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RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and method for prawn infective muscle necrosis virus by one-step process

An RT-PCR, detection kit technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve the problems of reducing experimental steps, shortening reaction time, cumbersome and time-consuming detection process, etc. Achieve the effect of reducing experimental steps, shortening reaction time, and high promotion and application value

Inactive Publication Date: 2011-11-23
LUDONG UNIVERSITY
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Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of complicated and time-consuming detection process in the existing nested RT-PCR detection method, to provide a one-step RT-PCR detection kit for shrimp infectious myonecrosis virus, and to provide A detection method for detection, the detection method is sensitive and specific, and can shorten the reaction time and reduce the experimental steps

Method used

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  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and method for prawn infective muscle necrosis virus by one-step process
  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and method for prawn infective muscle necrosis virus by one-step process
  • RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit and method for prawn infective muscle necrosis virus by one-step process

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Embodiment 1

[0032] The present embodiment prawn infectious myonecrosis virus one-step RT-PCR detection kit and detection method

[0033] Primers: According to the full gene sequence of IMNV in GenBank (AY570982), RT-PCR primers were designed, the forward primer sequence 5'-CCAACATACGGTACAGTGGCAG-3', the reverse primer sequence 5'-ATCGGTTGCCCAGGATACAAG-3', and the product length was 123bp.

[0034] RT-PCR reaction system:

[0035]

[0036] RT-PCR reaction procedure:

[0037] cDNA synthesis and pre-denaturation: 45°C for 30 minutes, 94°C for 2 minutes, 1 cycle;

[0038] PCR amplification: Denaturation at 94°C for 15s, annealing at 55°C for 30s, cDNA synthesis at 72°C for 1min, 40 cycles;

[0039] Final extension: 5 min at 72°C, 1 cycle;

[0040] Store at 4°C.

[0041] Detection sensitivity: This method detects 100 copies of infectious myonecrosis virus. (See figure 1 )

[0042] Detection specificity: This method has good specificity for detecting infectious myonecrosis virus, only...

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Abstract

The invention relates to a detection kit and method for prawn infective virus, belonging to the technical field of marine organism pathogeny detection. The invention aims to provide a detection kit and method. The detection method is more sensitive and specific, and can shorten the reaction time and reduce the experimental steps. The detection kit comprises (1) an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) buffer tube, (2) a primer tube, (3) an enzyme tube, (4) a deionized water tube, (5) a negative control tube and (6) a positive control tube, wherein the RT-PCR buffer tube is filled with a 2*RT-PCR buffer; the primer tube is filled with infective muscle necrosis virus forward / reverse primers; the enzyme tube is filled with reverse transcriptase and DNA polymerase; the deionized water tube is filled with sterile deionized water; the negative control tube is filled with SPF (Specific Pathogen Free) prawn nucleic acid; and the positive control tube is filled with infective muscle necrosis virus cDNA (complementary deoxyribonucleic acid) positive clone. The detection method comprises (1) an RT-PCR reaction system and (2) an RT-PCR reaction procedure.

Description

technical field [0001] The invention relates to a detection kit and a detection method for infectious viruses of prawns, and belongs to the technical field of detection of marine biological pathogens. Background technique [0002] Infectious myonecrosis is a new viral infectious disease that endangers the production of prawns found in Brazil in 2002. In 2004, Dr. Donald V. Lightner of the University of Arizona found that the disease was caused by a new virus and Name it Infectious myonecrosis virus (IMNV for short). The novel virus is a double-stranded RNA virus belonging to the family Holoviridae. The susceptible shrimp species of shrimp infectious myonecrosis virus is Penaeus vannamei, mainly juvenile shrimp infected 60-80 days old. The virus can cause muscle tissue necrosis in the whole body of susceptible prawns. Generally, pathological death occurs slowly and the mortality rate is not high, but there are deaths throughout the breeding process, and the cumulative morta...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 闫冬春刘鸿玲谢玮
Owner LUDONG UNIVERSITY
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