PCR-HRM primer and method for quickly distinguishing DHAV-1 from DHAV-3

A PCR-HRM and duck hepatitis A virus technology, which is applied in the field of primers for quickly distinguishing duck hepatitis A virus type 1 and type 3 PCR-HRM, can solve the problems of difficulty in differential diagnosis, sensitivity of test results, and restrictions on popularization and application. , to achieve the effect of shortening detection time, good specificity and low cost

Active Publication Date: 2016-02-17
梅州市桃花缘文化实业发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

According to research by domestic scholars, duck hepatitis A virus (DHAV-1 and DHAV-3) are the most prevalent, and the epidemiology, clinical symptoms, and pathological changes of the two are similar. There have been reports of mixed infection of the two, which is clinically very Difficult to make a differential diagnosis
Traditional methods for pathogen detection of duck hepatitis A virus at home and abroad mainly include neutralization test, agar diffusion test, agglutination test, fluorescent antibody technology, enzyme-linked immunosorbent assay and monoclonal antibody technology. These methods have cumbersome procedures in practical application. , poor sensitivity and stability of test results, long time-consuming, etc.
Although domestic scholars have established a double RT-PCR detection method for the identification of DHAV-1 and DHAV-3, this method requires the design of 2 pairs of specific primers, and gel electrophoresis is required to determine the results, so this method is being carried out High-throughput detection tasks will be time-consuming and laborious, which limits its clinical application

Method used

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  • PCR-HRM primer and method for quickly distinguishing DHAV-1 from DHAV-3
  • PCR-HRM primer and method for quickly distinguishing DHAV-1 from DHAV-3
  • PCR-HRM primer and method for quickly distinguishing DHAV-1 from DHAV-3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 distinguishes the screening of duck hepatitis A virus type 1 and type 3 PCR-HRM primers

[0059] PCR-HRM primers:

[0060] After screening a large number of designed primers, it was found that the base sequences of the primers SEQIDNO: 1 and SEQ ID NO: 2 have the best effect on the PCR-HRM method for distinguishing DHAV-1 and DHAV-3, and the base sequences are as follows.

[0061] DHAV-P1: 5'-TAGTGTTGTGGGATACCC-3' (SEQ ID NO: 1);

[0062] DHAV-P2: 5'-GTGGGTGTTTTACGTGTACTC-3' (SEQ ID NO: 2).

Embodiment 2

[0063] Embodiment 2 Establishment of a kind of PCR-HRM method for distinguishing duck hepatitis A virus type 1 and type 3

[0064] (1) Extraction and reverse transcription of DHAV-1 and DHAV-3 RNA

[0065] Use the kit TakaraMiniBESTViralRNA / DNAExtractionKitVer.4.0 to extract the RNA in the disease sample, and use TakaraReverseTranscriptaseM-MLV to reverse transcribe the RNA into cDNA.

[0066] (2) Preparation of positive plasmid samples

[0067] The purified DNA of DHAV-1 and DHAV-3 were respectively connected to the pMD-18T vector with a kit from Takara Company, and the positive clones obtained by screening through ampicillin resistance screening, colony PCR and sequencing were positive plasmid samples.

[0068] (3) PCR-HRM operation steps for positive plasmid samples

[0069] Using the three positive plasmid samples obtained above as DNA templates, the PCR-HRM amplification reaction and analysis were carried out respectively;

[0070] Vazyme2×TaqPlusMasterMix5μl

[007...

Embodiment 3

[0081] Embodiment 3 distinguishes the specificity test of duck hepatitis A virus type 1 and type 3 PCR-HRM method

[0082] (1) Extraction of viral RNA or DNA from the sample and reverse transcription: the method is the same as in Example 1 (1) Viral nucleic acid extraction method and RNA reverse transcription.

[0083] (2) Use the established PCR-HRM method to amplify the cDNA of DHAV-1 and DHAV-3 by PCR, and use the nucleic acids of GPV, MDPV, MDRV, NDV, DEV, and DTMUV as control samples to test the specificity of the method . Amplification reaction of PCR-HRM: the method is the same as the reaction system and reaction procedure in Example 1 (2).

[0084] (3) Analysis of specificity test PCR-HRM results

[0085] The PCR amplification product was analyzed with Rotor-GeneQ analyzer, and the results were as follows: image 3 shown.

[0086] From image 3 It can be seen from the peak-shaped melting curve graph shown that the DHAV-1 and DHAV-3 positive samples have the same...

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Abstract

The invention discloses a PCR-HRM primer and method for quickly distinguishing DHAV-1 from DHAV-3. According to the method, plasmid samples of corresponding target fragments of DHAV-1 and DHAV-3 are established firstly to serve as positive plasmids, then virus RNA is extracted from the samples and converted into cDNA through inverse transcription, and then cDNA is taken as a template and subjected to PCR-HRM amplification by means of the designed specific primer; HRM analysis is conducted on the amplification product, and virus type is determined. According to the method, operation is easy, and fluorescence saturable dye only needs to be added before ordinary PCR reaction; detection is quick, flux is high, the whole operation process lasts for about 3.5 h for PCR product detection of a 96/384 pore plate, and detection time is shortened greatly; cost is low, and it does not require multiple primers and specific probes; sensitivity is high, specificity and repeatability are high, and the PCR-HRM primer and method are suitable for being applied and popularized clinically.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a PCR-HRM primer and a method for quickly distinguishing duck hepatitis A virus type 1 and type 3. Background technique [0002] The pathogens of duck viral hepatitis (Duckviral hepatitis, DVH) include duck hepatitis A virus (DuckhepatitisAvirus, DHAV) (including DHAV-1, DHAV-2, DHAV-3) and duck astrovirus (Duckastrovirus, DAstV) (including DAstV-1 and DHV-3). According to research by domestic scholars, duck hepatitis A virus (DHAV-1 and DHAV-3) are the most prevalent, and the epidemiology, clinical symptoms, and pathological changes of the two are similar. There have been reports of mixed infection of the two, which is clinically very Difficult to make a differential diagnosis. Traditional methods for pathogen detection of duck hepatitis A virus at home and abroad mainly include neutralization test, agar diffusion test, agglutination test, fluorescent ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/706C12Q2527/107C12Q2563/107
Inventor 董嘉文张建峰孙敏华李林林刘志成
Owner 梅州市桃花缘文化实业发展有限公司
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