Method and kit for simultaneous detection of human coronavirus 229E, OC43 and NL63

A technology of human coronavirus and coronavirus, applied in the field of biotechnology applications, can solve the problems of difficult detection of trace nucleic acid and accurate quantification, long time-consuming, cumbersome operation, etc.

Inactive Publication Date: 2014-08-20
崔淑娟
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the classic method of detecting coronavirus at home and abroad is the isolation and cultivation of the virus, but its operation is cumbersome and time-consuming; electron microscopy, immunohistochemistry, EL1SA, conventional PCR, etc. have their own outstanding advantages, but it is difficult to detect trace amounts of nucleic acid and Accurate quantification, the real-time fluorescent quantitative PCR technology (Real-time fluorescent quan

Method used

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  • Method and kit for simultaneous detection of human coronavirus 229E, OC43 and NL63
  • Method and kit for simultaneous detection of human coronavirus 229E, OC43 and NL63
  • Method and kit for simultaneous detection of human coronavirus 229E, OC43 and NL63

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Embodiment 1

[0031] Embodiment 1. Human coronavirus 229E, OC43, NL63 corresponding N gene-specific primers and probe design and synthesis

[0032] 1.1 Selection of the corresponding target gene N gene and determination of the conserved region of representative strains of human coronavirus 229E, OC43, and NL63

[0033] The sequences used in this study were selected from NCBI GenBank (bttp: / / www.ncbi.nlm.nih.gov), the main selection basis is: (a) strains with a relatively recent age; (b) full-length sequences and main sequences (c) Select a representative strain after comparing the sequence with the subtype. The information of the corresponding N genes of the selected human coronavirus 229E, OC43, and NL63 representative strains is shown in Tables 1-1, 1-2, and 1-3.

[0034] In this study, the corresponding N genes of the selected representative strains of human coronavirus 229E, OC43, and NL63 were input into the DNAssist software for homologous comparison, and the sequence conserved regio...

Embodiment 2

[0047] Example 2. The establishment of a one-step multiple fluorescent RT-PCR detection system for human coronavirus 229E, OC43, and NL63

[0048] 2.1 According to Invitrogen's nucleic acid extraction kit ( Viral RNA Mini Kit) to extract sample nucleic acid.

[0049] 2.2 Preparation of human coronavirus 229E, OC43, NL63 one-step multiplex fluorescent RT-PCR detection system

[0050] Ambion’s AgPath-IDTM One-step RT-PCR Kit (P / N: 4387424) was used to prepare reagents, and the system was 50 μl:

[0051] 2×RT-PCR buffer 25μl

[0052] 25×RT-PCR Enzyme 2μl

[0053] Detection Enhancer 3μl

[0054] Primers and probes are added at the same time as shown in SEQ NO: 1, 2, 4, 5, 7, 8, 10, 11

[0055] 0.5 μl of each primer, the final primer concentration is 200nM; add such as SEQ NO: 3,

[0056] 0.5 μl each of the probe primers indicated in 6, 9, and 12, and the final concentration of the probe primers is 100 nM

[0057] Template 6 μ...

Embodiment 3

[0068] Example 3. Specific identification of human coronavirus 229E, OC43, NL63 one-step multiple fluorescent RT-PCR detection method

[0069] Utilize the multiplex fluorescent RT-PCR reaction system that embodiment 2 establishes respectively to human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, influenza A virus, influenza B virus, influenza C virus, parainfluenza The positive nucleic acids of virus, respiratory syncytial virus, enterovirus, adenovirus, and boca virus were detected, and the results showed that only human coronavirus 229E, OC43, and NL63 showed corresponding specificity in the detection channels of FAM, JOE, and ROX, respectively. There was no cross-reaction, but the other 8 viruses had no amplification curve except the internal quality control CY5 channel, and the other three channels had no amplification curve, indicating that the method has strong specificity (see Fig. 1).

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Abstract

The invention belongs to the field of biological technical application, and relates to a TaqMan probe based multiple fluorescent RT-PCR method for simultaneous detection of human coronavirus 229E, OC43 and NL63 and containing internal quality control. The invention designs specific primers and probed aiming at conserved sequences of N gene of human coronavirus representative strains 229E, OC43 and NL63, and establishes a one-step multiple fluorescent RT-PCR rapid detection method containing internal quality control; the method has simple and quick operations, overcomes the complexity of single hole single detection in a conventional single fluorescent RT-PCR method, simplifies the operation process, saves test cost, and provides a powerful technical support for on-site virus detection, health assessment and clinical diagnosis by using high specificity, sensitivity, efficiency and stability.

Description

technical field [0001] The invention relates to the application field of biotechnology, in particular to the simultaneous detection and identification of human coronavirus 229E, OC43 and NL63. Background technique [0002] Human coronaviruses (Human coronaviruses) belong to the Coronaviridae family and are a large family of viruses. They are named for the crown-like shape of virus particles under the electron microscope. The shape of coronavirus particles is round, oval or mildly pleomorphic, with a diameter of 100nm to 120nm, and its genome is a single-stranded positive-strand RNA, which is the largest of the currently known RNA viruses (the size is between 27kb and 32kb) ), the genome contains about 7 to 10 functional genes, 4 to 5 of which encode structural proteins, and from the 5' end to the 3' end are 5'-polymerase-HE-S-E-M-N-3'. Coronaviruses have strict host specificity and only infect natural hosts or closely related hosts. Coronavirus infections are widely distrib...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/686
Inventor 崔淑娟石伟先黄芳田丽丽张新陈萌孙玉兰王海滨张玉松
Owner 崔淑娟
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