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Fluorescence quantitative PCR (polymerase chain reaction) detection method of duck hepatitis B virus, and reagent

A hepatitis B virus, fluorescence quantitative technology, applied in the direction of microbial determination/inspection, fluorescence/phosphorescence, biochemical equipment and methods, etc., can solve the problems of lack, lack of versatility, complex operation, etc., to achieve strong immunogenicity , the effect of strong versatility and specificity

Inactive Publication Date: 2013-05-22
THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Quantitative detection of DHBV DNA has become an important indicator of the application of this model. In the past, methods such as Southern blot hybridization and dot hybridization were used to quantify DHBV DNA. Due to the low sensitivity, poor stability and complicated operation of these methods, they have been replaced by quantitative PCR methods.
However, due to the lack of standard experimental animals and uniform and universal DHBV DNA quantitative detection methods, researchers from all over the world often choose DHBV infected by local shelduck ducks, and establish detection methods for different regions of the DHBV gene
Due to the differences in DHBV genes infected by different regions and different duck species, the experimental results of the researchers are not comparable, and there are uncertainties in drawing on existing methods
[0003] In previous studies, primers were designed for the P gene or S gene of DHBV, and the DHBV PCR detection method was established, which lacked versatility, because DHBV has different subtypes, and the S gene and P gene encoding outer membrane protein and polymerase protein are easy to mutate

Method used

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  • Fluorescence quantitative PCR (polymerase chain reaction) detection method of duck hepatitis B virus, and reagent
  • Fluorescence quantitative PCR (polymerase chain reaction) detection method of duck hepatitis B virus, and reagent
  • Fluorescence quantitative PCR (polymerase chain reaction) detection method of duck hepatitis B virus, and reagent

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: the design of duck hepatitis B virus fluorescent quantitative PCR diagnostic method primer, probe

[0070] A total of 44 duck hepatitis B virus genome sequences were retrieved in GenBank, from China (including Beijing, Hubei, Shanghai, Chongqing, Sichuan, Guilin, Henan, Guangdong, etc.) and foreign countries (the United States, Germany, India, Canada, South Africa, Australia, etc.), through the comparison of DNASIS software, there are many differences in the DHBV gene sequences in different regions, but the core region sequence bp241-414 reported in different regions is basically the same, with a minimum of 98% and a maximum of 100%.

[0071] Therefore, use this region as a template to design primers, and match multiple pairs of primers with all DHBV Core region nucleic acid sequences to obtain the most matched primers and probes. The designed primer sequences and fluorescent

[0072] The photoprobe sequence is as follows:

[0073] Upstream primer F: DHB...

Embodiment 2

[0084] Embodiment 2. Preparation of PCR standards:

[0085] (1). Construction of the pGEM-T / DHBV Core recombinant plasmid: Based on the gene sequence of the DHBVCore region provided by GenBank, the forward and negative primers were designed using the principle of negative strand extension, and BamHI and Xhol enzymes were added to both ends of the positive and negative primers respectively Cutting site and protective base G, the sequence is as follows (the underline is the introduced enzyme cutting site):

[0086] F1:G GGATCC ATATCAATGCTTCTAG;

[0087] R1:G CTCGAG TTATTTTCCTAGGCGAG;

[0088] PCR amplifies the DHBV Core gene of the pBR322 / 2DHBV plasmid, and the 100 μl reaction system is as follows:

[0089] 10×buffer 10μl, 10mM dNTP2μl, 1U / μl HotStart Taq Polymerase1μl, 25mM MgCl 2 6 μl, 50 mM F primer 1 μl, 50 mM R primer 1 μl, DEPC-H 2 O 77μl, DHBV plasmid (10-fold dilution) template 1μl.

[0090] PCR amplification conditions: first, pre-denaturation at 94°C for 30...

Embodiment 3

[0099] Embodiment 3: Optimization of FQ-PCR reaction system:

[0100]The matrix method was used to screen for the best optimization of primer and probe concentrations to obtain the lowest Ct value and higher fluorescence intensity. Optimize PCR amplification conditions, choose FAM (490nm) for fluorescence signal collection, and set data collection at the end of extension. Positive control, negative control, blank control and standards of different concentrations are set on the same machine.

[0101] The optimized FQ-PCR 25μl reaction system is:

[0102] 2×HotStart Taq PCR MasterMix12.5μl (including 0.1HotStart Taq Polymerase / μl, 500μM dNTP each, 20mM Tris-HCl (pH8.3), 100mM KCl, 3mM MgCl2), F primer 0.5μl (50mM), R primer 0.5μl (50mM), DHBV-Probe 0.5μl (10mM), DEPC-H2O 9μl, DHBV template 2μl.

[0103] Optimized PCR amplification conditions: firstly, pre-denaturation at 94°C for 2 minutes, followed by 40 cycles at 94°C for 15s, 49°C for 30s, and 72°C for 30s.

[0104] fig...

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Abstract

The invention discloses a fluorescence quantitative PCR (polymerase chain reaction) detection method of duck hepatitis B virus (DHBV), and a reagent. Nucleic acid sequences in a conservative region most consistent to a DHBV Core region are used as a template to design and synthesize a primer and a fluorescent probe. A pGEM-T / DHBV Core recombinant plasmid is established to prepare a PCR standard product. A reaction system and amplification conditions are optimized. The fluorescence quantitative PCR detection method provided by the invention has the advantages of strong versatility, high sensitivity, strong specificity, good reproducibility and the like, and is suitable for detection of DHBV DNA (deoxyribonucleic acid) of different duck species in different regions.

Description

technical field [0001] The invention belongs to the technical field of detection of duck hepatitis B virus, and relates to a fluorescent quantitative PCR detection method and reagents of duck hepatitis B virus. Background technique [0002] In 1980, Mason et al. discovered duck hepatitis B virus (Duck Hepatitis B Virus, DHBV) from domestic Peking ducks. DHBV belongs to avian hepadnavirus. Very similarly, at present, DHBV-infected Peking duck has become an important experimental animal model for HBV replication, infection mechanism research and treatment strategy evaluation]. Quantitative detection of DHBV DNA has become an important indicator of the application of this model. In the past, methods such as Southern blot hybridization and dot hybridization were used to quantify DHBV DNA. Due to the low sensitivity, poor stability and complicated operation of these methods, they have been replaced by quantitative PCR methods. . However, due to the lack of standard experimental...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 王亚文李义平刘正稳惠凌云刘希冯艾王威张琳杨广笑王全颖
Owner THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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