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One-step double-fluorescent quantitative RT-PCR detecting kit for duck tembusu virus and H9-subtype avian influenza virus
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A technology for duck Tembusu virus and avian influenza virus is applied in the field of PCR detection of viruses, which can solve the problems of detection and diagnosis that have not yet been seen, and achieve the effect of high detection sensitivity
Inactive Publication Date: 2015-04-08
GUANGXI VETERINARY RES INST
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At present, there is no report on the detection and diagnosis of duck Tembusu virus and H9 subtype avian influenza virus using one-step real-time dual fluorescent quantitative RT-PCR
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Embodiment 1
[0030] Embodiment 1, design and synthesis of primers and Taqman probes
[0031] According to the conserved sequences of duck Tembusu virus and H9 subtype avian influenza virus in GenBank, multiple sets of probes and primers were designed by using Primer Premier 5.0 software, and two pairs of specific primers were selected by analyzing the dimers between the primers. Primers and two Taqmam probes (Table 1).
[0032] Table 1 Primers and TaqMan probe sequences (5'-3')
[0042] Duck Tembusu virus and H9 subtype avian influenza virus were reverse-transcribed to synthesize cDNA, specifically as follows: the following reverse transcription system was established with a total reaction volume of 20 μL, and the above 1) duck Tembusu virus RNA and H9 subtype avian influenza Viral RNA is 2 μL (approximately 20 μg), 4 μL 8mM MgCl 2 (Dalian Bioengineering Co., Ltd., catalog number: DRR0019A), 2 μL 10×PCR buffer (Dalianbao Bioen...
Embodiment 3
[0071] Embodiment 3, the assembly of detection kit
[0072] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.
[0073] Liquid A: PCR amplification buffer, primer 1, primer 2, probe A, primer 3, primer 4, probe B; the final concentrations of primer 1, primer 2, and probe A in liquid A are all 0.25 μM, and primer 3 , Primer 4, and Probe B were all at a final concentration of 0.2 μM.
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Abstract
The invention discloses a one-step double-fluorescent quantitative RT-PCR detecting kit for a duck tembusu virus and an H9-subtype avian influenza virus. The one-step double-fluorescent quantitative RT-PCR detecting kit comprises aprimer group and a probe group, wherein the primer group comprises primers 1, 2, 3 and 4 which respectively have a base sequence as shown in SEQ.ID.No.1 and 2 as well as SEQ.ID.No.4 and 5 in a sequence table; and the probe group comprises a probe A and a probe B which respectively have a base sequence as shown in SEQ.ID.No.3 and 6 in the sequence table. Experiments prove that the one-step double-fluorescent quantitative RT-PCR detecting kit has the advantages of short detection time, high detection sensitivity and strong specificity and can be used for simultaneously detecting and identifying two pathogens including the duck tembusu virus and the H9-subtype avian influenza virus and quantifying the corresponding pathogen contents of the duck tembusu virus and the H9-subtype avian influenza virus, so that the one-step double-fluorescent quantitative RT-PCR detecting kit can be used for evaluating the curative effects of vaccines and drugs for the duck tembusu virus and the H9-subtype avian influenza virus and researching the pathogenesis of the duck tembusu virus and the H9-subtype avian influenza virus, and furthermore the one-step double-fluorescent quantitative RT-PCR detecting kit has important significance for preventing and controlling the duck tembusu virus and the H9-subtype avian influenza virus.
Description
technical field [0001] The invention belongs to the technical field of virus detection by PCR, and in particular relates to a one-step dual fluorescence quantitative RT-PCR detection kit for duck Tembusu virus and H9 subtype avian influenza virus. Background technique [0002] Duck Tembusu Virus (DTMUV) was first discovered in Fujian, Zhejiang, Jiangsu and other major laying duck breeding areas in the east coast of China. It is a new pathogenic flavivirus. DTMUV infection of ducks can lead to a serious decline in egg production of laying ducks, and the egg production rate can drop from 90% to less than 10%, or even extinction; infection of ducklings can cause ducklings to appear unstable, falling down and other nerves Symptoms, which in turn lead to an increase in the elimination rate of ducklings, even as high as 80% in severe cases. Avian influenza virus (AIV) belongs to the Orthomyxoviridae Influenza A virus genus. According to the degree of virulence, AIV can cause diff...
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