Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection

Abstract

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

Fig 1. The amplification plot of various sample buffers with DNA polymerases.

The amplification plots were obtained using MM4 in combination with K4pol_L329A (red, triangle), RTX (green, cross), or M1pol_Tth (blue, open circle). Here, 10⁴ copies of synthetic RNA were used as the template. The sample buffers used were as follows, A. Buffer 1, B. Buffer 2, C. Buffer 3, D. Buffer 4, and E. Buffer 5.

Fig 2. Analysis of the detection limit with each buffer.

The amplification plot of each buffer with MM4 andM1pol_Tth was obtained. Standard synthetic RNA was used as the template at 10⁵, 10⁴, 10³, 10², 10, and 5 copies per reaction. The sample buffers used were as follows, A. Buffer 1, B. Buffer 2, C. Buffer 3, D. Buffer 4, and E. Buffer 5.

Fig 3. Log-log plot analysis of the MoCO kit compared to the commercial kit.

A. The MoCO kit with the CDC N1 primers and probe set was compared to the Takara Kit with the CDC N1 and N2 primers and probes. B. The MoCO kit with the CDC N2 primers and probe set was compared to the Takara Kit with the CDC N1 and N2 primers and probes. C. The MoCO kit with the CDC N1 primers and probe set was compared to the MoCO kit with the CDC N2 primers and probe set (n = 213).