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Reverse transcription pcr detection and typing kit for dengue virus and its detection method

A dengue virus, reverse transcription technology, applied in the field of biological science, to achieve the effect of rapid detection, convenient operation and high sensitivity

Inactive Publication Date: 2015-11-25
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, there is a lack of a highly sensitive, specific, rapid detection kit and detection method for reverse transcription PCR detection and typing of dengue virus

Method used

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  • Reverse transcription pcr detection and typing kit for dengue virus and its detection method
  • Reverse transcription pcr detection and typing kit for dengue virus and its detection method
  • Reverse transcription pcr detection and typing kit for dengue virus and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The invention provides a kind of test kit of reverse transcription PCR detection and typing dengue virus, comprise primer, probe and FRET-PCR standard item, described primer is upstream primer and downstream primer; Described probe is 6- FAM probe and Cy5 probe;

[0058] Upstream primer: 5'-GACTAGTGGTTAGAGGAGACCCCTCC-3'SEQ ID No.1, the primer sequence is completely consistent with DENV1, and has the same mismatched base with DENV2, DENV3 and DENV4;

[0059] 6-FAM probe: 5'-CTGTAGAGACAGCAGGATCTCTGGTC-6-FAM-3'SEQ ID No.2; 6-FAM probe sequence for reverse transcription PCR: 5'-CTGTAGAGACAGCAGGATCTCTGGTC-6-FAM-3'(26bp), this sequence It is the symmetrical chain nucleotide sequence of the sequence obtained in the figure. This FAM probe sequence is completely consistent with all dengue virus serotypes;

[0060] Cy5 probe: 5'-Cy5-CTCCCAGCGTCAATATGCTGTTT-phosphate group-3'SEQ ID No.3; Cy5 probe sequence of reverse transcription PCR: 5'-Cy5-CTCCCAGCGTCAATATGCTGTTT-phosphate gr...

Embodiment 2

[0089] Transcription method verifies the PCR system of the present invention

[0090] GenScript (GenScript Biotechnology, Nanjing, China) synthesized the plasmid pUC57 containing the dengue virus type 1 target fragment DNA and the T7 promoter, and used the appropriate restriction endonuclease (SacI, Baobio, Dalian) to Carry out enzyme digestion; Increase the concentration of target DNA by PCR amplification; Use commercial transcription kit ( Kit, bylife The United States) transcribed the PCR amplification product and obtained the RNA product; after the transcription was completed, the transcription product was directly used for reverse transcription PCR to amplify.

[0091] (1) Dilution of plasmid

[0092] Centrifuge the microcentrifuge tube containing the synthetic plasmid at 4000 rpm for 2 minutes, then add 40 μl of double distilled water or TE buffer (PH8.0) to make a solution with a plasmid concentration of 100 ng / μl;

[0093] (2) Digestion of plasmid

[0094] Dige...

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Abstract

The invention discloses a kit capable of detecting and parting dengue viruses by reverse transcription PCR and a detection method of the kit. The kit capable of detecting and parting the dengue viruses by the reverse transcription PCR comprises primers, probes and an FRET-PCR standard substance and is characterized in that the primers comprise a forward primer and a reverse primer; the probes comprise a 6-FAM probe and a Cy5 probe; the forward primer has a nucleotide sequence expressed by SEQ ID No.1; the 6-FAM probe has a nucleotide sequence expressed by SEQ ID No.2; the Cy5 probe has a nucleotide sequence expressed by SEQ ID No.3; the reverse primer has a nucleotide sequence expressed by SEQ ID No.4. The kit is convenient to operate, is suitable for detecting a large quantity of samples and is capable of quickly detecting and parting the serotypes of all the dengue viruses with high sensitivity and specificity.

Description

technical field [0001] The invention relates to the technical field of biological sciences, in particular to a kit for detecting and typing dengue virus by reverse transcription PCR and a detection method thereof. Background technique [0002] Dengue fever (Dengue fever, DF) is an acute infectious disease caused by dengue virus (DENV), mainly transmitted by Aedes aegypti and Aedes albopictus. Dengue fever is an acute infectious disease, its prevention in endemic areas, especially the diagnosis and control of outbreaks in endemic areas is particularly important. The main clinical manifestations are high fever, headache, muscle and joint pain, rash, enlarged lymph nodes and leukopenia, etc. In severe cases, hemorrhage or shock may occur, and even death. The World Health Organization divides dengue fever into three types: typical dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (Dengue shock syndrome, DSS). The latter two types are severe and have a high ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2531/113C12Q2561/113C12Q2563/107C12Q1/68
Inventor 王成明张继垒陆光武
Owner YANGZHOU UNIV
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