An Optimal Transport Medium for SARS-CoV-2 Detection in the Direct Method of Rapid Microfluidic PCR System

Background Recently developed rapid real-time reverse transcription PCR (RT-PCR) systems adopting microfluidic thermal cycling technology are ideal forpoint-of-care (POC) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because the RNA extraction step before real-time RT-PCR is rate-limiting, a direct RNA extraction method (direct method) that adopts chemical viral lysis and eliminates RNA purification steps is preferable for rapid real-time RT-PCR. In the direct method, selecting the transport medium is essential because it may be introduced into subsequent real-time RT-PCR steps, but might inhibit PCR. However, the influence of transport medium on the combination of the direct method and rapid realtime RT-PCR has been yet unstudied. In the present study, we examined the influence of various transport mediums when combining the direct method and rapid real-time RT-PCR of GeneSoC® (GeneSoC® RT-PCR), the recently developed compact PCR system that adapts novel microfluidic thermal cycling technology. Methods To explore the influence of the transport medium on the GeneSoC® RT-PCR, the concordance of the RNA extraction and direct method was evaluated in the clinical samples collected in viral transport medium (VTM) or eSwab®. The sensitivity of GeneSoC® RTPCR combined with the direct method was assessed using spiked samples in generic (H2O and PBS) or commercially available transport media (VTM and eSwab®). Analytical sensitivity was examined using clinical specimens collected from the VTM and eSwab®. The inhibitory effect of PCR inhibitors on clinical specimens was assessed using clinical samples diluted 1,000 times. Results While only 1 copy/reaction of RNA was detected in H2O and eSwab® of the spiked samples, a minimum of 5 copies/reaction was detected in PBS (-) and VTM. Among the clinical specimens tested using the direct method, the detection of viral RNA was unstable in the samples containing less than 100 copies/reaction viral RNA in VTM, whereas less than 10 copies/reaction viral RNA were detected in eSwab®. The positive, negative, and overall concordance between the RNA extraction and the direct method was 84%, 100%, and 85%, respectively, in eSwab® samples, whereas the values were 35%, 100%, and 38%, respectively, in VTM samples. When the clinical samples were diluted 1,000 times, GeneSoC® RT-PCR could detect as low as 1.15 copies/reaction RNA using direct method, and the sensitivity was comparable to that of RNA extraction. Conclusion The combination of the direct method and microfluidic rapid PCR machine GeneSoC® has a high sensitivity for detecting SARS-CoV-2 RNA in clinical samples with eSwab® transport medium.

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書誌情報

en : Yonago Acta Medica

巻 67, 号 4, p. 293-302, ページ数 10, 発行日 2024-11-27

出版者

Tottori University Medical Press

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ISSN

収録物識別子タイプ

PISSN

収録物識別子

05135710

ISSN

収録物識別子タイプ

EISSN

収録物識別子

13468049

書誌レコードID

収録物識別子タイプ

NCID

収録物識別子

AA00892882

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2025-01-22 00:06:09.691231

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