and spotted snakeheads as the main cultured species. However, since July 2012, the epide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 刘春曾伟伟李凯彬王庆王芳常藕琴梁慧丽梁惜梅吴淑勤
Owner 湖南振业动物检验有限公司
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