Method, primer group and kit for capturing novel coronavirus whole genome

A coronavirus and genome technology, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem that the new coronavirus cannot achieve large-scale promotion, rapid diagnosis, and long detection time (requires 24- 72 hours, affecting the efficiency of the whole genome of pathogens, etc., to achieve the effects of sequencing economy, reducing the risk of cross-contamination, and low sample quality requirements

Active Publication Date: 2020-05-08
北京微未来科技有限公司
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Problems solved by technology

However, the differences in application scenarios and many complex factors jointly affect the success rate of obtaining the whole genome of pathogens. At the same time, there are also problems such as time, manpower, and high sequencing costs for front-line personnel under the heavy task of testing.
Therefore, pathogenic metagenomic sequencing (mNGS) can be used for the diagnosis of negative samples, but it is not easy to obtain the whole genome information, and there are reasons such as complicated operation and relatively long detection time (requiring 24-72 hours). It is impossible to achieve the purpose of large-scale promotion and rapid diagnosis
As a technical supplement, the genome capture me

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  • Method, primer group and kit for capturing novel coronavirus whole genome
  • Method, primer group and kit for capturing novel coronavirus whole genome
  • Method, primer group and kit for capturing novel coronavirus whole genome

Examples

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Example Embodiment

[0037] Example 1 Database construction and sequencing based on the ultra-sensitive novel coronavirus whole genome capture method

[0038] The present invention specifically uses multiple PCR technology to perform targeted amplification for the gene sequence of SARS-CoV-2, which increases the depth of sequencing, effectively reduces subsequent sequencing costs and shortens research time. A key point of multiplex PCR technology is to design a reasonable multiplex PCR primer pair combination, ensure that there are no overlapping amplicons in the primer combination, and reduce the interaction between primers. In this method, 98 pairs of primers were designed, and the Invitrogen SurSprit III ONE-STEP RT-PCR amplification system was used. A small amount of RNA was taken to quickly amplify the whole genome of the coronavirus. After the amplification reaction, the PCR reaction will get Coronavirus gene fragments of different sizes can be directly used on the computer for second-generatio...

Example Embodiment

[0068] Example 2 Comparison of the ultra-sensitive novel coronavirus whole genome capture method of the present invention with other methods

[0069] The capture method of the present invention can amplify samples with high virus content such as virus strains, as well as samples with relatively low virus content such as throat swabs and sputum. As long as the samples are used for new coronavirus qPCR, the quantitative CT value is below 27. Both can amplify the whole genome sequence.

[0070] The method for capturing the whole genome of the ultra-sensitive novel coronavirus of the present invention is compared with other existing methods, as shown in Table 2. The results show that the existing methods such as the direct library building method using RNA and the direct library building after reverse transcription cannot obtain the whole genome sequence well, and the capture method after library building and the operation time of the method of direct RNA library building are all relat...

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Abstract

The invention relates to a super-sensitivity method, primer group and kit for capturing whole genome of novel coronavirus. The method can conveniently and quickly amplify the whole genome of the novelcoronavirus by using a small amount of RNA, and can directly dock with a second-generation sequencing library-building reagent and a third-generation sequencing platform so as to obtain a whole genome sequence of the novel coronavirus.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a novel coronavirus whole genome capture method, primer set and kit. Background technique [0002] In late December 2019, a patient with viral pneumonia caused by an unknown microbial pathogen was reported in Wuhan, China, and a novel coronavirus was subsequently identified as the causative pathogen. The study showed that the 10 genome sequences of the novel coronavirus initially obtained from 9 patients were extremely similar, with a sequence identity of more than 99.98%. It is worth noting that this novel coronavirus is closely related (88% concordance) to two SARS-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in Zhoushan, eastern China in 2018, but not closely related to SARS. - CoV (about 79%) and MERS-CoV (about 50%) are more distant. This novel coronavirus is sufficiently different from SARS-CoV to be considered a novel human-infecting oronas...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/701C12Q2531/113C12Q2535/122C12Q2521/107Y02A50/30
Inventor 张滋婷韩月婷陈文兵
Owner 北京微未来科技有限公司
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