Kim Jong-Min, Kim Hye-Ryung, Baek Ji-Su, Kwon Oh-Kyu, Kang Hae-Eun, Shin Yeun-Kyung, Park Choi-Kyu
College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University, Daegu 41566, Republic of Korea.
DIVA Bio Incorporation, Daegu 41519, Republic of Korea.
Pathogens. 2023 Jul 8;12(7):921. doi: 10.3390/pathogens12070921.
Despite its many advantages, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay has yet to be developed for canine parainfluenza virus 5 (CPIV5). In this study, a visual RT-LAMP (vRT-LAMP) assay was developed for the rapid detection of CPIV5 in clinical samples. At a constant reaction temperature of 62 °C, the assay was completed within 40 min, and the results could be directly detected with the naked eye using a hydroxynaphthol blue (HNB) metal indicator without any additional detection apparatuses. The assay specifically amplified CPIV5 RNA with a limit of detection of 10 RNA copies/reaction, which was 10-fold more sensitive than the previously reported conventional reverse-transcription polymerase chain reaction (cRT-PCR) assay and was comparable to the previously reported real-time RT-PCR (qRT-PCR) assay. In a clinical evaluation using 267 nasopharyngeal swab samples collected from hospitalized dogs with respiratory symptoms, the CPIV5 detection rate using the vRT-LAMP assay was 5.24% (14/267), which was higher than that of the cRT-PCR assay (4.49%, 12/267) and consistent with that of the qRT-PCR assay, demonstrating 100% concordance with a kappa coefficient value (95% confidence interval) of 1 (1.00-1.00). The discrepancies in the results of the assays were confirmed to be attributed to the low sensitivity of the cRT-PCR assay. Owing to the advantages of a high specificity, rapidity, and simplicity, the developed vRT-LAMP assay using an HNB metal indicator will be a valuable diagnostic tool for the detection of CPIV5 in canine clinical samples, even in resource-limited laboratories.
尽管逆转录环介导等温扩增（RT-LAMP）检测法有诸多优点，但针对犬副流感病毒5型（CPIV5）的该检测法尚未开发出来。在本研究中，开发了一种可视化RT-LAMP（vRT-LAMP）检测法，用于快速检测临床样本中的CPIV5。在62℃的恒定反应温度下，该检测法在40分钟内完成，并且无需任何额外检测设备，使用羟基萘酚蓝（HNB）金属指示剂即可直接用肉眼检测结果。该检测法特异性扩增CPIV5 RNA，检测限为10个RNA拷贝/反应，比先前报道的传统逆转录聚合酶链反应（cRT-PCR）检测法灵敏10倍，与先前报道的实时RT-PCR（qRT-PCR）检测法相当。在一项临床评估中，使用从有呼吸道症状的住院犬采集的267份鼻咽拭子样本，vRT-LAMP检测法的CPIV5检出率为5.24%（14/267），高于cRT-PCR检测法（4.49%，12/267），且与qRT-PCR检测法一致，kappa系数值（95%置信区间）为1（1.00 - 1.00），表明二者完全一致。经证实，各检测法结果的差异归因于cRT-PCR检测法的低灵敏度。由于具有高特异性、快速性和简便性等优点，所开发的使用HNB金属指示剂的vRT-LAMP检测法将成为犬临床样本中CPIV5检测的有价值诊断工具，即使在资源有限的实验室也是如此。
