Multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and kit

A technology of multiple fluorescence quantification and duck hepatitis A, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of difficult differential diagnosis and rare differential detection methods, and achieve Improve detection time and efficiency, high accuracy, and avoid false positive effects

Active Publication Date: 2014-12-24
WUHAN CHOPPER BIOLOGY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The diseases caused by the three genotypes are relatively similar in clinical symptoms and pathological changes, which brings great difficulties to differential diagnosis
Especially the outbreak and prevalence of DHAV-C in many areas of our country, because the relevant DHAV-C vaccine and antibody have not been developed and put into actual production, so the rapid and accurate detection of DHAV-C is very important for the prevention of duck viral hepatitis. important to control
[0003] Regarding the molecular rapid detection method of DHAV, at present, most reports at home and abroad are mainly for the detection of DHAV-A, and there are few methods for the identification and detection of DHAV-C and DHAV-A, especially the real-time fluorescent quantitative PCR method. Detection of DHAV-C and DHAV-A is rarely reported

Method used

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  • Multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and kit
  • Multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and kit
  • Multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Establishment of multiple fluorescent quantitative PCR method for detection of duck hepatitis A virus and identification of genotype A and C

[0051] (1) Primer probe design

[0052] Download all currently known DHAV genome sequences of different genotypes from the GenBank database, use BioEdit software for homology sorting analysis and comparison, and determine the DHAV3D region as the design region for detecting DHAV universal primers and probes; select DHAV-A and DHAV respectively The conserved region in the 5'UTR sequence of DHAV-C is used as the design region for DHAV-A and DHAV-C detection primers and probes. Finally, the Beacon Designer7.0 biological software is used to design multiple groups that meet the requirements of fluorescent quantitative PCR for the above regions. For the primers and probes, the specificity of the primers and probes was firstly analyzed by BLAST theory, and then the best combination of primers and probes was further screened ou...

Embodiment 2

[0095] Embodiment 2 detects genotype A duck hepatitis A virus infection

[0096] (1) Test sample

[0097] The selected detection samples are dead duck liver tissue (S1) infected with type A duck hepatitis A virus, healthy duck liver tissue (S2), chicken embryo tissue poisoned by type A duck hepatitis A virus (S3), healthy chicken embryo tissue (S4), A Duck hepatitis A virus chicken embryo attenuated allantoic fluid virus (S5), positive standard (P).

[0098] (2) Extraction of viral RNA

[0099] The extraction method of viral RNA refers to the above method, and the liquid nitrogen method is used to grind the tissue virus to prevent the heat generated during the grinding process from degrading the viral RNA. The nucleic acid extraction method of the tissue homogenate after grinding is the same as that of chicken embryo allantoic fluid virus, and the RNA is extracted by the traditional Trizol method.

[0100] (3) Reverse transcription and multiplex fluorescent quantitative PCR...

Embodiment 3

[0107] Embodiment 3 detects genotype C duck hepatitis A virus infection

[0108] (1) Test sample

[0109] The selected detection samples are dead duck liver tissue (Y1) infected by type C duck hepatitis A virus, healthy duck liver tissue (S2), duck embryo tissue poisoned by type C duck hepatitis A virus (Y2), healthy duck embryo tissue (D4), C Type Duck Hepatitis A Virus Duck Embryo Attenuated Allantoic Fluid Virus (Y3), Positive Standard (P).

[0110] (2) Extraction of viral RNA

[0111] The extraction method of viral RNA refers to the above method, and the liquid nitrogen method is used to grind the tissue virus to prevent the heat generated during the grinding process from degrading the viral RNA. The nucleic acid extraction method of the tissue homogenate after grinding is the same as that of chicken embryo allantoic fluid virus, and the RNA is extracted by the traditional Trizol method.

[0112] (3) Reverse transcription and multiplex fluorescent quantitative PCR detec...

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Abstract

The invention discloses a multi-fluorogenic quantitative PCR (polymerase chain reaction) method for detecting a duck hepatitis A virus (DHAV) and identifying DHAV-A and DHAV-C and a kit, and belongs to the technical field of biological detection. Aiming at a DHAV3D area, the invention designs a pair of conservative amplification primers (SEQIDNO:1 and SEQIDNO:2) and a conservative probe (SEQIDNO:5) to detect different gene types of DHAVs. A pair of specific primers (SEQIDNO:3 and SEQIDNO:4) and two specific probes (SEQIDNO:6 and SEQIDNO:7) designed by aiming at DHAV5'UTR are used to identify DHAV-A and DHAV-C. The method has excellent specificity, repeatability and sensitivity, is convenient, simple and quick in operations, is used for identifying and diagnosing the infection of DHAV as well as DHAV-A and DHAV-C in scientific research and clinical application, and is also used for the epidemiological investigation of DHAV.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a multiple fluorescence quantitative PCR method and a kit for detecting duck hepatitis A virus and identifying gene type A duck hepatitis A virus and gene type C duck hepatitis A virus. Background technique [0002] Duck viral hepatitis is an important infectious disease that endangers the duck industry. Its main pathogenic factor is duck hepatitis A virus (DHAV) belonging to the family Picornaviridae and the genus Avian Hepavirus. According to the whole genome sequence analysis, duck hepatitis A virus can be divided into three independent genotypes, gene type A duck hepatitis A virus (DHAV-A), gene type B duck hepatitis A virus (DHAV-B) and Genotype C duck hepatitis A virus (DHAV-C); according to the serological neutralization test, these 3 genotypes correspond to 3 different serotypes because there is no serum crossover, serotype 1 duck hepatitis A virus (DHAV-1), d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/70C12Q1/706C12Q2531/113C12Q2537/143C12Q2561/101
Inventor 孙庆歌薛霜赖志肖爱芳马慧慧朱薇廖园园祝春花漆世华谢红玲冯钊
Owner WUHAN CHOPPER BIOLOGY
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