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Integrated molecular diagnosis system and application thereof in tracing animal source of beta coronavirus

A technology for detecting coronaviruses and animals, which is applied in the field of molecular biology and can solve problems such as fewer products

Pending Publication Date: 2020-08-14
泰州蕾灵百奥生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] my country is also continuing to develop and promote in this field, but currently there are few products on the market, especially the detection methods for tracing beta coronavirus need to be further strengthened

Method used

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  • Integrated molecular diagnosis system and application thereof in tracing animal source of beta coronavirus
  • Integrated molecular diagnosis system and application thereof in tracing animal source of beta coronavirus
  • Integrated molecular diagnosis system and application thereof in tracing animal source of beta coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Design of primers and probes

[0045] Coronaviruses are currently classified into four genera, alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses, replacing the traditional three-group classification, ie, classes 1 to 3. The betacoronavirus genus is further divided into lineages A to D. Human coronavirus OC43 (HCoVOC43) and human coronavirus HV1 (HCoV HKU1) belong to betacoronavirus lineage A; severe acute respiratory syndrome-associated virus (SARS-CoV) belongs to betacoronavirus lineage B; Middle East respiratory syndrome (MERS -CoV) belongs to beta coronavirus lineage C (Lau SK, Feng Y, Chen H, et al. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination. J Virol. 2015 Oct ; 89(20):10532-10547.). After analysis and research, it is concluded that the coronaviruses that can induce human infectious severe respiratory syndrom...

Embodiment 2

[0059] Embodiment two: optimum reaction condition and procedure

[0060] By analyzing the Mn in the reaction system 2+ The screening of different concentrations of ions, different final concentrations of each set of primers and probes SEQ ID NO.1-15 and the characteristics of DNA polymerase determine the best reaction premix and the best reaction program. The composition of the optimal reaction master mix is: RNA-direct realtime PCR master mix (1×, Toyobo Biotechnology Co., Ltd.), Mn 2+ 2.5mM, primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.10, SEQ ID NO.11 each 0.3μM; primers SEQ ID NO.7, SEQ ID NO. ID NO.8, SEQ ID NO.13, SEQ ID NO.14 each 0.2 μM; probes SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.12 each 0.2 μM, probes SEQ ID NO.9 , SEQ ID NO.15, 0.15 μM each. The optimal reaction program is: 90°C for 30s, 61°C for 20min; 95°C for 30s, 40 cycles (95°C for 0s, 60°C for 45s).

[0061] The standard positive plasmid was reacted with classical swine fever virus ...

Embodiment 3

[0062] Example 3: Determination of internal standard gene

[0063] By comparing the full-length 18S rRNA sequences of almost all available mammals and birds in GenBank (sequence numbers include: XR_004135034.1; NR_046261.1; XR_003587981.1; NR_046271.1; EU823286.1; 1; X00640.1; XR_002778881.1; NR_036642.1; XR_004246958.1; NR_146166.1; NR_145820.1; XR_004069086.1; NR_046237.1; FM165414.1; Evolution tree such as Figure 10 As shown, it was found that the identity of 18S rRNA sequences among the species human, pig, cow, sheep, horse, rabbit, chicken, duck, rat, guinea pig, macaque, gorilla, gibbon, camel, deer, bat, and whale ranged from 93.3 % to 99.9% (eg Figure 11 (as shown), indicating that the gene is conserved in mammals and birds, and can be used as an ideal internal standard gene. And further find out the conserved region of the gene through sequence comparison, and exclude the region with complex advanced structure in the sequence, select the primer design region used...

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Abstract

The invention provides a primer and probe combination for detecting beta coronavirus. The primer and probe combination comprises primers and probes aiming at a beta coronavirus A general system, a beta coronavirus B general system, a beta coronavirus C general system, a beta coronavirus C general system MERS and / or a beta coronavirus D general system. The invention also provides an application ofa 18S rRNA sequence as a reference gene in virus detection of mammals and poultry. The invention further provides a kit for integrally detecting the beta coronavirus in animals, and the kit comprisesa reaction solution packaged by the kit, and further comprises a cracking extracting solution and a cleaning solution. According to the invention, through the design of the primers and probes, animal-derived beta coronaviruses such as novel coronaviruses SARS-CoV-2, SARS viruses and MERS viruses can be simultaneously detected in one reaction, so that complete coverage of three major high-infectivity respiratory system pathogens is realized in one reaction, and the kit and the method have relatively high application value for tracing animal souces of the three major high-infectivity respiratorysystem pathogens.

Description

Technical field: [0001] The invention belongs to molecular biology, in particular to the detection of viruses, and more specifically to the detection of betacoronavirus traceability. Background technique: [0002] For the detection that the pathogen is a novel coronavirus (SARS-CoV-2), scientists have provided detection primers and probes (http: / / www.chinaivdc.cn / ). The epidemic is serious. Finding the source of infection and clarifying the route of transmission are important issues that all sectors of society pay attention to. Research by Shi Zhengli's team at the Wuhan Institute of Virology, Chinese Academy of Sciences showed that the sequence identity of the new coronavirus (SARS-CoV-2) with a coronavirus isolated from a bat was as high as 96% (Zhou P, Yang XL, WangXG, et al. Discovery of a novel coronavirus associated with the recentpneumonia outbreak in humans and its potential bat origin.bioRxiv 2020), the natural host can be basically locked in bats. However, it is ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/101
Inventor 王永强陈蕾
Owner 泰州蕾灵百奥生物科技有限公司
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