Comparison of analytical sensitivity of SARS-CoV-2 molecular detection kits

Abstract

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a significant impact on global public health systems, making nucleic acid detection an important tool in epidemic prevention and control. Detection kits based on real-time reverse transcriptase PCR (rRT-PCR) have been used widely in clinics, but their analytical sensitivity (limit of detection, LOD) remains controversial. Moreover, there is limited research evaluating the analytical sensitivity of other molecular detection kits.

Methods: In this study, armored ribonucleic acid reference materials developed in-house were used to evaluate the analytical sensitivity of SARS-CoV-2 detection kits approved by the National Medical Products Administration. These were based on rRT-PCR and other molecular detection assays.

Results: The percentage retesting required with rRT-PCR kits was as follows: 0%, 7.69%, 15.38%, and 23.08% for samples with concentrations ranging from 50 000 to 781 copies/ml. In total, 93% of rRT-PCR kits had a LOD <1000 copies/ml. Only one kit had an LOD >1000 copies/ml. The LOD of other molecular detection kits ranged from 68 to 2264 copies/ml.

Conclusions: The study findings can help pharmaceutical companies optimize and improve detection kits, guide laboratories in selecting kits, and assist medical workers in their daily work.

Keywords: Analytical sensitivity; Limits of detection; Molecular detection; SARS-CoV-2; rRT-PCR.

Graphical abstract

Figure 1

Flow chart of the analytical sensitivity evaluation of the SARS-CoV-2 detection kits. (a) Preparation of the evaluation sample. (b) Evaluation of the LOD of the detection kits. (c) Statistical analysis. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; LOD, limit of detection; PEG, polyethylene glycol; VLP, virus-like particles; ddPCR, droplet digital PCR; qPCR, quantitative PCR.

Figure 2

Summary of the testing status of the SARS-CoV-2 rRT-PCR detection kits. (a) Retest rates of samples with different concentrations according to the rRT-PCR detection kits. (b) Measured LOD of each target gene and claimed LOD of each rRT-PCR kit. (c) Measured and claimed LODs of each rRT-PCR kit. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; LOD, limit of detection.

Figure 3

Estimated limit of detection (LOD) of the molecular detection assay kits using probit regression analysis. (a)–(m) Analysis graphs of probit regression for each of the 13 commercial detection kits investigated.

Figure 4

Estimated limit of detection (LOD) of the other detection assays using probit regression analysis. (a) Measured and claimed LODs of five other detection assay kits. (b)–(e) LOD obtained via probit regression analysis using 21 replicates of four-fold serially diluted samples.