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Kit and detection method for inverse transcription PCR detection of chikungunya virus

A chikungunya virus, reverse transcription technology, applied in the field of biological science, to achieve the effect of high accuracy and high sensitivity

Inactive Publication Date: 2015-04-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] At present, there is a lack of a highly sensitive, specific and rapid reverse transcription PCR detection kit and detection method for chikungunya virus

Method used

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  • Kit and detection method for inverse transcription PCR detection of chikungunya virus
  • Kit and detection method for inverse transcription PCR detection of chikungunya virus
  • Kit and detection method for inverse transcription PCR detection of chikungunya virus

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Embodiment 1

[0060] The invention provides a kit for detecting chikungunya virus by reverse transcription PCR, comprising primers, probes and FRET-PCR standard items, wherein the primers are upstream primers and downstream primers; the probes are 6-FAM probe and LCRed640 probe;

[0061] like Figure 1 to Figure 4 as shown, figure 1 It is a schematic diagram of the upstream primer of the reverse transcription PCR of the present invention; the reverse transcription PCR upstream primer sequence: 5'-CGGCTTCTTCAATATGATGCAGATG-3' (25bp). The sequence of this primer is completely consistent with the nucleic acid sequence of all genotypes of chikungunya virus.

[0062] figure 2 It is a schematic diagram of the 6-FAM probe of the reverse transcription PCR of the present invention; the sequence of the 6-FAM probe of the reverse transcription PCR: 5'-GACACAATGGCAGTCACAGGCAGT-6-FAM-3' (24bp), which is the sequence obtained in the figure The symmetrical chain nucleotide sequence. The FAM probe se...

Embodiment 2

[0118] The transcription method verifies the PCR system of the present invention:

[0119] GenScript (GenScript Biotechnology, Nanjing, China) synthesized the plasmid pUC57 containing the target fragment DNA of Chikungunya virus Asian genotype CHIKV-Asian and the T7 promoter, and used an appropriate restriction endonuclease (Sac I , Treasure Biotechnology, Dalian) to digest the plasmid; increase the concentration of the target DNA by PCR amplification; use a commercial transcription kit ( Kit, by life The United States) transcribed the PCR amplification product and obtained the RNA product; after the transcription was completed, the transcription product was diluted and used for reverse transcription PCR to amplify.

[0120] (1) Dilution of plasmid

[0121] Centrifuge the microcentrifuge tube containing the synthetic plasmid at 4000 rpm for 2 minutes, then add 40 μl of TE buffer solution with a pH of 8.0 to make a solution with a plasmid concentration of 100 ng / μl;

[0...

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Abstract

The invention discloses a kit and detection method for inverse transcription PCR detection of chikungunya virus, the kit for inverse transcription PCR detection of chikungunya virus comprises primers, probes and a FRET (fluorescence resonance energy transfer)-PCR standard substance, the primers are an upstream primer and a downstream primer; the probes comprise 6-FAM (carboxyfluorescein) probe and a LCRed640 probe; the upstream primer has a nucleotide sequence of SEQ ID No.1, the 6-FAM probe has a nucleotide sequence of SEQ ID No.2; the LCRed640 probe has a nucleotide sequence of SEQ ID No.3, and the downstream primer has a nucleotide sequence of SEQ ID No.4. The inverse transcription PCR system can effectively amplify RNA, namely can fast and efficiently amplify chikungunya virus RNA nucleic acid in clinical samples.

Description

technical field [0001] The invention relates to the technical field of biological sciences, in particular to a kit for detecting chikungunya virus by reverse transcription PCR and a detection method thereof. Background technique [0002] Chikungunya fever Chikungunya fever, CHIK is caused by Chikungunya virus Chikungunya virus, CHIKV, and is mainly transmitted by blood-sucking insects such as Aedes aegypti, Aedes albopictus, and Aedes africana Vector, an acute viral vector-borne infectious disease with fever, rash and severe joint pain as the main clinical symptoms, with an incubation period of 3-12 days. It is mainly prevalent in Africa and Southeast Asia, but in recent years it has been prevalent in the coasts of East Africa, islands in the Indian Ocean, and islands in the Americas and the Caribbean. At present, there are mainly imported cases of infection in my country, but some scholars have detected Chikungunya virus antibodies and isolated the pathogen from human seru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q1/68
Inventor 王成明张继垒陆光武成大荣
Owner YANGZHOU UNIV
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