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Novel coronavirus detection kit and method based on amplicon capture

A detection kit and coronavirus technology, which is applied in biochemical equipment and methods, microbe measurement/inspection, and resistance to vector-borne diseases. It can solve the problems of low sensitivity and missed detection of mutant strains, and achieve easy operation. Fast, uniform regional amplification, and shortened isolation time

Pending Publication Date: 2022-05-27
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a novel coronavirus detection reagent based on amplicon capture in order to solve the problems of low sensitivity and easy missed detection of mutant strains in the existing novel coronavirus detection methods box and method

Method used

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  • Novel coronavirus detection kit and method based on amplicon capture
  • Novel coronavirus detection kit and method based on amplicon capture
  • Novel coronavirus detection kit and method based on amplicon capture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] In the P3 laboratory of a disease control center, the sputum samples that are positive for viral nucleic acid detection are tested using the kit provided by the present invention.

[0117] 1. Experimental process:

[0118] (1) Sample pretreatment: For sputum samples Cov8041 (ct value 30) and Cov5118 (ct value 29): take 300ul sample and add 300ul sputum digestive juice, and incubate at 37°C for 30 minutes after mixing to make the sputum sample Homogenize by vortexing every 10 minutes.

[0119] (2) Nucleic acid extraction: Mix the pretreated samples and put them into the module of the ultrasonic breaker (Jingxin), set the ultrasonic frequency to 500w and the time to 30 minutes. The sonication steps were then run: 4 degrees with 1 minute intervals for every 10 minutes of operation. Then the sample was transferred to a grinding tube pre-packed with glass beads (0.1 mm & 0.5 mm), the tube cap was tightened and then fixed on the adapter of the grinding apparatus (Jingxin), ...

Embodiment 2

[0135] We used this kit to dilute 10-fold and 100-fold samples (COV-XS) with a Ct value of 32 determined by a common clinical detection kit (labeled as COV-XS-1, COV-XS-10, COV-XS), respectively. XS-100), to perform a concentration gradient detection,

[0136] The detection process is the same as the first embodiment,

[0137] Library Qualification and Mixing

[0138] Take 5ul of the prepared library and use the Caliper LabChip GX instrument for quality inspection. The normal library fragment size is about 300bp, and there are no detailed dimer and non-specific bands. Then use the Qubit4.0 fluorometer to accurately quantify the library concentration, and mix the library according to the quantitative results of the library.

[0139] The number of copies of the new coronavirus contained in the sample is calculated as follows:

[0140]

[0141] It shows that the detection limit of the kit provided by the present invention can reach 2 copies, which is the lowest among all si...

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Abstract

The invention discloses a novel coronavirus detection kit and method based on amplicon capture. The kit comprises the following components: 1) a primer library for detection; 2) a pair of barcode amplification primers; 3) identifying a diagnosis primer library; (4) an IVC-COV control system is adopted; (5) amplifying the premixed solution; (6) preparing a premixed solution used during library construction; and 7) obtaining analysis software of the data. The detection method comprises the following steps: step 1, sample treatment and nucleic acid extraction; 2, preparing a reverse transcription reaction system; step 3, reverse transcription reaction; step 4, preparing a primary amplification system; 5, carrying out primary amplification; step 6, first area analysis of products; step 7, preparing a secondary PCR system; step 8, carrying out secondary amplification; step 9, purifying a secondary PCR product; step 10, library quality inspection and electrophoresis quality inspection; and step 11, sequencing requirement and data volume. The method has the advantages that virus particles in a detection sample are directly obtained, and differential diagnosis is achieved.

Description

technical field [0001] The present invention relates to a virus detection kit and method, in particular to a novel coronavirus detection kit and method based on amplicon capture. Background technique [0002] Currently, novel coronavirus pneumonia (COVID-19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleic acid testing is by far the most sensitive method for detecting viruses. Ultra-high-sensitivity nucleic acid detection methods are an important line of defense to filter the source of new coronary pneumonia infection and completely block the spread of imported viruses from abroad. All current detection methods are limited by the amount of nucleic acid that can be effectively detected. For those patients with early infection or asymptomatic infection, the virus level in their samples may be lower, resulting in false negative results of the test, resulting in clinical diagnosis and disease. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6869
CPCC12Q1/701C12Q1/6869C12Q2531/113C12Q2521/107C12Q2535/122C12Q2537/165Y02A50/30
Inventor 崔银秋于湘晖吴东林谭行之
Owner JILIN UNIV
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