Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus)

A technology for Muscovy duck parvovirus and duck hepatitis A virus, applied in the field of PCR detection, can solve complex problems

Inactive Publication Date: 2015-09-02
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the establishment of a multiplex PCR method is much more complicated than that of a single PCR method, and it has higher requirements on reagents and primers, and at the same time, it is necessary to ensure that there is no mutual interference between different primers

Method used

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  • Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus)
  • Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus)
  • Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Design and synthesis of primers

[0033] According to the conservative sequences of DHAV-1 3C gene, DHAV-3 VP0 gene and MDPV NS gene in GenBank, DNAStar software was used for multiple sequence alignment, and primer 5.0 was used to design primers in the conservative region. The primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. (Table 1).

[0034] Table 1 Primer information

[0035]

Embodiment 2

[0036] Example 2. Multiplex PCR detection

[0037] 1. Extraction of nucleic acid and reverse transcription of RNA

[0038] Refer to the DNA / RNA extraction kit instructions to extract the DNA of Muscovy Duck Parvovirus, Duck Circovirus, and Duck Plague Virus, and perform RNA analysis of H5, H9, Duck Paramyxovirus, Duck Hepatitis Virus, Muscovy Reovirus Extract and reverse transcription of RNA into cDNA according to the reverse transcription instructions. The specifics are as follows: Using a 10μL system, add 2μL of 5× reverse transcription buffer, 10mmol / L dNTP 1μL, 5U / μLAMV 0.5μL to the EP tube. , 20U / μL RNase inhibitor 0.5μL, free primer 0.5μL, total RNA template to be tested 2μL, make up the volume to 10μL with RNase-free sterile water, put it in the PCR machine after instant separation, 42℃1h, 85℃ Store the prepared cDNA at -30°C for 5 minutes.

[0039] 2. Establishment of multiple PCR amplification system

[0040] The total reaction volume is 25μL, of which 2×PCR Master Mix 12.5...

Embodiment 3

[0053] Example 3. Assembling of the detection kit

[0054] According to the research results of Examples 1 and 2, the detection kit was assembled for ease of use.

[0055] The kit includes a primer set, PCR buffer and water; the primer set includes three pairs of specific primers, namely primers 1 and 2, primers 3 and 4, primers 5 and 6, the concentrations of which are all 25μmol / mL (in the PCR reaction system The final concentration in the chromosome is 0.5μmol / mL, 0.3μmol / mL, 0.7μmol / mL); PCR buffer is 2×Taq PCR Mix.

[0056] Other test solutions in PCR detection can be configured on site or directly use commercial reagents.

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Abstract

The invention discloses a multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus). The detection kit contains three pairs of specific primers. Experimental results show the multiple PCR detection kit for duck hepatitis A virus 1 and 3 as well as MDPV can be applied to detecting the duck hepatitis A virus 1 and 3 and the MDPV. The multiple PCR detection kit has the advantages of being convenient to operate, high in sensitivity, strong in specificity and the like; furthermore, the multiple PCR detection kit can be clinically used for distinguishing and detecting the duck hepatitis A virus 1 and 3 and the MDPV, the time cost can be saved, and the pollution can be reduced.

Description

Technical field [0001] The invention belongs to the technical field of PCR detection, and particularly relates to a multiple PCR detection kit for duck hepatitis A virus type 1, 3 and Muscovy parvovirus. Background technique [0002] Duck hepatitis A virus can cause liver swelling and hemorrhage in ducklings within 3 weeks of age, and the fatal rate of ducklings is as high as 90%. It is one of the most serious pathogens that endanger the development of duck industry. Duck hepatitis A virus can be divided into duck hepatitis A virus type 1 (DHAV-1), duck hepatitis A virus type 2 (DHAV-2) and duck hepatitis A virus type 3 (DHAV-3) according to its serotype, respectively corresponding to the traditional serotype I , Taiwan's new type and South Korea's new duck hepatitis virus. Currently, the prevalent duck hepatitis A viruses in my country are mainly DHAV-1 and DHAV-3. Muscovy parvovirus (MDPV) mainly affects ducklings less than 3 weeks old. After infecting ducklings, ducklings ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2561/101Y02A50/30
Inventor 谢芝勋赵虹谢丽基刘加波罗思思谢志勤邓显文黄莉黄娇玲曾婷婷
Owner GUANGXI VETERINARY RES INST
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