Droplet-type digital PCR kit for COVID-19 nucleic acid test and application of kit

Abstract

The invention provides a droplet-type digital PCR kit for COVID-19 nucleic acid test and an application of the kit. A reaction solution in the kit contains primers and a probe for detecting a COVID-19ORF1ab gene and a COVID-19 N gene, and primers and a probe for detecting human RNA reference genes, and two ends of each probe are labeled with fluorescent groups. The kit fully utilizes the characteristic of a small sample volume of microfluidic treatment, and realizes rapid heating and cooling of a sample, the maximum heating and cooling rate of the system can reach 10 DEG C / s, at the same time, the detection sensitivity to COVID-19 is 1 to 2 orders of magnitude higher than that of a traditional qPCR, and under the condition of a low virus concentration, the detection repeatability is better than that of the traditional qPCR.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a droplet digital PCR kit for nucleic acid detection of novel coronavirus and its application. Background technique [0002] Coronavirus is a type of RNA virus with an envelope and a linear single-stranded positive strand genome, with a diameter of about 80-120nm. Currently, it only infects humans, mice, pigs, cats, dogs, and poultry vertebrates. The novel coronavirus (COVID-19) has been confirmed as a new variant, which can cause viral pneumonia, with fever and dry cough in mild cases, dyspnea and even shock in severe cases. The method widely used in the market to detect the current novel coronavirus (COVID-19) is the method of nucleic acid extraction and purification followed by quantitative PCR. Its detection sensitivity is limited, and it is easily inhibited by different chemical substances from the patient's body in the sample, thereby affecting the dete...

AI Technical Summary

Problems solved by technology

Such a system has the following disadvantages: 1) The user needs to manually transfer the droplets to a 96-well plate after the system has generated the droplets to perform the next PCR reaction, which will cause loss or rupture of the droplets; 2 ) Due to the limitation of the heat transfer rate (the sample volume in the test tube is thicker, and the heating starts from the outside of the test tube), the reaction speed of PCR in the test tube is relatively slow, so the entire PCR process takes a long time (150 minutes)

[0004] Problems in the current clinical detection process of the new coronavirus: 1) COVID-19 patients with clinical symptoms and imaging studies are highly suspected, but the nucleic acid test is negative; 2) Some patients need to repeat several tests to determine the nucleic acid The test results are negative and positive; 3) Some patients after treatment, the viral load in the body has dropped significantly, but has the virus in the body been completely cleared, and can they be discharged from the hospital? The main reasons are that RT-PCR is not sensitive enough for low-load virus detection, difficult sampling sites for patients, and some viral RNAs are lost during extraction and transportation.

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