Viral nucleic acid extraction or preservation reagents, primer probe combinations, virus amplification reagents, kits and their applications

Abstract

The invention relates to the field of virus detection, in particular to virus nucleic acid extraction or preservation reagents, primer probe combinations, virus amplification reagents, kits and applications thereof. The virus detection device of the present invention provides a simple and easy virus nucleic acid extraction method, and the whole process takes about 5-15 minutes to recover and purify the nucleic acid, which can be used for the detection of the virus nucleic acid. Including PCR, NASBA, LAMP, RPA, etc. Compared with the traditional virus extraction method, the method has a high recovery rate of viral nucleic acid, takes less time, is convenient to operate, and is easy to promote clinically. The invention relates to isothermal amplification primers, probe combination sequences and reaction buffer for simultaneous detection of novel coronavirus COVID‑19 N and ORF genes and human reference genes in a single tube. The system has good specificity and high sensitivity (50cp / mL) , high specificity, only 20 minutes of detection time, the fastest can report positive in about 10 minutes.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to virus nucleic acid extraction or preservation reagents, primer probe combinations, virus amplification reagents, kits and applications thereof. Background technique [0002] The new coronavirus is the third to appear in humans in the past 20 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) that broke out in 2002 and the Middle East respiratory syndrome coronavirus (MERS-CoV) that broke out in 2012 Highly pathogenic coronavirus. Nucleic acid detection is a basis for pathogen diagnosis, and it is also an important basis for the current diagnosis of new crown infection. Therefore, carrying out nucleic acid virus detection is also an important means for virus infection screening and virus disease prevention and control. In the process of clinical and practical application, not only the specificity, sensitivity and accuracy of the virus detection method have hi...

AI Technical Summary

Problems solved by technology

[0005] (1) At present, the commonly used nucleic acid extraction technology has complex extraction process, long time and low nucleic acid extraction efficiency, which cannot meet the needs of clinical testing; commonly used viral nucleic acid extraction and purification technologies include Trizol method, spin column method, magnetic bead method, etc.

The Trizol method separates proteins and nucleic acids by using the difference in the distribution positions of DNA, RNA, and proteins in the organic layer and the aqueous layer. In the process of obtaining nucleic acids, phenol, chloroform, isoamyl alcohol, isopropanol, and ethanol are used, which are harmful to the human body. The volatile organic solvents present great health hazards to operators, and the Trizol method for nucleic acid extraction is cumbersome, and the reproducibility of different sample extraction results is not good. It takes at least 1.5 hours from sample lysis to nucleic acid acquisition. Time-consuming, not suitable for clinical testing

Conventional centrifugal adsorption column and magnetic bead method need to use ethanol or isopropanol for binding when extracting nucleic acid, and need to add ethanol for cleaning. This method of adding alcohol for cleaning makes the operation of nucleic acid extraction reagents relatively complicated and time-consuming. , if ethanol is not removed cleanly, it will also inhibit the subsequent reaction

[0006] (2) The amplification efficiency of the isothermal amplification system is extremely high. During the amplification process of the target, unnecessary non-specific amplification is inevitably introduced, making it difficult to judge the result. For non-specific amplification, non-specific fluorescent dyes The incorporation method can't do anything about the specificity of the product, and further analysis of the product is required, such as cas enzyme digestion identification, etc.

However, it involves opening the cover, which can easily cause environmental pollution such as aerosol pollution, which is not conducive to clinical testing.

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