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Hantavirus integrated nucleic acid detection kit and detection method

A detection kit, Hantavirus technology, applied in the field of biotechnology detection, can solve problems such as time delay, and achieve the effect of facilitating long-term storage and transportation

Inactive Publication Date: 2015-09-30
宋锋林 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If positive samples cannot be obtained in time to explore the reaction composition and reaction conditions, the above time will be significantly delayed or not tested at all

Method used

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  • Hantavirus integrated nucleic acid detection kit and detection method
  • Hantavirus integrated nucleic acid detection kit and detection method
  • Hantavirus integrated nucleic acid detection kit and detection method

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Embodiment 1

[0035] Hantavirus integrated nucleic acid detection kit, including Hantavirus nested PCR first round and nested PCR second round reaction system, in which the PCR first round reaction system includes a pair of universal primers HTV-MFO, HTV-MRO; The second round of PCR reaction system includes Seoul-type primers SEO-MF, SEO-MR and Hantan-type PCR primers HMF, HMR. The primer sequences are as follows:

[0036]

[0037] The first round of PCT is a RT-PCR reaction system with a total volume of 50 μL, including Enzyme Mix 2.0 μL, 2×Buffer 25.0 μL, template RNA 2-14 μL, primer HTV-MFO (20 μM) 2.0 μL, primer HTV-MRO (20 μM) 2.0 μL, supplemented with RNase Free H 2 O to bring the total volume to 50 μL;

[0038] The total volume of the Seoul-type reaction system for the second round of PCR is 50 μL, including 2.0 μL of DNA Polymerase (1U / μl), 5.0 μL of 10×PCR Buffer, 5.0 μL of 4×dNTP, 4.0 μL of template (RT-PCR), primers SEO-MF (20 μM) 2.0 μL, primer SEO-MR (20 μM) 2.0 μL, add dd...

Embodiment 2

[0041] Hantavirus integrated nucleic acid detection method, required reagents, sample RNA extraction, nucleic acid detection and result judgment are described as follows:

[0042] RNase Free H 2 O, 6×loading buffer, agarose, etc. were purchased from Treasure Bioengineering (Dalian) Co., Ltd., 50×TAE buffer was purchased from Beijing Solaibao Biotechnology Co., Ltd., and GelRed nucleic acid dye was purchased from Shanghai Open Biotechnology Co., Ltd.

[0043] RNA extraction: extract the RNA of the sample to be tested, the RNA of the Hantaan virus of Seoul type and the positive sample of Hantaan virus of Hantaan type respectively, and the extraction steps are as follows:

[0044] Place lung and other tissue samples in a 1.5 mL centrifuge tube and number them; add 1 mL of Trizol reagent, grind to a homogenate, and place at room temperature for 5 min; add 200 μL of chloroform, shake vigorously for 15 s, place at room temperature for 3 min, and place at 4°C at 12,000 rpm Centrifug...

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Abstract

The invention discloses a hantavirus integrated nucleic acid detection kit. Specifically, a hantavirus nest PCR first-round (RT-PCR) reaction system and a nest PCR second-round (a Seoul type and a Hantaan type) reaction system and reaction conditions are included. According to the detection kit, Enzyme Mix or DNA Polymerase needed for hantavirus detection, a reaction buffer solution 2*Buffer or 10*PCR Buffer, nucleotide 4*dNTP, and primers HTV-MFO, HTV-MRO, SEO-MF, SEO-MR, HMF and HMR needed for RT-PCR and PCR (a Seoul type and a Hantaan type) are integrated into a 200-microliter PCR tube, and an integrated RT-PCR (PCR) tube is formed through vacuum drying. The hantavirus integrated nucleic acid detection kit has the advantages of being high in sensitivity, high in specificity, good in stability and the like. When the hantavirus integrated nucleic acid detection kit is used for detecting rats and other samples, the detecting period is remarkably shortened, work procedures are simplified, workloads are reduced, cost is reduced, and a user can conveniently use the detection kit.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a hantavirus nucleic acid detection kit. Background technique [0002] Vector-borne diseases are an important class of infectious diseases. In the past 20 years, many of the more than 30 infectious diseases newly discovered in the world are transmitted by vector organisms. At present, there are 36 kinds of legal infectious diseases in my country, of which 13 diseases such as plague, hemorrhagic fever with renal syndrome, malaria, dengue fever and dengue hemorrhagic fever are insect-borne diseases. This kind of disease accounts for about 5%-10% of the total incidence of infectious diseases in my country every year, and the number of deaths accounts for 30%-40% of the total deaths of infectious diseases. [0003] Hantavirus is the causative agent of hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS). Virus. At present, there a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6848C12Q1/70C12Q2549/119C12Q2545/101Y02A50/30
Inventor 宋锋林高玉峰程晓兰姜陆万雪
Owner 宋锋林
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