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Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method

A duck hepatitis A virus, fluorescence quantification technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Low, good specificity, good reproducibility

Inactive Publication Date: 2016-01-20
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are many domestic and foreign reports on the detection methods of DHAV, including serum neutralization test, agar diffusion test and ELISA. Clinically, it often causes huge economic losses due to the inability to make correct diagnosis and effective prevention and control in time.

Method used

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  • Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method
  • Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method
  • Type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Fluorescence quantitative RT-PCR detection kit for detecting duck hepatitis A virus type 3

[0019] According to the nucleotide sequence of DHAV-3 (accession number EU877916.1), sequence alignment was performed through NCBI and MEGA5.2, and primers and probes were designed using Primer5.0. The primers and probes were all provided by Shanghai Jikang Biotechnology Co., Ltd. Synthesized by the company, the primers and probe sequences are as follows:

[0020] Standard primer:

[0021] Upstream primer F1 (DHAV-3-F1): 5'-gatgtagttatggtgcttagacgc-3' (SEQ IDNO.1),

[0022] Downstream primer R1 (DHAV-3-R1): 5'-acgaaccagccattgacg-3' (SEQ ID NO. 2),

[0023] Quantitative probes and matching primers:

[0024] Upstream primer (DHAV-3-F3): 5'-gtgcttagacgctggcagatt-3' (SEQ ID NO. 3),

[0025] Downstream primer (DHAV-3-F4): 5'-ttcgattgaaaactatctgaaaccta-3' (SEQ ID NO. 4),

[0026] Specific probe sequence (DHAV-3-FAM): 5'-FAM-tcagtgggctaacacagtgacccctg-BHQ-3' (SEQ ID NO. 5).

[0027] The f...

Embodiment 23

[0033] Example 23 Establishment of a fluorescent quantitative RT-PCR detection method for duck hepatitis A virus

[0034] 1. Material OneStepPrimerScript TM RT-PCRKit (PerfectRealTime) and RNAisoPlus were purchased from Bao Biology (Dalian Co., Ltd.).

[0035] 2. Methods and results

[0036] 2.1 Preparation of RNA standard template

[0037] (1) Cloning of VP3: Use Bao Biological RNAisoPlus to extract type 3 duck hepatitis A virus RNA according to the kit instructions, and transcribe viral RNA into cDNA according to Bao Biological reverse transcription kit. Using standard primers F1 and R1, using in vitro transcribed cDNA as a template for ordinary PCR amplification to obtain the VP3 target fragment. The target fragment was ligated with pGEM-T vector to construct a recombinant plasmid pGEM-T-VP3.

[0038] (2) Prepare RNA standard template: linearize the recombinant plasmid pGEM-T-VP3 with single enzyme digestion, and perform in vitro transcription according to the instructions of The...

Embodiment 3

[0061] Example 3 Fluorescence quantitative RT-PCR detection of DHAV-3 clinical disease materials

[0062] Collect 20 duck hepatitis disease materials, suspect duck hepatitis disease materials and 10 duck embryo disease materials artificially infected with DHAV-3 from 2005 to 2015 from all over the country, extract their RNA, and perform fluorescence quantitative RT-PCR with the kit of the present invention For the reaction, set up a negative control at the same time, and use the above-mentioned optimal reaction system and procedures for reaction. The results showed that 14 were positive and 16 were negative, which was consistent with the results of virus isolation and identification.

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Abstract

The invention discloses a type-3 duck hepatitis A virus TaqMan fluorescent quantitation RT-PCR detection reagent kit and method. The reagent kit comprises a primer pair composed of an upstream primer and a downstream primer and a probe composed of nucleotide sequences shown in the SEQ ID NO.5, wherein the upstream primer is composed of nucleotide sequences shown in the SEQ ID NO.3 and the downstream primer is composed of nucleotide sequences shown in the SEQ ID NO.4. The adopted reagent kit can be used for widely detecting the type-3 duck hepatitis A virus, the detection time is short, the detection cost is low, the repeatability is good, and the sensitivity is high. The detected target point has the single specificity, and the provided primer pair and the probe have no amplification signals for samples containing no target genes and have the good specificity; in addition, the sensitivity of the primer pair and the probe for detecting RNA can reach 48 copies / microliter and the good sensitivity is achieved.

Description

Technical field [0001] The invention belongs to the technical field of biological detection, and specifically relates to a TaqMan fluorescent quantitative RT-PCR detection kit and method for type 3 duck hepatitis A virus. Background technique [0002] Duck viral hepatitis is an important infectious disease that harms the duck industry. The main pathogenic factor is picornavirus, that is, duckhepatitis Avirus (DHAV) of the genus avian hepatitis of the Picornaviridae family. According to the analysis of the whole genome sequence, duck hepatitis A virus can be divided into 3 independent genotypes, gene A duck hepatitis A virus (DHAV-A), gene B duck hepatitis A virus (DHAV-B) and gene Duck hepatitis A virus type C (DHAV-C); according to serological neutralization experiments, these three genotypes have no serum crossover, so they correspond to 3 different serotypes, serotype 1 duck hepatitis A virus (DHAV-1) , Serotype 2 duck hepatitis A virus (DHAV-2) and serotype 3 duck hepatitis ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/706C12Q2531/113C12Q2545/113C12Q2561/101
Inventor 汪铭书胡琴程安春杨乔朱德康陈舜贾仁勇孙昆峰刘马蜂吴英陈孝跃
Owner SICHUAN AGRI UNIV
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