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A nucleic acid composition, kit and detection method for simultaneous detection of Sindbis virus, Getha virus and Yunnan orbivirus

A technology of nucleic acid composition and Geta virus, which is applied in biochemical equipment and methods, recombinant DNA technology, measurement/inspection of microorganisms, etc., to achieve the effects of easy results, good repeatability, and fast detection speed

Active Publication Date: 2022-06-14
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem that Sindbis virus, Geta virus and Yunnan orbivirus cannot be rapidly detected simultaneously in the prior art, the application provides a simultaneous detection of Sindbis virus Nucleic acid composition, kit and detection method of Getha virus and Yunnan orbivirus

Method used

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  • A nucleic acid composition, kit and detection method for simultaneous detection of Sindbis virus, Getha virus and Yunnan orbivirus
  • A nucleic acid composition, kit and detection method for simultaneous detection of Sindbis virus, Getha virus and Yunnan orbivirus
  • A nucleic acid composition, kit and detection method for simultaneous detection of Sindbis virus, Getha virus and Yunnan orbivirus

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Experimental program
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Effect test

Embodiment 1

[0065] Design of synthetic primers and probes for the nucleic acid sequences of three viruses

[0066] According to the nucleic acid sequences of Sindbis virus, Geta virus and Yunnan orbivirus published by NCBI, three sets of specific primers and probes were designed using Oligo7.0 primer design software. The sequences of primers and probes are shown in Table 1.

[0067]

[0068] The 5' end of the probe sequence designed in this application is modified with a fluorescent reporter group, and the 3' end of the probe sequence is modified with a fluorescent quencher group. The fluorescent reporting group is selected from one of FAM, JOE, VIC, HEX, ROX, TEXAS RED and CY5; the fluorescent quenching group is selected from one of BHQ1 and BHQ2.

[0069] Preferably, the 5' end of the Sindbis virus probe is modified with a FAM group, and the 3' end is modified with a BHQ1 group; the 5' end of the Geta virus probe is modified with a JOE group, and the 3' end is modified with a BHQ1 g...

Embodiment 2

[0073] A kit for simultaneously detecting Sindbis virus, Geta virus and Yunnan orbivirus, including three sets of primer probes in Example 1, hot start Taq enzyme, c-MMLV reverse transcriptase, RNasin, 10×PCR Buffer , dNTPs, MgCl 2 .

[0074] The final concentration of each reagent in the above kit in the final reaction amplification system is shown in Table 3:

[0075]

[0076] Specifically, taking the 25 μL reaction system as an example, the specific addition amount of each component in the above kit is shown in Table 4:

[0077]

[0078] Adopt the PCR reaction condition of above-mentioned reaction system as follows:

[0079] 40℃~50℃ reverse transcription 10min~30min; 93℃~95℃ pre-denaturation 2min~15min; 93℃~95℃ denaturation 10s~30s, 55℃~60℃ annealing, extension, signal acquisition 30s~60s, cycle 40 times ~45 times.

[0080] Preferably, the PCR amplification reaction program is: reverse transcription at 50°C for 15 minutes; pre-denaturation at 95°C for 15 minutes; ...

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Abstract

The application discloses a nucleic acid composition, a kit and a detection method for simultaneously detecting Sindbis virus, Getha virus and Yunnan orbivirus. The nucleic acid composition includes forward and reverse primers and probes against Sindbis virus, forward and reverse primers and probes against Getha virus, and forward and reverse primers and probes against Yunnan orbivirus. In addition to the above three sets of primers and probes, the kit also includes enzyme mixture and PCR reaction reagents. The present application also provides a method for simultaneous detection of the above three viruses. Firstly, the RNA of the test sample is extracted, and then the RNA is subjected to real-time fluorescent PCR reaction using the above kit, and finally the negative or positive of the sample to be tested is judged according to the intensity of the fluorescent signal. The application can simultaneously detect three viruses, and has the advantages of high sensitivity, good repeatability, simple operation, fast detection speed, easy interpretation of results and the like.

Description

technical field [0001] The present application relates to the technical field of in vitro molecular diagnosis, in particular to a nucleic acid composition, kit and detection method for simultaneous detection of Sindbis virus, Getha virus and Yunnan orbivirus. Background technique [0002] Arboviruses are a class of arthropod vector viruses. A variety of arbovirus antibodies induced by arboviruses have been detected in humans and livestock. Arboviruses can cause severe illness and death in humans and livestock, resulting in Huge social impact and hazards require extra attention. In the past, most arboviruses were only distributed in a certain region or continent. However, with the development of society and economy, the communication and interaction between humans have become more frequent, the continuous development of forests and other primitive resources, and the acceleration of urbanization have all promoted the arboviruses. The spread and spread of the virus to the whol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 付士红王环宇许松涛马力敏杨海芳聂凯李樊殷启凯何英
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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