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Duck Tembusu virus detection kit

A technology of duck Tembusu virus and detection kit, which is applied in the determination/inspection of microorganisms, microorganisms, biochemical equipment and methods, etc., can solve the problems of high cost, economic loss and easy pollution of Taqman probes, and achieve detection The process is convenient and simple, the accuracy and speed are improved, and the effect of optimizing reaction conditions

Active Publication Date: 2013-12-25
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Duck Tembusu virus is a new virus discovered in recent years. There is no vaccine and effective drug to prevent the virus. It mainly depends on comprehensive control measures to control it
Since 2010, the disease first broke out in southeastern my country (Shanghai, Zhejiang, Jiangsu, Fujian and other provinces), and then spread to Shandong, Henan, Hubei and other provinces. The society has brought a heavy burden and caused great economic losses
[0004] Currently, the gene detection techniques reported mainly include RT-PCR, nested PCR, RT-LAMP, Taqman RT-PCR, etc. These detection techniques (except Taqman RT- PCR) is not sensitive enough, has a low positive rate, is easy to contaminate, needs post-processing, and cannot accurately provide the level of virus content in the body. For Taqman RT-PCR detection technology, it not only requires high primer and probe design, but also Taqman probes High cost, not easy to detect a large number of samples

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The known duck Tembusu virus sequence was used to compare the E gene sequence to find out its conserved region, and then use Primer Express 3.0 software to design fluorescent quantitative PCR primers. The primer sequences were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The upstream and downstream primers are:

[0041] SEQ1: (RT-EF) 5'-3' ccg cta gat tcg tga taa c

[0042] SEQ2: (RT-ER) 5'-3' cat ggt aag ttg aga tca tg.

[0043] Embodiment 2. The preparation of standard substance

Embodiment 2

[0044] (1) Design and synthesis of upstream and downstream primers for amplifying the full-length E gene

[0045] Utilize Oligo 6 to design the upstream and downstream primers SEQ3 and SEQ4 for amplifying the full-length E gene, and add Eco RI restriction site, the 5' end of the downstream primer of SEQ4 is added xho Site Ⅰ, the primer sequence was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The upstream and downstream primers are:

[0046] SEQ3: (PEF) 5'-3' cga att cgc cac cat gat agc ccc agcgta cag ctt cag

[0047] SEQ4: (PER) 5'-3' cat ctc gag cta ctt gtc gtc atc gtc ttt gta gtc ggc att gac att tac tgc cag ga.

[0048] (2) Construction of E gene and pBSK recombinant plasmid and linearization of recombinant plasmid

[0049] The pathologically injured tissue with clinically confirmed duck Tembusu virus was taken, RNA was extracted by Trizol and reverse-transcribed into cDNA, the target fragment was amplified by PCR, and the target fragment w...

Embodiment 3

[0054] According to the copy number obtained after quantification, the standard sample was made into 2.5×10 2 —2.5×10 7 (copies / μl) and other 6 serially diluted detection standards, in order to reduce the error and improve the repeatability of the experiment, each dilution sample is provided with three replicate holes to determine the Ct value (Ct is the cycle threshold, which refers to the cycle threshold in the reaction tube). The number of cycles experienced when the fluorescence signal reaches the set threshold value), so as to determine the standard curve with the best amplification effect.

[0055] (1) Fluorescent quantitative PCR amplification

[0056] The amplification system is 25μl, including dNTP Mixture, Mg 2+ , SYBR Green I, RNase Inhibitor, reverse transcriptase, Ex Taq HS, ROX Reference Dye II, upstream and downstream primers SEQ1 and SEQ2, etc., and 2 μl of diluted standard samples.

[0057] Agilent Technologies-Mx3005pPCR amplification instrument was used f...

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Abstract

The invention discloses a one-step SYBR Green fluorescence quantitative PCR detection kit used for detecting duck Tembusu virus. The duck Tembusu virus detection kit comprises two specific primers SEQ1 and SEQ2 designed according to conservative regions of duck Tembusu virus E gene. With the detection kit provided by the invention, operation steps are simplified, reaction time can be shortened, pollution possibility is reduced, and detection accuracy and speed are improved. A detection reaction can be completed within 2 hours. With fluorescence quantitative PCR, after amplification is finished, initial virus copy number can be directly quantified through a standard curve, and reliable information can be provided for epidemiological investigations. Also, the fluorescence quantitative PCR kit provided by the invention has the advantages of simple and fast operation, and relatively low cost. Therefore, the kit can satisfy requirements of field sample testing.

Description

technical field [0001] The invention relates to a medical detection kit, specifically, the invention is a one-step SYBR Green fluorescent quantitative PCR detection kit for duck Tembusu virus. Background technique [0002] Duck disease caused by duck Tembusu virus has now formed a large-scale outbreak. Its main clinical symptoms are sudden drop in feed intake, elevated body temperature, green loose stools, paralysis of some ducks, and depression of individual ducks. Soon the egg production dropped sharply from more than 90% to less than 10%. Autopsy showed follicle rupture and bleeding, yolk peritonitis, spleen hemorrhage, pancreas hemorrhage and necrosis, and liver enlargement and hemorrhage. The disease is contagious and can occur throughout the year. The incidence rate is as high as 90%, and the mortality rate can reach 5%-30%. [0003] Duck Tembusu virus is a new virus discovered in recent years. At present, there is no vaccine and effective drug to prevent the virus, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 朱启运付钰广刘宗梁吉艳红郭建宏才学鹏
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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