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Isothermal amplification detection kit and detection method for chikungunya fever virus

A technology for constant temperature amplification detection and chikungunya fever, which is applied in the determination/testing of microbes, biochemical equipment and methods, and resistance to vector-borne diseases, etc. It can solve problems such as difficulty in widespread use and achieve high repeatability , high sensitivity, and the effect of reducing pollution

Inactive Publication Date: 2016-01-20
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current laboratory detection technology for chikungunya virus is mainly based on nucleic acid detection technology for viral RNA (such as fluorescent quantitative PCR, etc.), but it is difficult to be widely used due to the need for expensive fluorescent quantitative PCR instruments

Method used

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  • Isothermal amplification detection kit and detection method for chikungunya fever virus
  • Isothermal amplification detection kit and detection method for chikungunya fever virus
  • Isothermal amplification detection kit and detection method for chikungunya fever virus

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Experimental program
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Embodiment 1

[0038] Composition and preparation of embodiment 1 kit of the present invention

[0039] a) RNA extraction reagent: Germany QIAGEN RNA extraction kit (Qia-74104);

[0040]b) Chikungunya virus nucleic acid constant temperature PCR amplification reaction solution: two peripheral primers (10pmol), two probes (20pmol) and two cross primers (40pmol), 1×Thermolbuffer, MgSO 4 (7mmol), dNTPs solution (0.1mmol each), BstDNA polymerase (8U), AMV reverse transcriptase (5U) and sterile double distilled water, the total reaction volume is 25 μ l;

[0041] The forward peripheral primer is: 5′-CTCTCCTCTCTCACAGGTGTA-3′;

[0042] The reverse peripheral primer is: 5′-CGCAGTCTATGGAGATGTGC-3′;

[0043] The sequences of the two probes are:

[0044] Forward 5' end Biotin labeled probe 5'-Biotin-ATGGGAGAAGAAAATATCTGAGGC-3';

[0045] Reverse 3' end Fitc labeled probe 5'-Fitc-TGGTTCAGTGACTGGGTCAG-3';

[0046] The two amplification cross primers are:

[0047] amplified forward primer

[0048] 5'...

Embodiment 2

[0055] Embodiment 2 uses kit of the present invention to detect the specific method of chikungunya virus nucleic acid

[0056] a) Use the RNA extraction solution in the Chikungunya virus constant temperature amplification detection kit to extract RNA from the specimen to be tested (nucleic acid extraction from suspected case specimens must be performed in a biosafety laboratory) as specimen RNA.

[0057] b) Take the sample RNA as a template and add it to the PCR tube containing the Chikungunya fever virus constant temperature amplification reaction solution, in which the sample RNA is 3μl, the reaction solution is 22μl, and the amplification reaction is 35 minutes at 60°C; the positive and negative control PCR tubes A positive template (chikungunya virus nucleic acid (shown in SEQ ID NO.1)) and a negative template (sterile double-distilled water) were respectively added in.

[0058] c) Put the reacted PCR tube into the nucleic acid test strip anti-pollution detection device (N...

Embodiment 3

[0060] Embodiment 3 detects the specificity of chikungunya fever virus with the kit of the present invention

[0061] Detect dengue type I nucleic acid RNA, dengue type II nucleic acid RNA, dengue type III nucleic acid RNA, dengue type IV nucleic acid RNA, yellow fever virus nucleic acid RNA, Japanese encephalitis virus nucleic acid RNA according to the method of Example 2, the results are as follows figure 2 . From figure 2 The test results show that the detection of chikungunya virus nucleic acid by the kit of the present invention has strong specificity.

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Abstract

The invention discloses an isothermal amplification detection kit and a detection method for chikungunya fever virus. The isothermal amplification detection kit comprises an RNA extraction reagent, isothermal PCR amplification reaction liquid, a positive control and a negative control. The isothermal amplification detection kit is good in specificity and high in sensitivity; the process from virus RNA reverse transcription to strand displacement amplification is finished at a constant temperature, the isothermal amplification can be finished within only 35 minutes, the detection result of a nucleic acid test strip can be acquired within only 2 minutes, the whole detection process from sample acceptation to result acquisition can be finished within only about 1 hour, and furthermore, only one isothermal instrument is required in the whole reaction process; the isothermal amplification detection kit is particularly applicable to the rapid detection of infection medium monitoring of the chikungunya fever virus.

Description

(1) Technical field [0001] The invention relates to a rapid detection technology for constant temperature amplification of chikungunya virus nucleic acid, which is suitable for qualitative detection of chikungunya virus. (2) Background technology [0002] Chikungunya fever (Chikungunyafever, referred to as CHIK) is an acute infectious disease caused by the CHIK virus transmitted through the bite of Aedes mosquitoes. Clinically, it is characterized by fever, joint pain, rash, and mild bleeding. The disease is mainly prevalent in summer and autumn, and is mainly distributed in Africa, South Asia and Southeast Asia. From 2005 to 2007, it was widely prevalent in Indian Ocean islands, India and Southeast Asia, causing millions of people to become ill. The first imported case was discovered in my country in 2008, and an outbreak occurred in Guangdong in 2010. In the past two years, the epidemic area of ​​the disease has continued to expand, and the number of cases has continued ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/70
CPCC12Q1/6844C12Q1/701Y02A50/30
Inventor 冯燕徐昌平严菊英卢亦愚须周恒
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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