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

IF 0.9 4区医学 Q4 MEDICINE, RESEARCH & EXPERIMENTAL Yonago acta medica Pub Date : 2023-05-01 DOI:10.33160/yam.2023.05.007

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

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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.

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Genesoc®微流控PCR系统对甲型和乙型流感病毒的快速多重RT-PCR检测。

背景:快速抗原检测被广泛用于流感的诊断。然而，尽管这些检测方法简单，周转时间短，但灵敏度相对较低，人们正在寻找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快速多重检测甲型和乙型流感的方案，使用基于微流控热循环技术的快速实时PCR系统GeneSoC®。方法:采用流感A/B、人偏肺病毒和呼吸道合胞病毒培养的病毒株验证该方法的特异性。通过体外转录合成的连续稀释RNA和从因上呼吸道和一般症状合并就诊的连续患者收集的鼻咽拭子样本，评估分析灵敏度。通过对流感阳性临床标本进行平行检测，将GeneSoC®与传统实时RT-PCR和快速抗原检测相比较，进行交叉验证。结果:GeneSoC®检测分别在最低浓度为38拷贝/µL和65拷贝/µL时检测到甲型流感和乙型流感的目标序列。对于临床标本的分析，GeneSoC®RT-PCR与传统实时RT-PCR之间的阳性、阴性和总体一致性在所有情况下均为100%，而GeneSoC®RT-PCR与快速抗原检测之间的比较，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®RT-PCR的平均时间为16 min 29 s(95%置信区间为16 min 18 s ~ 16 min 39 s)。结论:微流控实时PCR系统GeneSoC®具有与传统实时RT-PCR相当的分析性能，周转时间快，是诊断甲型和乙型流感的快速抗原检测的有希望的替代方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Yonago acta medica MEDICINE, RESEARCH & EXPERIMENTAL-

CiteScore

1.60

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： Yonago Acta Medica (YAM) is an electronic journal specializing in medical sciences, published by Tottori University Medical Press, 86 Nishi-cho, Yonago 683-8503, Japan. The subject areas cover the following: molecular/cell biology; biochemistry; basic medicine; clinical medicine; veterinary medicine; clinical nutrition and food sciences; medical engineering; nursing sciences; laboratory medicine; clinical psychology; medical education. Basically, contributors are limited to members of Tottori University and Tottori University Hospital. Researchers outside the above-mentioned university community may also submit papers on the recommendation of a professor, an associate professor, or a junior associate professor at this university community. Articles are classified into four categories: review articles, original articles, patient reports, and short communications.