Fluorescence PCR kit for rapidly quantitative detection of gene C-type duck hepatitis A virus

A quantitative detection technology for duck hepatitis A, which is applied in the field of diagnosis of poultry infectious diseases, can solve the problems of unseen kits and literature reports, and achieve the effects of fast detection speed, accurate quantification and simple steps

Inactive Publication Date: 2012-12-19
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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Also do not see that the corresponding test kit that can make detectio

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  • Fluorescence PCR kit for rapidly quantitative detection of gene C-type duck hepatitis A virus

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Embodiment 1

[0029] The fluorescent PCR kit composition of embodiment 1 gene type C duck hepatitis A virus

[0030] A) Reagent composition:

[0031] RNA Lysis Solution, RNase Inhibitor, Reverse Transcriptase, 5×PrimeScript Buffer, primers Random6mers and SYBR Premix Ex TaqTM II was purchased from Dalian Bao Biological Company;

[0032] B) SYBR Premix Ex TaqTM II contains TaKaRa Ex Taq HS, dNTP Mixture, Mg 2+ , SYBR Green I;

[0033] C) Reverse transcription reaction solution: 5×PrimeScript Buffer 2μL, Random 6 mers 2μL, sterile double distilled water 3.5μL;

[0034] D) Fluorescence quantitative reaction solution: each 0.4 μL (10 μmol / L) of forward primer and reverse primer, 7.2 μL of sterile double distilled water;

[0035] E) Self-prepared reagents: chloroform, isopropanol, DEPC-treated sterile double-distilled water, and 75% ethanol prepared with DEPC-treated sterile double-distilled water.

Embodiment 2

[0036] Embodiment 2 to the detection of clinical sample

[0037] A). Dilute the positive standard 10-fold serially, take 8 μL of each fluorescent quantitative reaction solution, and SYBR Premix Ex TaqTM II each 1 μL, each positive standard template 2 μL, each positive standard template for three parallel controls, respectively added to different PCR reaction tubes, PCR detection in parallel on a fluorescent quantitative PCR instrument. The reaction conditions were: pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s; annealing at 60°C for 31s, and a total of 30 cycles.

[0038] Take the logarithm of the dilution of the positive standard as the abscissa and the Ct value as the ordinate to establish a standard curve. It can be seen from the standard curve that at 1.68×10 4 ~1.68×10 9 There is a good linear relationship in copies / μL, the correlation coefficient is 0.998, the standard equation is: Y=-3.348X+26.310, and the amplification efficiency is 97.9%.

[003...

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Abstract

The invention relates to a fluorescence PCR kit for rapidly quantitative detection of gene C-type duck hepatitis A virus. The rapidly qualitative and quantitative detection of the gene C-type duck hepatitis A virus comprises: a) RNA lysate, b) RNA enzyme inhibitor, c) reverse transcriptase, d) reaction solution of the reverse transcription, e) fluorescence quantitative reaction solution, f) SYBRP remix Ex Taq TM II, and g) a standard positive template, wherein the d) the reaction solution of the reverse transcription, e) the fluorescence quantitative reaction solution and f) the SYBRP remix Ex Taq TM II comprises TaKaRa Ex Taq HS, dNTP Mixture, Mg<2+> and SYBRGreen I. The sensitivity of the kit is up to 3.36 x 10<3> copies per reaction and can totally meet requirements of the rapidly quantitative detection of the gene C-type duck hepatitis A virus.

Description

technical field [0001] The invention relates to the diagnosis of poultry infectious diseases, in particular to the technical field of a fluorescent quantitative RT-PCR method for gene type C duck hepatitis A virus. Background technique [0002] In recent years, duck viral hepatitis caused by gene type C duck hepatitis A virus (DHAV-C) has been widely prevalent in South Korea and China. The virus mainly affects ducklings within 3 weeks of age. The development of duck industry. Duck viral hepatitis caused by DHAV-C is very similar to duck hepatitis caused by genotype A duck hepatitis A virus (DHAV-A) in epidemiology, clinical symptoms and pathological changes, and can only be identified with the help of laboratory diagnosis. Isolation and identification is a classic method for the etiological diagnosis of infectious diseases, but this method takes at least 1 to 2 weeks to confirm the diagnosis, which is time-consuming and laborious, and cannot meet the needs of early diagnosi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 汤承岳华黄秋雪
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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