HRM detection method for quickly distinguishing goose type I astrovirus from goose type II astrovirus and primer of HRM detection method

An astrovirus and detection method technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effects of high accuracy, improved efficiency and strong specificity

Active Publication Date: 2019-09-17
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are differences between goose astrovirus type 1 and goose astrovirus type 2, but there is no report on the differential diagnosis method for the two genotypes of goose astrovirus at home and abroad. The detection method of astrovirus (GAstV-1) and goose type 2 astrovirus (GAstV-2) is of great significance

Method used

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  • HRM detection method for quickly distinguishing goose type I astrovirus from goose type II astrovirus and primer of HRM detection method
  • HRM detection method for quickly distinguishing goose type I astrovirus from goose type II astrovirus and primer of HRM detection method
  • HRM detection method for quickly distinguishing goose type I astrovirus from goose type II astrovirus and primer of HRM detection method

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Experimental program
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Effect test

Embodiment 1

[0033] Example 1 A HRM detection method for rapidly distinguishing goose type 1 astrovirus and goose type 2 astrovirus

[0034] Primer

[0035] According to the nucleic acid sequences of goose type 1 astrovirus and goose type 2 astrovirus, a large number of primers were designed and screened, and it was found that the base sequences of the primers of this application, SEQ ID NO: 1 and SEQ ID NO: 2, can be distinguished by PCR-HRM method The effect of goose type 1 astrovirus and goose type 2 astrovirus is the best. This set of primers has high sensitivity and strong specificity, and their base sequences are shown below.

[0036] Primer P1: TATGATGAKTWCTGGCTKRTKCAA (SEQ ID NO: 1),

[0037] Primer P2: TCATCTKCAATCATMTCWTCATACC (SEQ ID NO: 2).

Embodiment 2

[0038] Example 2 A HRM detection method for rapidly distinguishing goose type 1 astrovirus and goose type 2 astrovirus

[0039] PCR-HRM analysis of standard samples

[0040] 1) Extraction of Goose Astrovirus Standard Nucleic Acid:

[0041] Add 3mL of PBS hydrochloric acid buffer solution to the diseased goose tissue samples (liver and spleen) infected by goose type 1 astrovirus and goose type 2 astrovirus, and grind the homogenate at 4000×g for 8min, and draw the centrifuge The supernatant was stored at -20°C for later use. Take 200 μL of the supernatants of the two grinding solutions, and extract nucleic acids from the samples according to the instructions of TAKARA's MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0.

[0042] 2) PCR-HRM operation steps for positive standard samples

[0043] In order to verify the ability of the designed primers to identify clinical samples, this study used goose type 1 astrovirus (SS strain) and goose type 2 astrovirus (QY strain) as standard...

Embodiment 3

[0051] Example 3 Clinical Sample PCR-HRM Analysis

[0052] 1) Extracting viral nucleic acid from the samples: 2 g of tissue samples (liver, spleen) suspected to be infected with goose type 1 astrovirus and 2 g of tissue samples (liver, spleen) suspected of being infected with goose type 2 astrovirus. Add 3 mL of PBS hydrochloric acid buffer solution for grinding, centrifuge the ground homogenate at 4000×g for 8 min, and absorb the centrifuged supernatant to -20°C for future use. Take 200 μL of tissue sample homogenate for nucleic acid extraction according to the instructions of TAKARA's MiniBEST Viral RNA / DNA Extraction KitVer.4.0.

[0053] 2) take the extracted viral nucleic acid as template, carry out RT-PCR amplification (referring to One Step RT-PCR Kit kit instructions), the amplification reaction system is:

[0054]

[0055] The Syto9 is a fluorescent saturating dye

[0056] The amplification reaction program was: reverse transcription at 50°C for 30 min; pre-dena...

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Abstract

The invention discloses an HRM detection method for quickly distinguishing goose type I astrovirus from goose type II astrovirus and a primer of the HRM detection method. The HRM detection method disclosed by the invention is simple to operate, only a pair of universal primers is needed, and before a PCR reaction, fluorescence saturable dye is added. The detection speed is high and the flux is high, and time for subtyping is shortened; a specific fluorescence labelling probe is not needed; the specificity is high, the accuracy is high, samples can be analyzed in a quick, accurate and high-flux manner, popularization and application of the HRM detection method in clinical practice are facilitated, and the HRM detection method has higher application value.

Description

technical field [0001] The invention relates to a method for identifying different genotypes of viruses, in particular to an HRM detection method for rapidly distinguishing goose type 1 astrovirus and goose type 2 astrovirus and primers thereof. Background technique [0002] Goose Astrovirus is a new poultry infectious disease. It is a highly pathogenic infectious disease of goslings mainly characterized by goose. The disease is characterized by severe urate deposition in viscera and joints. The incidence of goose flocks is Between 80 and 90%, the mortality rate is between 20 and 70%, which has caused huge economic losses to the goose industry. [0003] At present, the methods for molecular diagnosis of animal infectious pathogens include single-strand conformation polymorphism analysis (SSCP) method, denaturing gradient gel electrophoresis (DGGE) method, temperature gradient capillary electrophoresis (TGCE) method, denaturing high performance liquid chromatography ( DHPLC)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2527/107C12Q2563/107Y02A50/30Y02A40/70
Inventor 董嘉文张俊勤李林林孙敏华刘志成张建峰
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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