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Nucleic acid detection kit for Zika virus, dengue fever virus and Chikungunya virus, and application of the kit

A technology of chikungunya virus and dengue virus, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, and resistance to vector-borne diseases, can solve the problems of consuming manpower and material resources, prolonging the diagnosis time, etc. , strong repeatability, rapid and objective test results

Active Publication Date: 2018-07-27
GUANGZHOU CENT FOR DISEASE CONTROL & PREVENTION (GUANGZHOU HYGIENE INSPECTION CENT GUANGZHOU CENT FOR FOOD SAFETY RISK SURVEILLANCE & ASSESSMENT INST OF PUBLIC HEALTH OF GUANGZHOU MEDICAL UNIV) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Most of the existing in vitro diagnostic kits for Zika virus, dengue virus and chikungunya virus use a single-plex RT-PCR reaction. To complete the detection of the three viruses, one RT-PCR reaction is required separately. Large-scale sample testing will undoubtedly consume a lot of manpower and material resources, and will also prolong the diagnosis time. Therefore, there is an urgent need to establish a specific and sensitive test that can simultaneously detect Zika virus, dengue virus and chikungunya virus. The multiplex RT-PCR method is expected to be better and faster for the detection of Zika virus, dengue virus and chikungunya virus

Method used

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  • Nucleic acid detection kit for Zika virus, dengue fever virus and Chikungunya virus, and application of the kit
  • Nucleic acid detection kit for Zika virus, dengue fever virus and Chikungunya virus, and application of the kit
  • Nucleic acid detection kit for Zika virus, dengue fever virus and Chikungunya virus, and application of the kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Assembly of Zika virus, Dengue virus and Chikungunya virus nucleic acid detection kit

[0047] The kit includes the following components:

[0048] (1) RT-PCR reaction solution:

[0049] The RT-PCR reaction solution consists of RNase-removed water, 10×PCR buffer, 25mM Mg 2+ , 10mM dNTPs composition (Table 1).

[0050] Table 1 Composition of RT-PCR reaction solution

[0051]

[0052]

[0053] (2) Enzyme mixture

[0054] The enzyme mixture includes RNase inhibitor, DNA polymerase, reverse transcriptase, 10× buffer, and RNase water. Reverse transcriptase and DNA polymerase play a very important role in PCR amplification, including reverse transcription and amplification efficiency and specificity. In order to achieve better results, the present invention preferably uses M-MLV reverse transcriptase and Taq Hot start enzymes (Table 2).

[0055] Table 2 Composition of enzyme mixture

[0056] Reagent name

Add volume (μl) / 50 reaction

RNase inhibitor

12.5

Taq enzyme

12.5

...

Embodiment 2

[0077] Example 2 Detection test of clinical samples of the kit of the present invention

[0078] 1. Sample processing (RNA extraction)

[0079] 1.1 Take 200ul clinical sample, add 400ul Binding Buffer supplemented with Poly(A), mix well and transfer to high purification filter tube, centrifuge at 8000rpm for 15s, discard the waste liquid in the collection tube.

[0080] 1.2 Add 500ul Inhibitor Removal Buffer to the filter tube, centrifuge at 8000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0081] 1.3 Add 450ul Washing Buffer to the filter tube, centrifuge at 8000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0082] 1.4 Repeat step 3, and then centrifuge at high speed for 10 seconds. This step must remove the waste liquid.

[0083] 1.5 Add 50ul Elution Buffer to the filter tube, let it stand at room temperature for 2 minutes, centrifuge at 8000 rpm for 1 minute, and the solution obtained by centrifugation is the purified RNA.

[0084] 2. Re...

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Abstract

The invention discloses a nucleic acid detection kit for Zika virus, dengue fever virus and Chikungunya virus, and an application of the kit, wherein the kit includes: a RT-PCR reaction solution, an enzyme mixture liquid, a Zika virus / dengue fever virus / Chikungunya virus / endogenous reference gene quadruple reaction solution, a positive control, and a blank control. The kit can simultaneously detect the Zika virus, dengue fever virus and Chikungunya virus in one reaction and solves a problem that an in-vitro detection kit cannot simultaneously detect the Zika virus, dengue fever virus and Chikungunya virus in one reaction tube; besides, the detection kit is high in specificity and sensitivity, has simple operation, excellent repeatability and quick and objection detection results. The invention provides an effective technical means for in-vitro detection of the Zika virus, dengue fever virus and Chikungunya virus.

Description

Technical field [0001] The invention relates to an in vitro diagnostic kit for mosquito-borne pathogens, in particular to a nucleic acid detection kit for Zika virus, dengue virus and chikungunya virus and uses thereof, and belongs to the technical field of virus in vitro diagnosis. Background technique [0002] Zika Virus (ZIKV) belongs to the Flaviviridae (Flaviviridae) genus Flavivirus. It has a spherical shape with a diameter of about 40-70 nm and an envelope. The genome is a single-stranded positive-stranded RNA with a length of about 10.8 kb The genome includes 5'and 3'non-coding regions, with a single open reading frame in the middle. The encoded polypeptide chain can be cleaved and matured into 3 structural proteins and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A). , NS4B and NS5), the structural proteins are capsid protein (capsid, C), membrane protein precursor / membrane protein (Premembrane / membrane, prM) and envelope protein (Envelope, E). [0003] Zika virus ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2537/143C12Q2521/107C12Q2531/113C12Q2545/101Y02A50/30
Inventor 狄飚张颖苏文哲胡玉山朱娉婷杨静李丹刘中华王国强
Owner GUANGZHOU CENT FOR DISEASE CONTROL & PREVENTION (GUANGZHOU HYGIENE INSPECTION CENT GUANGZHOU CENT FOR FOOD SAFETY RISK SURVEILLANCE & ASSESSMENT INST OF PUBLIC HEALTH OF GUANGZHOU MEDICAL UNIV)
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