Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19)

Abstract

The severe acute respiratory coronavirus 2 (SARS-CoV-2), which emerged in December 2019 in Wuhan, China, has spread rapidly to over a dozen countries. Especially, the spike of case numbers in South Korea sparks pandemic worries. This virus is reported to spread mainly through person-to-person contact via respiratory droplets generated by coughing and sneezing, or possibly through surface contaminated by people coughing or sneezing on them. More critically, there have been reports about the possibility of this virus to transmit even before a virus-carrying person to show symptoms. Therefore, a low-cost, easy-access protocol for early detection of this virus is desperately needed. Here, we have established a real-time reverse-transcription PCR (rtPCR)-based assay protocol composed of easy specimen self-collection from a subject via pharyngeal swab, Trizol-based RNA purification, and SYBR Green-based rtPCR. This protocol shows an accuracy and sensitivity limit of 1-10 virus particles as we tested with a known lentivirus. The cost for each sample is estimated to be less than 15 US dollars. Overall time it takes for an entire protocol is estimated to be less than 4 hours. We propose a cost-effective, quick-and-easy method for early detection of SARS-CoV-2 at any conventional Biosafety Level II laboratories that are equipped with a rtPCR machine. Our newly developed protocol should be helpful for a first-hand screening of the asymptomatic virus-carriers for further prevention of transmission and early intervention and treatment for the rapidly propagating virus.

Keywords: COVID-19; Communicable diseases; Coronavirus; Diagnostic techniques and procedures; Emerging; Infectious disease; SARS virus.

Fig. 1

Instructions for collecting a pharyngeal swab.

Fig. 2

Schematic diagram of the low-cost, laboratory-safe protocol for SARS-CoV2.

Fig. 3

Experimental scheme for human tissue sampling and total RNA extraction. (A) Each volunteer collects his/her tissue sample through pharyngeal swab, following the detailed procedures in Fig. 1. (B) The collected human sample on polyester swab was dissolved in DMEM and transferred to a Trizol containing tube. (C) Following the Trizol-based total RNA extraction, the RNA was further processed.

Fig. 4

Determination of detection efficiency and limit of rtPCR system using Lentivirus. (A) A standard calibration curve of Ct vs. genomic copy number was generated based on rtPCR results from LTR targeting primer set and known genomic copy number of LTR. (B) rtPCR results for RNA extraction by QIAmp kit or Trizol-based method. The known number of Lentivirus was tested with the two extraction methods and compared. (C) The average Ct values are compared between QIAmp and Trizol methods.

Fig. 5

The domain map of SARS-CoV-2 form the sequence obtained from the first patient in Republic of Korea. The location of each primer set is indicated at corresponding sequence. The primer set from CDC is targeting nucleocapsid protein gene (N) to detect SARS-CoV-2. In-house (IBS) designed primer sets for detecting SARS-CoV-2 target RNA-dependent RNA polymerase gene (RdRP), spike protein gene (S), envelope protein gene (E) and N.

Fig. 6

Unexpected amplification during rtPCR with CDC primer sets in no-template control condition. (A) Quantitative rtPCR result by using all primer sets in Table 1 in no-template control. All primers in Table 1 were tested, and the data were represented as black traces, except CDC_N2 and CDC_N3 primer sets. (B) Gel electrophoresis result confirming the amplification by CDC_N2 and CDC_N3 primer sets in no-template control.

Fig. 7

Representative quantitative rtPCR result from IBS-designed SARS-CoV-2 detecting primer sets in (A) SARS-CoV-2 as a positive control; (B) Volunteer 11; and (C) no template. Left panels represent SARS-CoV-2 specific amplifying results and right panels represent human internal control amplifying results.