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. 2022 Mar 7:10:837838.
doi: 10.3389/fbioe.2022.837838. eCollection 2022.

One-Step RT-qPCR for Viral RNA Detection Using Digital Analysis

Hyuna Park  1 , Wonjong Jung  2 , Hyeongseok Jang  2 , Kak Namkoong  2 , Kwon-Young Choi  1
Affiliations

One-Step RT-qPCR for Viral RNA Detection Using Digital Analysis

Hyuna Park et al. Front Bioeng Biotechnol. .

Abstract

The rapid detection of viruses is becoming increasingly important to prevent widespread infections. However, virus detection via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is time-consuming, as it involves independent nucleic acid extraction and complementary DNA synthesis. This process limits the potential for rapid diagnosis and mass analysis, which are necessary to curtail viral spread. In this study, a simple and rapid thermolysis method was developed to circumvent the need for extraction and purification of viral RNA. The developed protocol was applied to one-chip digital PCR (OCdPCR), which allowed thermolysis, RT, and digital PCR in a single unit comprising 20,000 chambers of sub-nanoliter volume. Two viruses such as tobacco mosaic virus and cucumber mosaic virus were tested as model viral particles. First, the temperature, exposure time, and template concentration were optimized against tobacco mosaic viral particles, and the most efficient conditions were identified as 85°C, 5 min, and 0.01 μg/nL with a cycle threshold of approximately 33. Finally, the OCdPCR analysis yielded 1,130.2 copies/µL using 10-2 μg/nL of viral particles in a 30 min thermolysis-RT reaction at 70°C. This novel protocol shows promise as a quick, accurate, and precise method for large-scale viral analysis in the future.

Keywords: RT-qPCR; multiplex detection; one-chip digital PCR; thermolysis; virus detection.

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Conflict of interest statement

WJ, HJ, and KN were employed by Samsung Electronics Co.Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Determination of CT values depending on various TMV template concentration. (A) RT-PCR curves with CT numbers. ∆Rn threshold was set as 0.8. (B) Determination of CT values at template TMV particle concentrations of 0.1 to 0.01 μg/nL. Viral RNA was extracted using a commercial RNA extraction kit, followed by conventional separate RT and PCR reactions. All data were averaged and deviated (bars = S.D., n ≥ 3).
FIGURE 2
FIGURE 2
Determination of CT values by varying TMV template concentration and thermolysis conditions. (A) Thermolysis-based RT-qPCR protocol optimization by varying thermolysis temperature and time. (B) RT-qPCR curve comparison between different RNA isolation methods (RNA isolation kit and thermolysis isolation). CT values were obtained with the ∆Rn threshold set as 0.8. (C) Summary of obtained CT results under different thermolysis conditions. CT values were obtained by thermolysis-based RNA isolation, followed by conventional separate RT and PCR reactions. All data were averaged and deviated (n ≥ 3).
FIGURE 3
FIGURE 3
(A) Determination of the lowest concentration for viral particle detection using optimized thermolysis conditions at 85°C for 5 min. A specifically designed primer set (see section 2.5 PCR conditions and detection of TMV and CMV) was used for cDNA synthesis, and both RT and PCR reactions were conducted by a one-step process in a single tube. Each CT value was determined against a series of viral particle concentrations of 10−2, 10−5, 10−10, 10−20, and 10−30 μg/nL (black bar). (B) RT-qPCR curves from one-step thermolysis. A viral concentration of 10−6 μg/nL was used as a template, and the PCR cycle was programmed by adding two steps: thermolysis for 5 min at 85°C and an RT reaction for 60 min at 50°C prior to the PCR amplification cycles. The obtained CT value was included as slashed bar in Figure 3A for a comparison. All data were averaged and deviated (bars = S.D., n ≥ 3).
FIGURE 4
FIGURE 4
Determination of CT values to reduce total operational time by reducing the difference between the thermolysis and RT temperatures at a viral particle concentration of 10−6 μg/nL. (A) TMV and (B) CMV (Black bar: 5 min, grey bar: 10 min). The thermolysis temperature varied from 50 to 90°C for five or 10 min, followed by RT reaction at same temperature. All data were averaged and deviated (bars = S.D., n ≥ 3).
FIGURE 5
FIGURE 5
Digital PCR analytical protocols and CMV detection results with chip images and fluorescence intensity distribution. (A) Overall digital PCR analysis scheme compared to conventional RT-qPCR analysis. (B) Photograph of the QuantStudio 3D digital PCR chip (10 × 10 × 0.3 mm3) with 20,000 nanoscale through-hole PCR wells, and optical microscope image of through-hole PCR wells. The diagonal length of through-hole is about 60 μm and the width of sidewall is about 18 μm. (C) Digital PCR chip analysis images. CMV concentrations of 0, 10−2, and 10−3 μg/nL. Fluorescence intensity distribution at each CMV template concentration. For RNA isolation, a commercial RNA extraction kit was used, and RT reactions were conducted at 70°C for 60 min. (D) One-chip digital PCR results of CMV detection with dPCR chip analysis images. Simultaneous thermolysis and RT reactions were conducted at 70°C for either 30 min or 5 min, and CMV concentration were set at either 10−2 μg/nL or 10−4 μg/nL.
FIGURE 6
FIGURE 6
Summary of the developed thermolysis-based one-step qPCR protocol and comparison of operation time. Numbers in each bar indicate each operational time in min. Details of operational time and CT values are listed in Table 4.
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