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Primer group and kit for duplex PCR detection of fish rhabdoviridae and duplex PCR detection method of fish rhabdoviridae

A rhabdovirus and detection method technology, which is applied in the field of fish rhabdovirus double PCR detection, can solve the problem of lack of special literature and technology for virus identification, and achieves a reduction in the probability of false positives, high accuracy and fast speed. Effect

Pending Publication Date: 2020-09-25
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is currently no effective treatment, and there is currently no specific literature and technology for the identification of the virus

Method used

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  • Primer group and kit for duplex PCR detection of fish rhabdoviridae and duplex PCR detection method of fish rhabdoviridae
  • Primer group and kit for duplex PCR detection of fish rhabdoviridae and duplex PCR detection method of fish rhabdoviridae
  • Primer group and kit for duplex PCR detection of fish rhabdoviridae and duplex PCR detection method of fish rhabdoviridae

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Experimental program
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Effect test

Embodiment 1

[0024] The establishment of embodiment 1 double PCR detection method

[0025] 1. Experimental materials: The diseased fish samples were collected from the hybrid snakehead farm in Shunde City, Guangdong Province, and kept in the laboratory at -80°C.

[0026] Experimental reagents: TRIpure Reagent (Beijing Aidelai Biology); isopropanol, chloroform, 75% ethanol (Sinopharm Chemical Reagent Factory); DEPC water (Biosharp company product); Premix Taq (TaKaRa Version 2.0plusdye), reverse Transcriptase, DL2000 DNA Marker (product of TaKaRa Company); DNA Gel Recovery Kit (produced by Quanshijin Company); agarose (Biowest Agarose); 50×TAE buffer.

[0027] 2. Primer design and synthesis

[0028] According to the gene sequence related to snakehead vesicular virus published by NCBI (National Center for Biotechnology), using Clone Manager software, according to the conserved sequence of the nucleocapsid protein gene of snakehead vesicular virus. Two pairs of PCR primers (SHVV-1F, SHVV-1R...

Embodiment 2

[0039] Evaluation of embodiment 2 double PCR method

[0040] 1. Specific detection

[0041] The DNA or cDNA of snakehead vesicular virus (SHVV), infectious spleen and kidney necrosis virus (ISKNV), carp spring viremia virus (SVCV) and red sea bream iridescent virus (RSIV) were respectively used as detection templates, and ddH 2 O is a negative control, which is detected according to the double PCR detection method established by the present invention. The results showed that SHVV was positive, and two target bands of 845bp and 313bp were amplified. The experimental control samples ISKNV, SVCV, RSIV and ddH 2 O, no bands, negative (see figure 2 ).

[0042] 2. Sensitivity test

[0043] 1TCID 50 / mL virus solution was diluted 10 times (10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 ), extract RNA according to the detection method established by the present invention, and synthesize cDNA. Using this as a PCR template, double PCR amplification was carried out with corresponding s...

Embodiment 3

[0044] Embodiment 3 clinical sample detection

[0045] Select 21 clinical samples of diseased hybrid snakehead kidney tissue, set ddH 2 O is a negative control, which is detected according to the double PCR detection method established by the present invention. The results showed that specific bands were amplified in 21 samples, which were positive; the negative control ddH 2 O No band, negative (see Figure 4 ). The positive band gel was recovered and sequenced, and the gene sequence of the positive sample was consistent with the gene sequence of snakehead vesicular virus (SHVV) published by NCBI.

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Abstract

The invention discloses a primer group and kit for duplex PCR detection of fish rhabdoviridae and a duplex PCR detection method of the fish rhabdoviridae. The detection method comprises the followingsteps: extracting RNA from kidney tissue of to-be-detected sample fishes, and synthesizing cDNA with the RNA as templates; with the synthesized cDNA as templates, conducting duplex PCR amplification by adopting the synthesized primer group; and conducting agarose gel electrophoresis on products obtained from duplex PCR amplification, and placing gel in an imaging system to observe a result. According to the primer group and kit for duplex PCR detection of the fish rhabdoviridae and the duplex PCR detection method of the fish rhabdoviridae, related specific primers and suitable PCR reaction conditions are designed for a conserved gene sequence of snakehead fish vesicularvirus, the duplex PCR detection method of the snakehead fish vesicularvirus is built, and has the characters of high specificity, high sensitivity and the like, the snakehead fish vesicularvirus can be fast and accurately detected, an important technical means is provided for clinical diagnosis and prevention of the snakehead fish vesicularvirus, and a technical scheme is provided for research of implementation of identification, separation, epidemiological survey and the like of the snakehead fish vesicularvirus.

Description

technical field [0001] The invention relates to a primer set, a kit and a detection method for double PCR detection of fish rhabdovirus. Background technique [0002] PCR stands for Polymerase Chain Reaction technique, a process similar to DNA replication in reference cells. This technology uses DNA as a template, adding PCR enzymes and corresponding specific primers to perform programmed reactions in a PCR instrument, and copy and amplify to generate new complementary DNA fragments. The process consists of three steps: denaturation-annealing-extension. After multiple cycles, DNA amplification can be amplified millions of times within 2-3 hours. Because of its advantages of high amplification efficiency, strong specificity and high sensitivity, it has been widely used in the field of molecular biology research. [0003] Double PCR is an improved solution based on the common PCR method. The difference from the general PCR technology is that double PCR needs to add two pairs...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848C12Q2600/16C12Q2537/143C12Q2531/113
Inventor 曹攀刘晓丹张晓君孙威周紫城
Owner YANGZHOU UNIV
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