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Method for single-tube multiplex fluorescent polymerase chain reaction (PCR) detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS, and primers, probes and kit adopted by the method

A human coronavirus and detection kit technology, applied in the field of multiple fluorescent quantitative PCR detection, can solve the problems of simultaneous detection of human coronavirus, time-consuming and labor-consuming, cumbersome operation, etc., and achieve high sensitivity, simple and convenient operation, and low cost Effect

Inactive Publication Date: 2012-10-17
SUN YAT SEN UNIV
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AI Technical Summary

Problems solved by technology

[0005] However, the currently developed coronavirus typing reagents are basically single-plex fluorescent quantitative PCR detection reagents, which cannot simultaneously detect the five types of human coronavirus OC43, 229E, NL63, HKU1 and SARS; Carried out in 5 tubes, the operation is cumbersome, time-consuming and labor-intensive

Method used

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  • Method for single-tube multiplex fluorescent polymerase chain reaction (PCR) detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS, and primers, probes and kit adopted by the method
  • Method for single-tube multiplex fluorescent polymerase chain reaction (PCR) detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS, and primers, probes and kit adopted by the method
  • Method for single-tube multiplex fluorescent polymerase chain reaction (PCR) detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS, and primers, probes and kit adopted by the method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Primer, probe specific experiment

[0057] After using bioinformatics and biological software for designing primer probes to compare and analyze the sequence data of human coronavirus OC43, 229E, NL63, HKU1, and SARS in the existing database, the primers and probes shown in Table 1 were designed. Needle to realize one-tube multiplex fluorescent PCR detection of five types of human coronavirus OC43, 229E, NL63, HKU1 and SARS. Table 1 is the human coronavirus OC43, 229E, NL63, HKU1, SARS primer and probe sequences.

[0058] Table 1 Human coronavirus OC43, 229E, NL63, HKU1, SARS primer and probe sequences

[0059]

[0060] In order to verify the specificity of the designed primers and probes, a single-plex fluorescent PCR reaction was designed first, and the primers and probes were evaluated, that is, the specificity of the five sets of primers and probes for OC43, 229E, NL63, HKU1, and SARS were detected separately. sex. The experiment was carried out ...

Embodiment 2

[0066] Embodiment 2: the specificity experiment of multiple detection reagents of human coronavirus

[0067] Multiplex detection reagents using Fermentas' RevertAid TM First Strand cDNA Synthesis Kit' (cDNA First Strand Synthesis Kit) reverse-transcribes the RNA of coronavirus (OC43), coronavirus (229E), coronavirus (NL63), coronavirus (HKU1), and coronavirus (SARS) into cDNA , and then use the iQ Supermix in the 'Bio-Rad iQ TMSupermix kit' to prepare the reagents, prepare the primers and probes of the human coronavirus OC43, 229E, NL63, HKU1, and SARS in Table 1 into a tube of reaction solution, and prepare The reagent system is 18ul / reaction, the amount of each primer is 3-5pmol, the amount of each probe is 5-10pmol, 2ul is reserved for adding cDNA template, and the total reaction volume is 20ul.

[0068] Use single positive template (OC43, 229E, NL63, HKU1, SARS), double positive template (mixed OC43 and 229E, mixed OC43 and NL63, mixed OC43 and HKU1, mixed OC43 and SARS, ...

example 3

[0076] Example 3: Sensitivity experiment of human coronavirus multiplex detection reagent

[0077] In order to further test the sensitivity of the reagents, a 10-fold gradient dilution was performed on the positive samples of human coronavirus OC43, 229E, NL63, HKU1, and SARS. 105 times, compared with multiple human coronavirus detection reagents and single detection. Table 4 is the data table of the comparative experimental results of multiplex and single-weight human coronavirus quantitative PCR detection reagents for 10-fold serially diluted viruses

[0078] Table 4

[0079]

[0080]

[0081]From the above experimental results, the standard curves were made according to the multiples of dilution, and the respective linear relationships were all above 0.99, which was good. The multiple human coronavirus detection reagents can replace the single detection without affecting the experimental results.

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Abstract

The invention provides a method for single-tube multiplex fluorescent polymerase chain reaction (PCR) detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS. The method adopts primers having sequences of SEQ ID NO: 1-10, and probes having sequences of SEQ ID NO: 11-15. The invention also provides a kit for single-tube multiplex fluorescent PCR detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS. The kit contains the primers and the probes. The method provided by the invention adopts the primers which are specific primers of OC43, 229E, NL63, HKU1 and SARS, and the probes which are Taqman probes, utilizes TAMRA / CY5 / FAM / ROX / JOX multiple fluorescein labeling, realizes single-tube multiplex detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS, and has the advantages of strong specificity, high sensitivity, fast detection speed, simple and convenient operation and low cost. The primers and the probes can be used as detection reagents for a scientific research and clinical application.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a one-tube multiplex fluorescent quantitative PCR detection method for five types of human coronaviruses OC43, 229E, NL63, HKU1 and SARS, as well as primers, probes and kits thereof. Background technique [0002] Human coronavirus virus (HCOV) is a single-stranded positive-sense RNA virus belonging to the family Coronaviridae. Viruses are polymorphic and slightly spherical. There are three kinds of glycoproteins on the membrane surface: spike glycoprotein (S, Spike Protein), which is the receptor binding site, cytolysis and main antigenic site; small envelope glycoprotein (E, Envelope Protein) is smaller and bound to the envelope; Membrane glycoprotein (M, Membrane Protein) is responsible for the transmembrane transport of nutrients, the release of nascent virus budding and the formation of the outer envelope of the virus. [0003] Coronavirus is one of the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701
Inventor 曹开源黎孟枫徐霖吴珏珩张定梅许沙沙常彦敏郑芸何霞冯发深王铸关琳琳
Owner SUN YAT SEN UNIV
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