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Detection method and kit for infectious muscle necrosis of penaeus vanmamei

A muscle necrosis and detection method technology, applied in the biological field, can solve the problems of poor amplification effect and low sensitivity, and achieve the effects of improving detection efficiency, high repeatability and high resolution

Inactive Publication Date: 2014-01-29
中华人民共和国中山出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although OIE has released a fluorescent PCR detection method for the virus, according to the author's work practice results, its sensitivity is not high and the amplification effect is not good, so it is necessary to redesign a fast and accurate new detection method

Method used

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  • Detection method and kit for infectious muscle necrosis of penaeus vanmamei
  • Detection method and kit for infectious muscle necrosis of penaeus vanmamei
  • Detection method and kit for infectious muscle necrosis of penaeus vanmamei

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Embodiment 1

[0029](1) According to the literature method of Areerat Kunanopparat et al. (Journal of Virological Methods, 2010, 171(1): 141-148), Dalian Bao Biological Technology Co., Ltd. was entrusted to synthesize the amplification primers of CP-I. Yan Dongchun, School of Life Sciences, Ludong University Infectious Myonecrosis Virus (Infectious Myonecrosis Virus) IMNV cDNA donated by Dr. (IMNV cDNA has been published in the literature "Borsa M. et al., Detection of infectious myonecrosis virus in penaeid shrimps using immunoassays: usefulness of monoclonal antibodies directed to the viral maj or capsid protein. Arch Virol, published 2010-09-23" as a template, PCR (reaction system: 10 times PCR buffer 5 μl, 10 mM dNTPs 2 μl, 20 μM forward and reverse primers (forward primer: 5'-GCTGCAAAAGAGGGTGCTCG-3'; reverse Primers: 5'-TTGCATTGAACTCCACGAAAAC-3') 1 μl each, Taq polymerase 0.5 μl, cDNA 2 μl, with ddH 2 O to make up 50 μl. The reaction conditions were 94°C for 3 minutes; 34 cycles of 94...

Embodiment 2

[0048] Three sets of primer probe-specific fluorescent PCR detection:

[0049] Using pGMT-CPI plasmid, WSSV (shrimp white spot syndrome virus) DNA, and IHHNV (prawn infectious subcutaneous and hematopoietic necrosis) DNA as templates respectively (prawn white spot syndrome virus (WSSV DNA and IHHNV DNA in the literature "Yang Bing et al., Establishment of a PCR detection method for shrimp infectious subcutaneous and hematopoietic necrosis virus (IHHNV). Marine Aquatic Research, 2005, 26(2): 1-5 "open), and a negative control group was set at the same time, and double distilled water was used as the template in the negative control Alternative. Add 3 pairs of primers and probes designed above to prepare 25 μL reaction system, 12 groups in total. Among them, each 25 μL reaction system contains 10 × buffer (containing Mg 2+ ) 2.5 μl, dNTP 1 μl, Taq DNA polymerase 0.5 μL, the final concentration of the forward and reverse primers is 0.1 μmol / L, the final concentration of the probe...

Embodiment 3

[0052] Fluorescence PCR detection of sensitivity of three pairs of primers and probes:

[0053] (1) Amplification curves using CPI-603 and CPI-732 as primers, PCPI-712 as probe, and different concentrations of pGMT-CPI plasmid as template. Prepare a 25μL reaction system containing 10×buffer (containing Mg 2+ ) 2.5 μl, dNTP 1 μl, Taq DNA polymerase 0.5 μL, the final concentration of the primer is 0.1 μmol / L, the final concentration of the probe is 0.06 μmol / L, and the volume is adjusted to 25 μL with double distilled water. The reaction conditions of PCR were: 48°C for 5 min; 95°C for 5 min; 95°C for 15 s, 60°C for 15 s, 40 cycles.

[0054] Such as image 3 As shown, the primer probe sets of CPI-603, CPI-732, and PCPI-712 can effectively amplify the viral nucleic acid from as low as 0.000015ng to as high as 150ng, and can quantitatively reflect the concentration gradient of the template, and its detection sensitivity very high. In view of the fact that the Ct value is close...

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Abstract

The invention discloses a detection method and a kit for infectious muscle necrosis of penaeus vanmamei. cDNA of muscular tissue of the penaeus vanmamei is as a template; primers and a probe are added into a fluorescent PCR reaction system; and PCR is fluorescently quantified in real time so as to effectively detect whether the penaeus vanmamei is infected by infectious muscle necrosis viruses; a primer pair is as follows: 5'-GGGAGTAGATATAAATGTTCAG-3' and 5'-CAACCACCCAAATTCATA-3'; the probe is 5'-FAM-CACCTGCTACCACTTCTTCTCTT-TAMRA-3'. The kit implementing the method mainly comprises the primer pair and the probe. The method and the kit are accurate and fast, have the advantages of better specificity for detection results compared than the other detection technology, high detection sensitivity and higher reliability and adaptability and increase the detection efficiency so as to provide more effective detection means for the infectious muscle necrosis viruses of the penaeus vanmamei.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection method and a kit for infectious myonecrosis of Penaeus vannamei. The detection method is a real-time fluorescent quantitative PCR method. Background technique [0002] In recent years, the World Health Organization for Animals (OIE) has reported a newly discovered serious virus that mainly infects Penaeus vannamei, initially named it Infectious Myonecrosis Virus (IMNV). It entered Asia and was included in the disease surveillance list of the Asia-Pacific Quarterly Aquatic Animal Surveillance Report (QAAD), and OIE included the disease in the OIE key aquatic animal disease list. In 2002, infectious myonecrosis occurred in Brazil for the first time, mainly affecting Penaeus vannamei. The pathogen causing the disease is a double-stranded RNA virus referred to as IMNV, which is a brand new virus. Announcement No. 1125 issued by the Ministry of Agriculture of th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 岳巧云邱德义蔡先全王一飞闫冬春胡佳刘国雄汪小东魏晓雅刘德星
Owner 中华人民共和国中山出入境检验检疫局