Kit for joint detection on dengue fever virus and chikungunya virus on basis of fluorescent PCR method

A technology of dengue virus and chikungunya, which is applied in the field of kits for combined detection of dengue virus and chikungunya virus based on fluorescent PCR method, can solve problems such as trouble and waste of cost, avoid mutual interference and ensure accuracy Effect

Inactive Publication Date: 2017-05-31
广东省疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, rapid detection kits for the above two viruses have been developed using fluorescent quantitative PCR technology, but due to a single detection, the detection of the two viruses is both troublesome and costly

Method used

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  • Kit for joint detection on dengue fever virus and chikungunya virus on basis of fluorescent PCR method
  • Kit for joint detection on dengue fever virus and chikungunya virus on basis of fluorescent PCR method
  • Kit for joint detection on dengue fever virus and chikungunya virus on basis of fluorescent PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Design and synthesis of detection primers and probes of dengue virus and chikungunya virus nucleic acid joint detection kit (fluorescent PCR method):

[0040] Synthesize specific primers for dengue virus, dengue virus probes, specific primers for chikungunya viruses and chikungunya virus probes according to SEQ ID No.1-6, and label at the 5' of the dengue virus probes FAM fluorescent group, the 5'-labeled HEX fluorescent group of the Chikungunya virus probe, and the 3' end of both are labeled with BHQ1, and the primer probes for dengue fever and Chikungunya were synthesized. The primers and probe sequences are as follows :

[0041] Dengue virus:

[0042] Upstream primer: AGGACTAGAGGTTAGWGG (SEQ ID No.1)

[0043] Downstream primer: CAGCACCATTCCATTTTC (SEQ ID No.2)

[0044] Probe: 5' fluorescent reporter group-tccCagCgtCaaTatg-BHQ13' (SEQ ID No.5)

[0045] Chikungunya virus:

[0046] Upstream primer: ACTCAACCATCCTGGAYA (SEQ ID No.3)

[0047] Downstream primer: GAGTC...

Embodiment 2

[0052] Preparation of detection mixture of dengue virus and chikungunya virus nucleic acid joint detection kit (fluorescent PCR method):

[0053] Dengue virus upstream primer 0.5 μl / test; downstream primer 0.5 μl / test; probe 0.1 μl / test; chikungunya virus upstream primer 0.5 μl / test; downstream primer 0.5 μl / test; probe 0.1 μl / test; Internal control probe 0.1μl / test; primer concentration 20μM, probe concentration 10μM; RT-PCR MIX 12.5μl / test (OneStep RT-PCR Kit, QIAGEN); process water (ddH 2 O) Mix 3.2 μl / test to get the D&C nucleic acid fluorescent PCR detection mixture.

Embodiment 3

[0055] Sensitivity Analysis of Dengue Virus and Chikungunya Virus Nucleic Acid Joint Detection Kit (Fluorescent PCR Method):

[0056] 3.1 Sample preparation:

[0057] Take 1×10 7 Dengue virus plasmid (DFV-S1) of copies / ml (cloning the target amplified sequence to the pMD8-T vector by TA to obtain the DFV-S1 plasmid. The target amplified sequence: AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCTACAAGCATCATTCCAGGCACAGAACGCCA) (SEQ ID No.8), Diluted at 1:10, 1:100, 1:1000, 1:10000 to obtain DFV-S2 (1×10 6 copies / ml), DFV-S3 (1×10 5 copies / ml), DFV-S4 (1×10 4 copies / ml), DFV-S5 (1×10 3 copies / ml) were used as test samples.

[0058] Take 1×10 7 Chikungunya virus plasmid (CHIKV-S1) of copies / ml (cloning the target amplified sequence onto the pMD8-T vector by TA to obtain the CHIKV-S1 plasmid. Target amplified sequence: GAATGACCATGCTAATGCTAGAGCGTTCTCGCATCTAGCTATAAAACTAATAGAGCAGGAAATTGACCCCGACTCAACCATCCTGGATATCGGCAGTGCGCCAGCAAGGAGGATGATGTCGG) (SEQ ID No. 9), according to 1:10, 1:...

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Abstract

The invention relates to the field of PCR detection kits, in particular to a kit for joint detection on a dengue fever virus and a chikungunya virus on the basis of a fluorescent PCR method. The kit for joint detection on the dengue fever virus and the chikungunya virus comprises a specific primer for the dengue fever virus, a dengue fever virus probe, a specific primer for the chikungunya virus and a chikungunya virus probe, wherein the specific primer for the dengue fever virus comprises a forward primer and a reverse primer; the specific primer for the chikungunya virus comprises a forward primer and a reverse primer. The kit provided by the invention specifically relates to two pairs of specific PCR primers and probers for respectively specifically amplifying the dengue fever virus and the chikungunya virus, the two pairs of the specific PCR primers can amplify the viruses simultaneously in a same PCR reaction system, multi-PCR detection is achieved, and not only is operation simple and convenient, but also the detection time is greatly shortened.

Description

technical field [0001] The invention relates to the field of fluorescent PCR detection kits, in particular to a kit for combined detection of dengue virus and chikungunya virus based on a fluorescent PCR method. Background technique [0002] Dengue virus is a small flavivirus belonging to the Flavivirus genus that can cause a range of clinical symptoms, including life-threatening hemorrhagic shock syndrome and, less commonly, acute hepatitis with hepatic failure and encephalopathy. With Aedes aegypti and Aedes albopictus as the vector, it is widely prevalent in more than 60 tropical and subtropical countries and regions around the world. More than 100 million people are infected every year, the mortality rate is as high as 50%, and more than 2.5 billion people are threatened , the spread of the virus has become a serious public health problem in tropical and subtropical regions. Dengue has a mortality rate of approximately 5% in most countries, mostly in children and young ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2561/101C12Q2563/107Y02A50/30
Inventor 柯昌文管大伟肖红徐静邵俊斌朱勤玮王凯熊磊
Owner 广东省疾病预防控制中心
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