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Chikungunya virus testing method

A chikungunya virus and detection method technology, applied in the field of molecular biology detection, to achieve important social and economic benefits

Inactive Publication Date: 2008-09-24
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no effective drug to treat this disease, and mosquito control is the most important preventive work

Method used

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  • Chikungunya virus testing method
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  • Chikungunya virus testing method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, design and synthesis of primers and probes

[0030] The SeqMan program in the Lasergene software package was used to analyze the nucleotide sequences of 37 strains of Chikungunya virus obtained from Genbank, select non-structural protein genes with highly conserved nucleotides, and use Primer Express2.0 software to design specific primers and The probe design, primers and probe sequences are listed in Table 1.

[0031] Table 1

[0032]

[0033] The location of the primers and probes on the Chikungunya virus genome was based on the strain sequence with the GenBank sequence number AF490259.

Embodiment 2

[0034] Example 2. Preparation of positive control RNA template and extraction of viral RNA for specificity assessment

[0035] According to the sequence of Chikungunya virus (GenBank sequence number: AF490259), synthesize a DNA fragment with a size of 120bp including the fragment sequence detected by fluorescent RT-PCR, clone it on the T vector (TOPO TA cloning kit), and use plasmid DNA extraction reagent Purify the plasmid DNA with the cassette, and after sequencing confirms that the inserted fragment is correct, reverse transcription in vitro: first use Not I to digest the plasmid to linearize it to exclude downstream RNA products, then perform in vitro transcription with MAXIscript T7 Kit to generate the target RNA, and then use Turbo DNase DNase treatment was performed to remove the untranscribed plasmid DNA template, and finally ethanol precipitation was used to purify the RNA product, which was the positive control RNA template and stored in a -70°C ultra-low temperature ...

Embodiment 3

[0038] Embodiment 3, determination of amplification program

[0039] The kit used for TaqMan RT-PCR reaction is Ag-Path-ID TM One step RT-PCR Kit (product of Ambion, USA), amplification and detection were performed on Applied Biosystems 7500 Fast Real-Time PCR Systems, a fully automatic fluorescent quantitative instrument, or on instruments such as Stratagene Mx3005P real-time fluorescent quantitative PCR instrument.

[0040]According to the recommendations of the kit and the specific characteristics of the primers and probes, the reaction program on the instrument tested 3 temperatures and 3 times in the selection of annealing extension conditions: the reverse transcription reaction conditions were 50°C for 10 minutes, and 95°C hot start for 10 minutes. Minutes later, enter 45 two-step PCR cycles: 95°C for 10 seconds → (58 / 60 / 62)°C (30 / 40 / 60) seconds; ×RT-PCR buffer 10 μl, 20 μM forward primer CHIK-FP 1 μl, 20 μM reverse primer CHIK-RP 1 μl, 5 μM probe CHIK-Probe 0.5 μl, 25...

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Abstract

The invention provides a detection method of Chikungunya virus, which comprises the procedures as follows: pretreatment of test sample; RNA extraction of test sample to be detected; design and synthesis of primer and probe; determination of optimum fluorescence quantitative PCR reaction system; program amplification and adjudging results according to Ct value. The optimum fluorescence quantitative PCR reaction system is determined as follows: the CHIK-FP terminal concentration of positive primer is 500nM, the CHIK-RP terminal concentration of reverse primer is 1000nM, and the CHIK-Probe terminal concentration of probe is 250nM. The amplification program is 45 times of circulation of 10 minutes at 50 DEG C, 10 minutes at 95 DEG C, 10 seconds at 95 DEG C and 30 seconds at 62 DEG C. The invention establishes a detection method for Chikungunya virus, which can be applied on monitoring of the virus, and can further strengthen the monitoring work for preventing from spreading the disease into our country and has important social efficiency and economic efficiency.

Description

technical field [0001] This field belongs to the field of molecular biology detection, and in particular relates to a detection method for chikungunya virus. Background technique [0002] Chikungunya disease is an acute infectious disease caused by Chikungunya virus. The virus originated in Africa, with the first known outbreak occurring in Tanzania in 1952. After the 1960s, Chikungunya disease moved eastward to Southeast Asia. In 1965, a pandemic hit India and 300,000 people were infected. In March 2005, an outbreak of chikungunya disease broke out in French Reunion Island. By March 2006, 186,000 people had been infected, and 93 people died directly or indirectly from the disease. In March 2006, Chikungunya disease broke out in four island countries in the Indian Ocean (French Reunion Island, etc.), and more than 300,000 people were infected and became ill. About 40 people in mainland France were also infected with the virus. "Chikungunya" is a Swahili word that means "...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCY02A50/30
Inventor 郑夔黄吉城李小波洪烨郭波旋相大鹏幸芦琴师永霞钟玉清
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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