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RT-LAMP (reverse transcription loop-mediated isothermal amplification) nucleic acid detection primers and kit of Hantaan viruses

A technology of RT-LAMP and detection kit, which is applied in the application field of biological detection technology, can solve the problems of easy pollution of the environment, inconvenient to carry, increase costs, etc., and achieve the effects of high sensitivity, easy reaction results, and low cost of use

Inactive Publication Date: 2015-09-30
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of a turbidimeter not only increases the cost, but is also inconvenient to carry; some scholars use the method of adding SYBR Green I dye to the reacted system to detect the amplification product, but this observation must be opened for processing, LAMP amplification The high efficiency and high sensitivity make the product contain a large number of target fragments, and adding dyes after opening the cap is extremely easy to pollute the environment

Method used

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  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) nucleic acid detection primers and kit of Hantaan viruses
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) nucleic acid detection primers and kit of Hantaan viruses
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) nucleic acid detection primers and kit of Hantaan viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design and synthesis of LAMP primers

[0042] According to the sequence of S segment of Hantavirus 76-118 gene published by GenBank (GenBank: M14627.1), the relatively conserved region was analyzed by biological software BLAST, and the LAMP online design software PrimerExplorer V4 (http: / / primerexplorer.jp / e / ) 6 primers were designed for relatively conserved sequence regions, including two inner primers FIP and BIP, two outer primers F3 and B3, and two loop primers LF and LB, which respectively bind to 8 binding regions in the target sequence For matching, the primers were evaluated using Oligo 6 software and BLAST. See Table 1 for details.

Embodiment 2

[0043] Example 2 Tissue sample total RNA extraction

[0044] The total RNA of tissue samples was extracted by Trizol method and micro-extraction; the specific operation is as follows:

[0045] A. Take 100 μl of blood or 100 μg of tissue samples (ground and homogenized) in a 1.5ml centrifuge tube, add 500 μl of Trizol solution, vortex and mix for 1 min, then place at room temperature for 5-10 min, and place the above microcentrifuge tube at 12000 rpm at 4 °C Centrifuge for 15 minutes;

[0046] B. Take 500 μl of the supernatant and put it into a new 1.5ml centrifuge tube, and add 500 μl of water-saturated phenol:chloroform (1:1), shake vigorously by hand for 15 seconds, and then place it at room temperature for 10 minutes;

[0047] C. Centrifuge at 12000rpm for 10min at 4°C, then take 500μl of the supernatant and add it to a new 1.5ml centrifuge tube, add 500μl of isopropanol, mix it upside down, place it at -20°C for 1h, and then put it at 12,000rpm , centrifuge at 4°C for 10 m...

Embodiment 3

[0049] Example 3 RT-LAMP detection kit and detection method for detecting Hantavirus

[0050] The RT-LAMP detection kit that detects Hantavirus includes: a group of RT-LAMP primers for detecting Hantavirus, specifically, this group of primers includes the forward outer primer (F3 ), the reverse outer primer (B3) shown in the sequence listing SEQ ID No.2, the forward inner primer (FIP) shown in the sequence listing SEQ ID No.3, the reverse primer (B3) shown in the sequence listing SEQ ID No.4 Internal primer (BIP), the forward loop primer (LF) shown in the sequence table SEQ ID No.5, the reverse loop primer (LB) shown in the sequence table SEQ ID No.6; the primers are all entrusted to Shanghai Shenggong Synthesized by Biotech Ltd.

[0051] Further, the above-mentioned RT-LAMP detection kit for detecting Hantavirus also includes 10×ThermoPol buffer solution, dNTPs, MnCl 2 , MgSO 4 Strand displacement DNA polymerase (purchased from New England Biolabs, U.S.); AMV reverse trans...

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Abstract

The invention discloses RT-LAMP (reverse transcription loop-mediated isothermal amplification) nucleic acid detection primers and a kit of Hantaan viruses and in particular relates to a group of primers for detecting Hantaan viruses, a kit containing the primers and a detection method of the Hantaan viruses. The primers comprise base sequences shown in a sequence table SEQ ID No.1 to a sequence table SEQ ID No.6. The kit has high sensitivity and strong specificity, is low in cost and is simple and convenient to operate.

Description

technical field [0001] The invention relates to the application field of biological detection technology, in particular to primers and a detection kit for detection of Hantaan-type nucleic acid of Hantaan virus in blood samples. Background technique [0002] Hantavirus belongs to the Bunyaviridae family and is a negative-strand RNA virus with an envelope and segments. When the virus proliferates, negative-strand RNA is used as a template to generate positive-strand RNA and mRNA, and the progeny viral genome is copied from the positive-strand RNA, which is budded and released to obtain an envelope. The genome includes three segments, L, M, and S, which encode L polymerase protein, G1 and G2 glycoproteins, and nucleoproteins, respectively. Hantaan viruses include Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Dobraval virus ( Dobrava virus, DOBV) and the unknown virus (Sin Nombre virus, SNV) that causes Hantavirus pulmonary syndrome (HPS), New York virus (Ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/70C12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107
Inventor 谢远红刘艳华高璟瑜张红星李丹郭慧琳
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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