Takata Miyako, Nakamoto Masaki, Kitaura Tsuyoshi, Okada Kensaku, Tsuneki-Tokunaga Akeno, Yamasaki Akira, Kageyama Seiji, Burioka Naoto, Chikumi Hiroki
Department of Pathobiological Science and Technology, Graduate School of Medical Science, School of Health Science, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan.
Division of Infectious Diseases, School of Medicine, Faculty of Medicine, Tottori university, Yonago 683-8503, Japan.
Yonago Acta Med. 2023 Apr 29;66(2):223-231. doi: 10.33160/yam.2023.05.007. eCollection 2023 May.
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.
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 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.
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).
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.
快速抗原检测广泛用于流感诊断。然而，尽管这些检测方法操作简单、周转时间短，但其灵敏度相对较低，因此人们正在寻求灵敏度更高的分子检测方法。在本研究中，我们开发并临床评估了一种使用基于微流控热循环技术的快速实时PCR系统GeneSoC对甲型和乙型流感进行快速多重检测的方案。
使用甲型/乙型流感病毒、人偏肺病毒和呼吸道合胞病毒的培养病毒株验证所开发检测方法的特异性。通过对经转录合成的系列稀释RNA以及从因上呼吸道和全身症状前来就医的连续患者采集的鼻咽拭子样本进行检测，评估分析灵敏度。通过对流感阳性临床标本进行平行检测，与传统实时逆转录PCR和快速抗原检测进行比较，对GeneSoC进行交叉验证。
GeneSoC检测在反应中分别以最低浓度38和65拷贝/微升检测到甲型和乙型流感的靶序列。对于临床标本分析，GeneSoC逆转录PCR与传统实时逆转录PCR之间的阳性、阴性和总体一致性在所有情况下均为100%，而在GeneSoC逆转录PCR与快速抗原检测的比较中，阳性、阴性和总体结果的一致性分别为100%、90.9%和95.7%。完成GeneSoC逆转录PCR的平均时间为16分29秒（95%置信区间，16分18秒至16分39秒）。
微流控实时PCR系统GeneSoC具有与传统实时逆转录PCR相当的分析性能，周转时间快，是用于诊断甲型和乙型流感的快速抗原检测的一种有前景的替代方法。
