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COVID-19 detection kit

A COVID-19 and detection kit technology, which is applied in the field of COVID-19 detection kits, can solve the problems of cumbersome detection process, low library concentration, loss, etc., to shorten the detection time, overcome the large sample size and the effect of The effect of improving efficiency

Pending Publication Date: 2021-08-10
阿吉安(福州)基因医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the use of mNGS to detect COVID-19 has the following problems: 1. The abundance of pathogens in the sample is not high enough, the total amount of extraction and accounting is low, and the library cannot be built normally, which affects subsequent detection
2. COVID-19 is an RNA virus. When building a library for RNA, it needs to go through multiple steps such as high-temperature interruption, first-strand synthesis, second-strand synthesis, addition of adapters, and library amplification. Multiple purifications are required. A lot of target fragments are lost, resulting in a low concentration of the library, and there is a risk that it cannot be detected on the machine
3. Compared with RT-PCR, the detection process of mNGS is more cumbersome and the detection time is longer, which cannot meet the current demand for rapid detection of COVID-19

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A detection kit for COVID-19, including pretreatment reagents and library building reagents;

[0048] The pretreatment reagents include sputum liquefaction agents;

[0049] The reagents for building a library include reverse transcriptase and Tn5 transposase;

[0050] The reverse transcriptase is a mutant reverse transcriptase, and the mutation sites of the mutant reverse transcriptase are: P55I, D204E, T334S, E566Q, D587N and L607I;

[0051] The Tn5 transposase is a mutant Tn5 transposase, and the mutation sites of the mutant Tn5 transposase are: K54E, I64Y, K160R, K200R and S303G.

Embodiment 2

[0053] A detection kit for COVID-19, including pretreatment reagents and library building reagents;

[0054] The pretreatment reagents include sputum liquefaction agents;

[0055] The reagents for building a library include reverse transcriptase and Tn5 transposase;

[0056] The reverse transcriptase is a mutant reverse transcriptase, and the mutation sites of the mutant reverse transcriptase are: P55I, D204E, T334S, E566Q, D587N and L607I;

[0057] The Tn5 transposase is a mutant Tn5 transposase, and the mutation sites of the mutant Tn5 transposase are: K54E, I64Y, K160R, K200R and S303G;

[0058] The amino acid sequence of the reverse transcriptase is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse transcriptase is shown in SEQ ID NO.2;

[0059] The primers used for synthesizing the reverse transcriptase are as follows:

[0060] NZ55-F:tgattatcatcctgaaggc;

[0061] NZ55-R: gccttcaggatgataatca;

[0062] NZ204-F: gacccttttcgaagaggcc;

[0063] NZ204-R: gg...

Embodiment 3

[0085] A detection kit for COVID-19, including pretreatment reagents and library building reagents;

[0086] The pretreatment reagents include sputum liquefaction agents;

[0087] The library building reagents include reverse transcriptase, Tn5 transposase, reverse transcription buffer, Oligo (dT), random primers, Tn5 transposase reaction solution, primers, high-fidelity amplification reaction solution and high-fidelity amplification enzyme

[0088] The reverse transcriptase is a mutant reverse transcriptase, and the mutation sites of the mutant reverse transcriptase are: P55I, D204E, T334S, E566Q, D587N and L607I;

[0089] The Tn5 transposase is a mutant Tn5 transposase, and the mutation sites of the mutant Tn5 transposase are: K54E, I64Y, K160R, K200R and S303G;

[0090] The amino acid sequence of the reverse transcriptase is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse transcriptase is shown in SEQ ID NO.2;

[0091] The amino acid sequence of the Tn5 t...

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Abstract

The invention discloses a COVID-19 detection kit, and belongs to the technical field of biological detection. The detection kit comprises a pretreatment reagent and a library building reagent; the pretreatment reagent comprises a sputum liquefying agent; and the library building reagent comprises inverse mutant transcriptase and mutant Tn5 transposase. The COVID-19 detection kit provided by the invention comprises the sputum liquefying agent, the mutant reverse transcriptase and the mutant Tn5 transposase, when the kit is used for detecting COVID-19, the amount of nucleic acid required for library building is lower than that of a general commercially available kit, and the invention overcomes the problems that: the abundance of pathogens is generally not high enough in a COVID-19 sample, the total extraction accounting amount is low, and a database cannot be established normally; and moreover, the kit is used for detecting COVID-19, the time required by library building is short, the detection time is greatly shortened, and an accurate detection report can be given in a short time.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a detection kit for COVID-19. Background technique [0002] COVID-19 is an RNA virus. At present, no matter whether RT-PCR (reverse transcription PCR) or next-generation sequencing is used for detection, it is necessary to first reverse-transcribe its RNA into cDNA before it can be detected. Clinically, RT-PCR is often used to qualitatively analyze specific nucleic acid fragments to determine whether or not to be infected with COVID-19. The RT-PCR method requires sequence-specific primers to detect, but COVID-19 mutates very quickly as an RNA virus, and RT-PCR cannot detect the mutated COVID-19, so this method is not suitable for the study of COVID-19. Research on the origin, virulence and transmission of COVID-19. [0003] Metagenomics next-generation sequencing (mNGS) can directly perform high-throughput sequencing of nucleic acids in clinical samples without rel...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6806C12R1/93
CPCC12Q1/701C12Q1/6806
Inventor 薛怡婷郭文浒浦世珺杨露
Owner 阿吉安(福州)基因医学检验实验室有限公司
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