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Reagent kit and nucleotide sequence for detecting three kinds of fish Rhabdovirus

A nucleotide sequence and rhabdovirus technology, applied in the field of inspection and quarantine, can solve the problems of unmentioned, inaccurate, and inability to completely eliminate the mutual interference of probes, so as to reduce workload, improve work efficiency, and improve specificity. sexual effect

Inactive Publication Date: 2009-05-27
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

) reported a method for the simultaneous detection of IHNV, VHSV and SVCV by multiple real-time fluorescent RT-PCR based on Taqman probes. They mixed three different concentrations of viral RNA as templates and carried out interference experiments. Exclude the mutual interference between the probes, and due to the possible errors in the operation, it is impossible to guarantee the same Ct value for the same template amount, so it is not accurate to judge the influence of the template concentration on the efficiency.
Zongxiao Liu et al. applied the multiple real-time fluorescent RT-PCR method designed by them to the analysis of clinical detection samples, and the effect was good. However, the detection of disease materials in their article did not mention the detection of the presence of several viruses at the same time, so It also cannot fully explain the feasibility and effect of this method in the practical application of simultaneous detection of several viruses.

Method used

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  • Reagent kit and nucleotide sequence for detecting three kinds of fish Rhabdovirus
  • Reagent kit and nucleotide sequence for detecting three kinds of fish Rhabdovirus
  • Reagent kit and nucleotide sequence for detecting three kinds of fish Rhabdovirus

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Design and synthesis of Taqman MGB probes and primers

[0039] According to the relevant gene sequences of the three viruses IHNV, VHSV and SVCV registered in Genbank, DNAMAN software was used for comparison and analysis, and the highly conserved regions in the genomes of the three viruses were respectively selected, and PrimerExpress V2. Taqman MGB probes and primers for the virus. The 5' ends of the three probes were labeled with three fluorescent reporter groups, FAM, VIC, and NED, as shown in Table 1.

[0040] Primers and probes were synthesized by Shanghai Jikang Biotechnology Co., Ltd.

Embodiment 2

[0041] Embodiment 2: the extraction of three kinds of viral RNAs of IHNV, VHSV and SVCV

[0042] RNAs of the three viruses were extracted from EPC cells infected with IHNV, VHSV and SVCV with TRIZOL lysate (purchased from INVITROGEN).

[0043] The specific operation is as follows:

[0044] ①Add 600μl TRIZOL Lysis Solution to the finger tube, then add 200μl virus-infected EPC cell suspension, mix well by pipetting

[0045] ②Add 200μl chloroform, shake and mix for 15sec, then centrifuge at 13,000rpm at 4℃ for 15min

[0046] ③ Transfer the supernatant to 500 μl of isopropanol, invert and mix well, then place at -20°C for 10 minutes to precipitate

[0047] ④ 4°C, 13,000rpm, centrifuge for 15min

[0048] ⑤ Discard the supernatant, add 600 μl of 75% ethanol to the vial, wash upside down

[0049] ⑥Centrifuge at 13,000 rpm for 10 min at 4°C, discard the supernatant

[0050] ⑦4,000rpm, centrifuge for 10sec, shake the residual liquid on the tube wall to the bottom of the tube, use ...

Embodiment 3

[0052] Example 3: The establishment of triple real-time fluorescent RT-PCR

[0053] The concentrations of probes and primers, magnesium ions and enzymes were optimized respectively, and a triple real-time fluorescent RT-PCR reaction system was established. Obtain the RNA of three kinds of viruses of IHNV, VHSV and SVCV according to the method of embodiment 2, with the mixed RNA of IHNV, VHSV and SVCV as template, each virus template concentration is about 10 4 -10 6 copy / microliter, perform triple real-time fluorescent RT-PCR, and optimize the reaction system.

[0054] (1) Optimization of probe and primer concentrations

[0055] The probes and primers of the three viruses were optimized separately. Primer concentrations increased by 0.1 μM from 0.1 μM to 0.8 μM and probe concentrations increased by 0.1 μM from 0.1 μM to 0.5 μM were cross-matched for real-time fluorescent RT-PCR to select the most suitable primers and probes concentration. The reaction system conditions ar...

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Abstract

The invention discloses a kit and a nucleotide sequence for inspecting three fish rhabdoviruses, which belongs to the field of inspection and quarantine. An oligonucleotide sequence for inspecting three fish rhabdoviruses is the oligonucleotide sequence shown from a sequence table SEQ ID No.1 to a sequence table SEQ ID No.9. The invention has the advantages that real-time fluorescent RT-PCR technique applied to the inspection of IHNV, VHSV and SVCV further improves the specificity and sensitivity of the inspection, reduces workload, improves work efficiency, and can perform quantitative inspection to a target fragment. Through the optimization of reaction conditions, the triple real-time fluorescent RT-PCR can inspect the virus quantity of 10<2> copy / reaction at least. The method also inspects virus mixed infection EPC (Epithelioma papulosum cyprini) cells and virus-challenge brachydanio rerio. Experimental results show that the method is a specific sensitive efficient virus inspection method. The technique has great application prospects in import-export sample quarantine, as well as the monitoring and diagnosis for clinical plague.

Description

technical field [0001] The present invention relates to a test kit and nucleotide sequence for detecting three kinds of fish rhabdoviruses, more specifically, a triple real-time fluorescent RT-PCR test kit for detecting three kinds of fish rhabdoviruses and primers thereof The probe and the probe belong to the field of inspection and quarantine. Background technique [0002] Infectious hematopoietic neCrosis virus (IHNV), fish viral hemorrhagic septicemia virus (VHSV) and Spring Viraemia of Carp Virus (SVCV) belong to the same genus Three fish viruses of the Rhabdoviridae family (Rhabdoviridae) are also three important viruses affecting the aquaculture industry. Among them, IHNV and VHSV belong to the genus Novirhabdovirus, and SVCV belongs to the genus Vesiculovirus, both of which are single-stranded negative-sense RNA viruses. These three viruses are widely distributed in many parts of Europe, America and Asia, and can infect many kinds of fish, especially fry and young ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张利峰许建明
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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