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Double fluorescent lyophilized microchip, kit and method for detecting novel coronavirus 2019-nCoV

A 2019-ncov, coronavirus technology, applied in the field of molecular biology detection of viruses, can solve the problems of long running time, easy pollution, long cycle, etc., to improve accuracy and reliability, high degree of automation, and prevent leakage. Check the effect

Active Publication Date: 2020-06-26
BEIJING YISEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high-throughput sequencing method takes too long due to the need for library construction and the long time on the machine
The first-generation sequencing method needs to be based on ordinary PCR. As a new type of molecular biology technology, ordinary PCR has been able to obtain a large number of target fragments through gene amplification in a short period of time due to its high specificity and sensitivity since its birth. , overcoming the shortcomings of traditional virus detection technology including the long cycle of virus isolation and identification test, providing a sensitive, fast and practical detection method for the early rapid detection of new coronavirus, but ordinary PCR still has cumbersome operation, long detection time and easy detection. Disadvantages such as pollution

Method used

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  • Double fluorescent lyophilized microchip, kit and method for detecting novel coronavirus 2019-nCoV
  • Double fluorescent lyophilized microchip, kit and method for detecting novel coronavirus 2019-nCoV
  • Double fluorescent lyophilized microchip, kit and method for detecting novel coronavirus 2019-nCoV

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0114] Experimental example 1 Specificity test of novel coronavirus (2019-nCoV) freeze-dried microchip fluorescent RT-PCR detection kit certificate

[0115] 1. Design primers and Taqman-MGB probes

[0116] According to the published novel coronavirus genes in China, find the specific conserved sequences of ORF 1a / b gene and N gene, and design multiple pairs of primers and probes. After comparison and screening, a set of optimal primers and Taqman-MGB probes were finally determined. The specific sequences are as follows:

[0117]ORF1a / b gene upstream primer: 5'-ATGTACGTGCATGGATTGGC-3',

[0118] ORF1a / b gene downstream primer: 5'-GGTGTATCAACATAACCTGTAGGTAC-3',

[0119] ORF1a / b gene Taqman probe:

[0120] 5'FAM-CCAATTTACCTTTACAGCTAG-MGB-3';

[0121] N gene upstream primer: 5'-TCTCCTGCTAGAATGGCTGG-3',

[0122] N gene downstream primer: 5'-TCAAGCAGCAGCAAAGCAAG-3',

[0123] N gene Taqman probe: 5'ROX-AATGGCGGTGATGCT-MGB-3'.

[0124] The 5' end of the Taqman probe of the O...

experiment example 2

[0137] Experimental example 2. New coronavirus 2019-nCoV freeze-dried microchip fluorescent RT-PCR detection kit and conventional commercially available Fluorescent RT-PCR reagents for sensitivity verification and comparison

[0138] Set the concentration to 1 x 10 8 Copies / mL of 2019-nCoV viral plasmids were serially diluted 10 times. take 10 3 Copies / mL of plasmid was diluted 2-fold. Take 1×10 8 copies / mL~10 3 Copies / mL, 500copies / mL, 250copies / mL The plasmid of 2019-nCoV virus in each gradient concentration was used as a template, and nuclease-free water was used as a negative control to perform freeze-dried microchip fluorescent RT-PCR reagents and conventional commercially available fluorescent RT-PCR reagents sensitivity test.

[0139] The freeze-dried microchip fluorescent RT-PCR system was prepared according to Example 1. Add 10× buffer dilution to 24 μL of nuclease-free water for shaking and mixing, then add 0.6 μL to each well, add 600 μL of mineral oil on t...

experiment example 3

[0147] Experimental Example 3: Preparation of novel coronavirus (2019-nCoV) freeze-dried microchip fluorescent RT-PCR detection kit and its detection

[0148] 1. Preparation of the kit:

[0149] Preparation of lyophilized microchips:

[0150] Prepare the freeze-dried microchip RT-PCR system according to the following reaction system:

[0151]

[0152]

[0153] The whole freeze-dried microchip loading hole of the AriaYSB microchip (Beijing Yisenbao Biotechnology Co., Ltd.) of fluorescence quantitative PCR is 30, and the above-mentioned fluorescence quantitative PCR system (comprising ORF 1a / b gene and N gene 2 sets of primer detection needle), add 1.2 μL to each microchip well.

[0154] The microchips coated with PCR reagents were frozen at -80°C for 1 h. The freeze-drying conditions of the equipment are as follows: in the pre-freezing stage, the temperature of the clapboard drops to -55°C, and the holding time is 1h during pre-freezing, and then the equipment is v...

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Abstract

The invention discloses a double fluorescent lyophilized microchip, a kit and a method for the novel coronavirus (2019-nCoV). The kit comprises the lyophilized microchip, a tube of mineral oil, a tubeof positive control, a tube of negative control, a tube of diluent and a tube of nuclease-free water, wherein the lyophilized microchip is coated with a primer, a probe, Taq enzyme, reverse transcriptase, trehalose, Tris-Cl, dNTP, Mg<2+>. According to the lyophilizing condition, the temperature of an insulator is reduced to negative 55 DEG C at a pre-freezing stage; pre-freezing is kept for 1 hour, and equipment is used for vacuumizing, and freeze-drying is kept for 1 hour; in an analytic drying stage, the temperature of the insulator is increased to negative 25 DEG C and kept for 1 hour, isincreased to 37 DEG C and kept for 2 hours and is reduced to 25 DEG C and kept for 1 hour to obtain the lyophilized microchip, and the diluent and sample nucleic acid are added during use. The doublefluorescent lyophilized microchip can be used for simultaneously detecting an ORF 1a / b gene and an N gene. The detection kit and detection method have high accuracy, high specificity, high sensitivityand short detection time.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection of viruses, in particular to a dual fluorescence freeze-dried microchip, kit and method for detecting novel coronavirus 2019-nCoV. Background technique [0002] Recently, an extremely serious new coronavirus infection broke out in the world. The causative virus is a new coronavirus, namely "2019-nCoV", which was named by the World Health Organization on January 12, 2020. The new coronavirus 2019-nCOV is a linear single-stranded RNA (ssRNA) virus with a genome length of about 29,903 nucleotides and a total of 10 genes. There are three main routes of transmission of the new coronavirus: airborne transmission through coughing or sneezing; close contact with sick people; touching contaminated surfaces and then touching the mouth, nose or eyes with dirty hands. Its infected population ranges from infants to the elderly over 80 years old. In addition, its infectivity is far higher ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2545/114C12Q2537/143C12Q2521/107C12Q2561/101Y02A50/30
Inventor 王新杰高姗姗孙晓明王真真胡祥钰
Owner BEIJING YISEN BIOTECH
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