Development of an automatic integrated gene detection system for novel severe acute respiratory syndrome-related coronavirus (SARS-CoV2)

Abstract

In December 2019, Wuhan, China suffered a serious outbreak of a novel coronavirus infectious disease (COVID) caused by novel severe acute respiratory syndrome-related coronavirus (SARS-CoV 2). To quickly identify the pathogen, we designed and screened primer sets, and established a sensitive and specific qRT-PCR assay for SARS-CoV 2; the lower limit of detection (LOD) was 14.8 (95% CI: 9.8-21) copies per reaction. We combined this qRT-PCR assay with an automatic integration system for nucleic acid extraction and amplification, thereby establishing an automatic integrated gene detection system (AIGS) for SARS-CoV 2. Cross reactive analysis performed in 20 other respiratory viruses and 37 nasopharyngeal swabs confirmed a 100% specificity of the assay. Using two fold diluted SARS-CoV 2 culture, the LOD of AIGS was confirmed to be 365 copies/ml (95% CI: 351-375), which was Comparable to that of conventional q RT-PCR (740 copies/ml, 95% CI: 689-750). Clinical performances of AIGS assay were assessed in 266 suspected COVID-19 clinical respiratory tract samples tested in parallel with a commercial kit. The clinical sensitivity of the AIGS test was 97.62% (95% CI: 0.9320-0.9951) based on the commercial kit test result, and concordance analysis showed a high agreement in SARS-CoV-2 detection between the two assays, Pearson R was 0.9623 (95% CI: 0.9523-0.9703). The results indicated that this AIGS could be used for rapid detection of SARS-CoV 2. With the advantage of simple operation and less time consuming, AIGS could be suitable for SARS-CoV2 detection in primary medical institutions, thus would do a great help to improve detection efficiency and control the spread of COVID-19.

Keywords: COVID-19; SARS-CoV2; automatic integrated gene detection system; qRT-PCR; rapid detection.

Figure 1.

Schematic of AIGS cartridge. The cartridge consists of the lysis area, washing area 1, washing area 2, and PCR amplification area; adjacent areas are separated by silicone oil and a plunger seal. The virus was lysed with detergent in the lysis area, and DNA/RNA bound to the magnetic beads under the high-salt conditions in the lysate. The magnet inside the instrument attracted the magnetic beads and pulled them into washing areas 1 and 2 for RNA/DNA extraction. Finally, the magnetic beads were dragged into the PCR amplification area for nucleic acids amplification and detection.

Figure 2.

AIGS flow chart. A: add 10ul magnetic beads; B: add 200ul sample to each cartriadge; C: Inserting the cartridge into detection site; D: result analysis. Thermal cycling conditions were as follows: reverse transcription, 56°C for15 min; initial denaturation, 94°C for1 min; and 40 cycles of 95°C for 10 s and 58°C for 30 s. The amplification curve was displayed on the screen in real time while the amplification programme ran.

Figure 3.

LOD test by conventional qRT-PCR and AIGS. A: Series of two fold diluted T7 transcripted RNA containing specific target genes were applied for conventional qRT-PCR assay. B: Series of two fold diluted RNA from cultured SARS-CoV 2 culture were applied for conventional qRT-PCR assay. C: Series of two fold diluted SARS-CoV 2 culture were applied for AIGS assay. The x-axis shows input RNA copies per reaction or virus per microtiter. The y-axis shows positive results in all parallel reactions performed, squares are experimental data points resulting from replicate testing of given concentrations (x-axis) in parallels assays (eight replicate reactions per point). Limits of detection are given in the panels headings. The inner line is a probit curve (dose-response rule). The outer dotted lines are 95% Confidence Interval.