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TaqMan real-time fluorescent quantitative RT-PCR kit and method for detecting double RNA viruses of micropterus salmoides

A technology of real-time fluorescence quantification and RNA virus, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of virus detection method publication, etc., to increase the income of farmers and companies, increase production, The effect of reducing losses

Pending Publication Date: 2021-02-02
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For this unknown aquatic animal diRNA virus, so far, no detection method for this virus has been published

Method used

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  • TaqMan real-time fluorescent quantitative RT-PCR kit and method for detecting double RNA viruses of micropterus salmoides
  • TaqMan real-time fluorescent quantitative RT-PCR kit and method for detecting double RNA viruses of micropterus salmoides
  • TaqMan real-time fluorescent quantitative RT-PCR kit and method for detecting double RNA viruses of micropterus salmoides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Materials and methods

[0031] 1.1 Cells and virus strains

[0032] The Chinese perch brain cell line (CPB) was established and preserved in our laboratory. Largemouth bass birnavirus (LBBV), grouper neuronecrosis virus (Nervous Necrosis Virus, NNV), carp herpesvirus II (Cyprinid herpesvirus II, CyHV-II), mandarinfish ranavirus (MRV) ), grouper iridovirus (SGIV), giant salamander iridovirus (Andrias davidianus iridovirus, ADIV), mandarin fish infectious spleen and kidney necrosis virus (ISKNV), mandarin fish bullet virus (Sinipercachuatsis rhabdovirus, SCRV) and Tilapia Lake Virus (TiLV) were isolated and preserved by our laboratory.

[0033] 1.2 Method

[0034] 1.2.1 Primer and probe design

[0035] According to the VP1 sequence of LBBV, use Primer 5.0 software to design VP1 gene amplification primers VP1-F and VP1-R (for plasmid construction), fluorescent quantitative primers TZ-F and TZ-R and probe probe (see Table 1) . The primer sequences were up...

Embodiment 2

[0056] Embodiment 2 experimental result

[0057] 2.1 Standard curve

[0058] The recombinant plasmid PMD-LBBV-VP1 was serially diluted 10 times. According to the copy number calculation formula, the copy number of the standard product is 4.15×10 10 (copy / μL). Choose the most stable concentration range (4.15×10 7 copy / μL~4.15×10 3 copy / μL), a standard curve was generated in this range. Taking the logarithm of the copy number as the abscissa, and the Ct value as the ordinate, the regression equation is obtained: y=-3.456x+39.257 (R 2 = 1). Based on this, it is speculated that when the detection sample contains 1 copy of virus, the detection Ct value is 39.257, therefore, the detection limit Ct value of this method is set to 39.257. The R of the regression equation 2 =1, indicating that the copy number of the standard equation has a very high correlation with the Ct value.

[0059] 2.2 TaqMan real-time fluorescent quantitative PCR specific detection

[0060] The establi...

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Abstract

The invention relates to the technical field of double-RNA virus detection, in particular to a TaqMan real-time fluorescent quantitative RT-PCR kit and method for detecting double RNA viruses of micropterus salmoides. The kit comprises a primer pair and a TaqMan probe, wherein the sequences of the primer pair and the TaqMan probe are as follows: TZ-F: 5'-AATCCAAAAACAACACGCTAAACA-3' ; TZ-R: 5'-GCGCCTCATGATTGAGTCAAG-3' ; Probe: 5'-(FAM)-ATGGGTTCAATCCCTTCAACGGCG-(Eclipse)-3'. The TaqMan real-time fluorescent quantitative RT-PCR kit and method are established for LBBV, and a rapid and sensitive detection means is provided for early warning and effectively prevention and control of virus diseases.

Description

technical field [0001] The invention relates to the technical field of double RNA virus detection, in particular to a TaqMan real-time fluorescent quantitative RT-PCR kit and method for detecting double RNA virus of largemouth bass. Background technique [0002] The largemouth bass (Micropterus salmoides) belongs to the family Centrarchidae and the genus Micropterus. The meat is delicious and tender, without intermuscular thorns, and rich in nutrition. It is a high-quality freshwater fish. [0003] Since the 19th century, there have been frequent outbreaks of diRNA virus disease in aquatic animals, and its representative species, infectious pancreatic necrosis virus (IPNV), has been prevalent in Shanxi, Gansu, Liaoning, Sichuan and other places in my country, and the sick juveniles died The rate is as high as 95%, which brings great damage to my country's aquaculture industry. Xu Ye, Rodriguez Saint-Jean and others have established a qPCR detection method for double RNA vir...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2561/101C12Q2563/107C12Q2561/113Y02A50/30
Inventor 李宁求付小哲罗明菊林强刘礼辉牛银杰罗霞梁红茹
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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