Isothermal amplification detection kit and detection method for Zaire type Ebola virus

A technology of constant temperature amplification detection and Ebola virus, which is applied in the field of constant temperature amplification rapid detection technology, can solve the problems of inability to obtain results quickly, insufficient sensitivity, complicated operation, etc., and achieves high sensitivity, high repeatability, and steps simple effect

Inactive Publication Date: 2015-01-28
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods are either complicated to operate, time-consuming, laborious, or not sensitive enough, requiring special ins...

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  • Isothermal amplification detection kit and detection method for Zaire type Ebola virus
  • Isothermal amplification detection kit and detection method for Zaire type Ebola virus
  • Isothermal amplification detection kit and detection method for Zaire type Ebola virus

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Embodiment 1

[0042] Composition and preparation of embodiment 1 kit of the present invention

[0043] a) RNA extraction reagent: Germany QIAGEN RNA extraction kit (Qia-74104);

[0044] b) Zaire type Ebola virus nucleic acid constant temperature PCR amplification reaction solution: two peripheral primers (10pmol), two probes (20pmol) and two cross primers (40pmol), 1×Thermol buffer, MgSO 4 (6mmol), dNTPs solution (0.4mmol each), Bst DNA polymerase (8U), AMV reverse transcriptase (5U) and sterile double distilled water, the total reaction volume is 25μl;

[0045] Peripheral forward primer: 5′-TGGTTCAAGTGCACAGTCAA-3′

[0046]Peripheral reverse primer: 5′-TGTCTGCTCTACGGTGATGT-3′

[0047] Forward 5' end Biotin-labeled probe:

[0048] 5′-Biotin-GCAAGGGTTGTCAGATGCG-3′

[0049] Reverse 5' Fitc labeled probe:

[0050] 5′-Fitc-ATAATACACCCGTGTATAAACTTGAC-3′

[0051] Amplify the forward primer:

[0052] 5′-AGGGATTGGGGACTCGTGGAGGGAAGGAAAGCTGCAGTGT-3′

[0053] Amplification reverse primer:

[0...

Embodiment 2

[0062] Embodiment 2 detects the concrete method of Zaire type Ebola virus nucleic acid with kit of the present invention

[0063] a) Use the RNA extraction solution in the Zaire-type Ebola virus constant temperature amplification detection kit to extract RNA from the specimen to be tested (nucleic acid extraction from suspected cases must be performed in a biosafety level 3 laboratory) as the specimen RNA.

[0064] b) Take the sample RNA as a template and add it to the PCR tube containing the Zaire Ebola virus constant temperature amplification reaction solution, including 5 μl of sample RNA, 20 μl of reaction solution, and 58 ° C amplification reaction for 35 minutes; positive and negative controls Add positive and negative templates to PCR tubes, respectively.

[0065] c) Put the reacted PCR tube into the nucleic acid test strip anti-pollution detection device (No. 3 device of Hangzhou Ustar Biotechnology Co., Ltd.) for detection, and interpret the result after 2 minutes. W...

Embodiment 3

[0067] Embodiment 3 detects the specificity of Zaire type Ebola virus with kit of the present invention

[0068] Detect West Nile virus nucleic acid RNA according to the method for embodiment 2; Chikungunya fever virus RNA; Dengue fever virus nucleic acid RNA; Ebola virus detection kit positive control substance, its result is as follows figure 2 . From figure 2 The test results show that the detection of Zaire-type Ebola 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 for Zaire type Ebola virus. The isothermal amplification detection kit comprises an RNA extraction agent, an isothermal amplification reaction solution, a positive control and a negative control. The isothermal amplification detection kit has high specificity and sensitivity; from virus RNA reverse transcription to strand displacement amplification, the temperature is constant all the time, isothermal amplification only needs 35min, a detection result of a nucleic acid test strip can be obtained just in 1-2min, the whole detection process from receiving a sample to obtaining the result only needs about 1h, and in addition, the whole reaction process only needs a constant temperature instrument, so that the isothermal amplification detection kit is particularly suitable for field rapid detection.

Description

(1) Technical field [0001] The invention relates to a rapid detection technology for constant temperature amplification of Zaire-type Ebola virus nucleic acid, which is suitable for qualitative detection of Zaire-type Ebola virus. (2) Background technology [0002] Ebola hemorrhagic fever is a highly contagious infectious disease caused by Ebola virus infection. After infecting humans, it can cause severe acute hemorrhagic fever, which is easily transmitted from person to person, with a mortality rate as high as 70-90%. Ebola virus has an envelope, and its genome is not segmented. It is a single-stranded negative-sense RNA and belongs to the genus Filovirus of the family Filoviridae of the order Mononegative-sense Virus. Ebola virus contains a linear RNA genome, about 19Kb in length, containing 7 genes, which sequentially encode nucleoprotein (NP), viral structural protein (VP35, VP40), glycoprotein (GP), additional viral structural protein (VP30, VP24 ), RNA-dependent RNA ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6844C12Q1/70C12Q2537/1376C12Q2521/107
Inventor 徐昌平卢亦愚冯燕陈寅高见
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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