Chikungunya virus isothermal amplification detection kit and primer thereof

A chikungunya virus and detection kit technology, which is applied in the field of temperature amplification rapid detection kits and primers, can solve problems such as unfavorable on-site diagnosis and basic detection, unfavorable rapid detection, long detection cycle, etc., and achieve optimized primers The effect of spacing and amplification efficiency, perfect primer design, and high yield

Inactive Publication Date: 2012-04-04
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation is currently the gold standard for detection, but due to the long detection cycle of this technology, it is not conducive to the requirement of rapid detection; serum antibody detection can quickly diagnose chikungunya virus, but because the virus antibody is easy to detect

Method used

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  • Chikungunya virus isothermal amplification detection kit and primer thereof
  • Chikungunya virus isothermal amplification detection kit and primer thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Chikungunya virus isothermal gene amplification rapid detection kit

[0060] The test kit of the present invention consists of the following components:

[0061] (1) Detection primers

[0062] (2) DNA polymerase: Bst DNA polymerase

[0063] (3) Reaction solution: 10mM dNTP (deoxynucleoside triphosphate), 10×ThermoPol Buffer (reaction buffer), 150mMMgSO4 (magnesium sulfate)

[0064] (4) Fluorescent dye: SYBR Green I

[0065] The detection primer sequence is the sequence shown in SEQ ID NO: 1-4:

[0066] Outer primer 1: CGCCCTCTTTAACGGACATG;

[0067] Outer primer 2: AATTCGGCGCTGGCTAAG;

[0068] Inner primer 1: TGCCTTTCTTGCTGGCTGCATATACCAGCCTGCACCCATT;

[0069] Internal primer 2: AGTGTGCGGTGCATTCGATGATGCAGCTGAGAATTCCCTTC;

[0070] Or the sequences shown in SEQ ID NO: 5-8:

[0071] Outer primer 3: GCTGAAAACACGCAGTTGAG;

[0072] Outer primer 4: TGGCCCCACAATGAATTTGG;

[0073] Inner primer 3: GCGGTATGAGCCCTGTATGCTGAAGCACATGTGGAGAAGTCC;

[0074] Inner prim...

Embodiment 2

[0085] Embodiment 2: the method for detecting chikungunya virus with a preferred embodiment of the kit of the present invention

[0086] Proceed as follows:

[0087] Step 1: Pretreatment of the sample to be tested: Extract sample RNA according to conventional methods.

[0088] Step 2: Loop-mediated constant temperature amplification (LAMP) reaction:

[0089] The kit selected in this embodiment contains primers of the following sequences, such as the sequences shown in SEQ ID NO: 1-4:

[0090] Outer primer 1: CGCCCTCTTTAACGGACATG

[0091] Outer primer 2: AATTCGGCGCTGGCTAAG

[0092] Inner Primer 1: TGCCTTTCTTGCTGGCTGCATATACCAGCCTGCACCCATT

[0093] Inner primer 2: AGTGTGCGGTGCATTCGATGATGCAGCTGAGAATTCCCTTC.

[0094] Prepare the reaction solution: the reaction system is 25uL: 1.6mM inner primer 1 and inner primer 2, 0.2mM outer primer 1 and outer primer 2, 1.6mM dNTPs, 6mM MgSO4, 1M betaine, 2uL sample RNA, 8U Bst DNA polymerase, 2U reverse transcriptase, add distilled water ...

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Abstract

The invention provides a chikungunya virus isothermal amplification detection kit and a primer thereof. The kit comprises the primer, Bst DNA polymerase, a reaction liquid and SYBR Green I fluorescent dye, wherein the reaction liquid is 10mM deoxyribonucleoside triphosphate, 10*reaction buffer liquid and 150mM MaSO4; the sequences of the primer are sequences represented by SEQ ID NO:1-4 or sequences represented by SEQ ID NO:5-8 or sequences represented by SEQ ID NO:9-12 or sequences represented by SEQ ID NO:13-16. The kit provided by the invention has the advantages of short detection time, strong specificity, high sensitivity, high detection accuracy and programmed reaction, is easy and convenient to qualify and is widely applied to conventional detection and epidemiological survey in clinic and ports.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a chikungunya virus isothermal amplification rapid detection kit and primers thereof. Background technique [0002] Chikungunya virus is the pathogen that causes chikungunya fever, which is mainly transmitted through the bite of Aedes mosquitoes. Chikungunya fever is a zoonotic disease, which is an acute viral infectious disease mainly characterized by fever, rash and joint pain. It is mainly prevalent in Africa and Southeast Asia. Areas with high vector density are prone to large-scale outbreaks and epidemics. Aedes mosquitoes are widely distributed in South China and Southwest my country, and become a potential cause of the outbreak of the disease. In 2006 alone, India reported more than 1.39 million suspected cases of chikungunya fever, and the incidence rate in some areas exceeded 45%. In 2008, the Guangdong Entry-Exit Inspection and Quarantine Bureau detected th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCY02A50/30
Inventor 李小波石磊师永霞鲁曦幸芦琴郑夔黄吉城
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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