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Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof

A detection kit and multiple fluorescent technology, applied in the field of multiple fluorescent PCR detection primers, can solve the problems of waste of cost, trouble, single detection, etc., and achieve the effects of avoiding mutual interference, easy operation, and shortening detection time

Inactive Publication Date: 2013-09-18
浙江国际旅行卫生保健中心 +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 three viruses have been developed using fluorescent quantitative PCR technology, but due to a single detection, the detection of the three viruses is both troublesome and costly

Method used

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  • Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof
  • Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof
  • Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Synthesis of primers and probes for multiplex fluorescent PCR detection of dengue fever, chikungunya and yellow fever virus

[0067] The 5'-labeled FAM fluorophore of the dengue virus probe, the 5'-labeled HEX fluorophore of the chikungunya virus probe, and the 5'-labeled Cal Red610 fluorophore of the yellow fever virus probe. Synthesis of primers for dengue, chikungunya and yellow fever viruses. probe.

[0068] Dengue virus:

[0069] Dengue virus upstream primer: 5'-TGGGAGAGACCAGAGATCCTG-3' (SEQ ID NO: 1)

[0070] Dengue virus downstream primer: 5'-ATTCAACAGCACCATTCCATTTTC-3' (SEQ ID NO: 2)

[0071] Dengue virus probe: 5'FAM-TCTCTACAGCATCATT-BHQ1 3' (SEQ ID NO: 3)

[0072] Chikungunya virus:

[0073] Chikungunya virus upstream primer: 5'-ACGAGGCAAGTTGTCTATTATGAG-3' (SEQ ID NO: 4)

[0074] Chikungunya virus downstream primer: 5'-GGTAGAGCGTTGACCCTACTG-3' (SEQ ID NO: 5)

[0075] Chikungunya virus probe: 5'HEX-AACAGCACACGGTCGCACGGTT-BHQ1 3' (SEQ ID NO: 6) ...

Embodiment 2

[0080] Example 2 Preparation of multiple fluorescent PCR detection mixture

[0081] Dengue virus upstream primer 0.5 μl / test; dengue virus downstream primer 0.5 μl / test; dengue virus probe 0.1 μl / test; chikungunya virus upstream primer 0.5 μl / test; chikungunya virus downstream primer 0.5 μl / test test; chikungunya virus probe 0.1 μl / test; yellow fever virus upstream primer 0.5 μl / test; yellow fever virus downstream primer 0.5 μl / test; yellow fever virus probe 0.1 μl / test; RT-PCR MIX 12.5 μl / test; 3.2μl / test of process water is mixed to get dengue fever, chikungunya, yellow fever virus nucleic acid multiple fluorescent PCR detection mixture.

Embodiment 3

[0082] Example 3 Sensitivity Analysis of Dengue Fever, Chikungunya and Yellow Fever Virus Multiplex PCR Detection Kit

[0083] 1. Experimental method

[0084] 1) Sample preparation:

[0085] The method for constructing the dengue virus plasmid: PCR method is used to amplify the nucleic acid of the dengue virus sample to obtain a nucleic acid fragment containing the target amplified sequence shown in SEQ ID NO: 10, and the primer sequences are shown in SEQ ID NO: 1-2. After the amplified fragment was purified, it was cloned into the pMD8-T vector by TA, identified by sequencing, and the recombinant vector with correct sequencing result was transformed into DH5α, amplified, and the dengue virus plasmid was obtained.

[0086] The construction method of the chikungunya virus plasmid: the nucleic acid of the chikungunya virus sample is amplified by the PCR method to obtain a nucleic acid fragment containing the target amplified sequence shown in SEQ ID NO: 11, and the primer seque...

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Abstract

The invention relates to the technical field of biological detection, and in particular relates to a primer, a probe and a kit used for detecting dengue fever, chikungunya and yellow fever virus through a single tube by the multiplex-fluorescence PCR (Polymerase Chain Reaction). The primer and probe used for detecting the dengue fever, chikungunya and yellow fever virus through the multiplex-fluorescence PCR have nucleotide sequences as shown in SEQ ID NO: 1-9; the multiplex PCR detection kit for detecting the dengue fever, chikungunya and yellow fever virus comprises multiplex-fluorescence PCR detection mixed liquid which contains the primer and probe mentioned above. The kit, primer and probe can quickly detect the dengue fever virus, chikungunya virus and yellow fever virus, have the advantages of being simple and convenient to operate, high in sensitivity and outstanding in specificity, and can timely find out and confirm a suspect case so as to improve the monitoring level on such diseases.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a multiple fluorescent PCR detection primer, probe, kit and application thereof for detection of dengue fever, chikungunya and yellow fever viruses. 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, with a mortality rate 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 about 5% in most c...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCY02A50/30
Inventor 吴忠华郑伟吕沁风杨永耀朱旭平熊磊高旭光杨双彪
Owner 浙江国际旅行卫生保健中心
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