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A primer set and detection kit for detecting new coronavirus based on rt-lamp technology

A technology of RT-LAMP and detection kit, applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of lack of new coronavirus and difficult application of LAMP detection technology, etc., to achieve strong specificity, Fast, efficient and accurate detection, the effect of improving accuracy

Active Publication Date: 2020-11-17
上海国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of specific amplification primers for the new coronavirus, it is difficult to apply the LAMP detection technology in the detection of the new coronavirus

Method used

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  • A primer set and detection kit for detecting new coronavirus based on rt-lamp technology
  • A primer set and detection kit for detecting new coronavirus based on rt-lamp technology
  • A primer set and detection kit for detecting new coronavirus based on rt-lamp technology

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Through the SARS-CoV-2 genome downloaded in GISAID, NCBI downloaded other common coronaviruses SARS-2003, bat-SL-CoVZC45, bat-SL-CoVZXC21, SARS coronavirus ZS-B, SARS coronavirus ZS-C, SARS coronavirus Sin3408L, SARS Coronavirus Sin3765V, SARS coronavirusTaiwan TC3 sequence, BioEdit and SnapGene sequence comparative analysis, obtained specific Orf1ab fragment and N fragment sequence, and designed RT-LAMP primer set based on this.

[0045] 1. Design and optimization of RT-LAMP primer set

[0046] 1) Using the online design software Primer ExplorerV5, design RT-LAMP primers for the 6 regions of the specific Orf1ab fragment, including 2 outer primers Orf1ab-F3 / Orf1ab-B3, 2 inner primers Orf1ab-FIP / Orf1ab-BIP, in Design Orf1ab-Loop-F primers and Orf1ab-Loop-B primers between F2 and F1C regions and between B2 and B1C regions, and use LAMP technology to detect recombinant plasmids containing Orf1ab fragments of SARS-CoV-2 (pUC57-Orf1ab, commissioned Suzhou Jinweizhi Biotechn...

Embodiment 2

[0063] The minimum detection limit of the LAMP detection method

[0064] 1) The recombinant plasmid (pUC57-Orf1ab) containing the Orf1ab target fragment was treated with ddH 2 O is used for gradient dilution, and the copy number is 10 in turn 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 Copies / μL, 10copies / μL, 1copies / μL. Detect with the LAMP detection method established in Example 1, and determine the minimum detection amount of the positive nucleic acid by the detection method. Depend on figure 1 It can be seen that with the decrease of the template concentration in the reaction system, the minimum detection limit of the LAMP detection method is 100copies / 20μL.

[0065] 2) Use ddH for the recombinant plasmid (pUC57-N) containing the N target fragment 2 O for serial dilution, the copy number is 10 in sequence 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 Copies / μL, 10copies / μL, 1copies / μL. Use the established LAMP detecti...

Embodiment 3

[0085] Specific detection of RT-LAMP detection method

[0086] With the recombinant plasmid containing Target-SARS-CoV-2-Orf1ab fragment, the recombinant plasmid of Target-SARS-2003-Orf1ab fragment (the nucleotide sequence of SARS-2003-Orf1ab gene fragment is SEQ ID No.14), Target-bat -The recombinant plasmid of the SL-CoVZC45-Orf1ab fragment (the nucleotide sequence of the bat-SL-CoVZC45-Orf1ab gene fragment is SEQID No.15) as the detection object, and ddH 2 O as negative control, utilize the LAMP detection method that the screening of embodiment 1 obtains, determine specificity detection result. Among them, the above three recombinant plasmids were commissioned to be synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0087] see results figure 2 . Depend on figure 2 It can be seen that only the plasmid RT-LAMP containing the Target-SARS-CoV-2-Orf1ab fragment has an "S"-shaped fluorescence curve, and the test result is positive, while the rest of the nucleic acid ...

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Abstract

The invention provides a primer set and a detection kit for detecting novel coronavirus based on RT-LAMP technology, belonging to the technical field of virus detection. The primer set includes two sets of primers, and the RT-LAMP primer set for detecting the Orf1ab gene of the new coronavirus includes 2 outer primers F3 / B3, 2 inner primers FIP / BIP and 2 loop primers Loop-F / Loop-B , the RT-LAMP primer set for detecting the N gene of the new coronavirus includes 2 outer primers F3 / B3, 2 inner primers FIP / BIP and 1 loop primer Loop-F. The detection kit includes the primer set. The kit is based on RT-LAMP technology to detect the new coronavirus, which has the characteristics of strong specificity, good accuracy and high sensitivity. Compared with the fluorescent RT-PCR detection method, the detection time is greatly shortened, and the purpose of fast and efficient detection of the new coronavirus is realized. .

Description

technical field [0001] The invention belongs to the technical field of virus detection, in particular to a primer set and a detection kit for detecting novel coronavirus based on RT-LAMP technology. Background technique [0002] The new coronavirus, also known as the new coronavirus, has been named "SARS-CoV-2" by the International Committee on Taxonomy of Viruses. The clinical manifestations of novel coronavirus pneumonia are mainly fever, fatigue, and dry cough, and upper respiratory symptoms such as nasal congestion and runny nose are rare. About half of the patients developed dyspnea more than a week later, and severe cases rapidly progressed to acute respiratory distress syndrome, septic shock, metabolic acidosis that was difficult to correct, and coagulation dysfunction. [0003] According to the latest guidelines, the use of fluorescent RT-PCR to detect positive nucleic acid of the new coronavirus is still the gold standard for diagnosis. However, more expensive ins...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107C12Q2563/107Y02A50/30
Inventor 张子龙田桢干张威水晶李深伟蒋原张晓航
Owner 上海国际旅行卫生保健中心
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