Application experience of a rapid nucleic acid detection system for COVID-19

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging worldwide. The COVID-19 outbreak caused severe threats to the life and health of all humans caused by SARS-CoV-2. Clinically, there is an urgent need for an in vitro diagnostic product to detect SARS-CoV-2 nucleic acid quickly. Under this background, commercial SARS-CoV-2 nucleic acid POCT products came into being. However, how to choose these products and how to use these products in a standardized way have brought new puzzles to clinical laboratories. This paper focuses on evaluating the performance of these commercial SARS-CoV-2 nucleic acid POCT products and helps the laboratory make the correct choice. At the same time, to standardize the use of this kind of product, this paper also puts forward corresponding suggestions from six elements of total quality management, namely, human, machine, material, method, environment, and measurement. In addition, this paper also puts forward some ideas on the future development direction of POCT products.

Keywords: COVID-19; Point-of-care testing; Quality control; SARS-CoV-2.

Fig. 1

Common detection principles of point-of-care testing in SARS-CoV-2. Since nucleic acid POCT technology integrates nucleic acid extraction, amplification, and detection and automatically completes detection and result analysis, it is more rapid than classic PCR. The principle of RT–PCR is that reverse transcriptase is used to convert RNA into its complementary cDNA, the specific region of the cDNA is amplified by polymerase chain reaction (A); LAMP-PCR first reverse transcribes the RNA genome of SARS-CoV-2 into cDNA, then designs four specially designed primers that can bind to six different regions of the target genome, and uses DNA polymerase with strand displacement activity instead of thermal denaturation to generate a single-stranded template (B); RPA-PCR mainly depends on three enzymes: recombinase, which can bind single-stranded nucleic acids, single-stranded DNA binding protein (SSB), and strand displacement DNA polymerase, is also an isothermal amplification method of nucleic acids (C); NEAR-PCR is driven by reverse transcriptase, nicking enzymes, and isothermal amplification DNA polymerase. The template hybridizes with the primer, the extension product is replaced by the next template, the complementary strand of the replacement product is extended to form the nicking enzyme recognition site, and the nicking enzyme recognizes and cuts the specific short sequence of one strand in the double-stranded DNA to form a gap. Constant temperature-amplified DNA polymerase extends nucleotides from the 3′ end of primers to synthesize short sequences, thus obtaining double-stranded NEAR amplification. The target DNA template is continuously amplified through cutting and extension cycles, and the molecular beacon is designed to generate fluorescent signals for quantification (D).

Fig. 2

Total quality management of point-of-care testing in SARS-CoV-2.