Rapid Multiplex RT-PCR for Influenza A and B by Genesoc®, a Microfluidic PCR System

Miyako Takata; Masaki Nakamoto; Tsuyoshi Kitaura; Kensaku Okada; Akeno Tsuneki-Tokunaga; Akira Yamasaki; Seiji Kageyama; Naoto Burioka; Hiroki Chikumi

doi:10.33160/yam.2023.05.007

Rapid Multiplex RT-PCR for Influenza A and B by Genesoc^®, a Microfluidic PCR System

Yonago Acta Med. 2023 Apr 29;66(2):223-231. doi: 10.33160/yam.2023.05.007. eCollection 2023 May.

Authors

Miyako Takata¹, Masaki Nakamoto², Tsuyoshi Kitaura², Kensaku Okada², Akeno Tsuneki-Tokunaga³, Akira Yamasaki⁴, Seiji Kageyama³, Naoto Burioka¹, Hiroki Chikumi²

Affiliations

¹ Department of Pathobiological Science and Technology, Graduate School of Medical Science, School of Health Science, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan.
² Division of Infectious Diseases, School of Medicine, Faculty of Medicine, Tottori university, Yonago 683-8503, Japan.
³ Division of Virology, Department of Microbiology and Immunology, School of Medicine, Faculty of Medicine, Tottori university, Yonago 683-8503, Japan.
⁴ Division of Respiratory medicine and Rheumatology, Department of Multidisciplinary Internal Medicine, School of Medicine, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan.

Abstract

Background: Rapid antigen tests are widely used to diagnose influenza. However, despite their simplicity and short turnover time, the sensitivity of these tests is relatively low, and molecular tests with greater sensitivity are being sought. In this study, we developed and clinically evaluated a protocol for the rapid multiplex testing of influenza A and B, using a rapid real-time PCR system, GeneSoC^®, that is based on microfluidic thermal cycling technology.

Methods: The specificity of the developed assay was validated using cultured viral strains of influenza A/B, human metapneumovirus, and respiratory syncytial virus. Analytical sensitivity was evaluated using serially diluted RNA synthesized via in vitro transcription and nasopharyngeal swab samples collected from consecutive patients seeking medical attention for a combination of upper respiratory and general symptoms. Cross-validation of GeneSoC^® based on comparisons with conventional real-time RT-PCR and rapid antigen tests was performed by parallel testing of influenza-positive clinical specimens.

Results: The GeneSoC^® assay detected the target sequences of influenza A and B at minimum concentrations of 38 and 65 copies/µL in reaction, respectively. For the analysis of clinical specimens, the positive, negative, and overall agreement between GeneSoC^® RT-PCR and a conventional real-time RT-PCR was in all cases 100%, whereas for the comparison between GeneSoC^® RT-PCR and the rapid antigen test, the agreements for positive, negative, and overall findings were 100%, 90.9%, and 95.7%, respectively. The mean time for completing GeneSoC^® RT-PCR was 16 min 29 s (95% confidence interval, 16 min 18 s to 16 min 39 s).

Conclusion: The microfluidic real-time PCR system, GeneSoC^®, has an analytical performance comparable to that of conventional real-time RT-PCR with rapid turnover time, and represents a promising alternative to rapid antigen tests for diagnosing influenza A and B.

Keywords: GeneSoC®; antigen detection test; influenza A; influenza B; realtime-PCR.