College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University, Daegu 41566, Republic of Korea.
Foot-and-Mouth Disease Research Division, Animal and Plant Quarantine Agency, Gimcheon, Gyeongsangbuk-do 39660, Republic of Korea.
J Virol Methods. 2018 Oct;260:6-13. doi: 10.1016/j.jviromet.2018.06.017. Epub 2018 Jun 30.
A sensitive and specific swarm primer-based reverse transcription loop-mediated isothermal amplification (sRT-LAMP) assay for the detection of serotype O foot-and-mouth disease virus (FMDV) was developed and evaluated. The assay specifically amplified the VP3 gene of serotype O FMDV, but did not amplify the VP3 gene of other serotype FMDVs or any other viruses. The limit of detection of the assay was 10 TCID/mL or 10 RNA copies/μL, which is 100 times lower than that of the RT-LAMP assay without swarm primers. The new assay is 10 times more sensitive than reverse transcription-polymerase chain reaction (RT-PCR) and is comparable to the sensitivity of real time RT-PCR (qRT-PCR). Evaluation of the assay using different serotypes of FMDV strains showed 100% agreement with the RT-PCR results. The previously reported serotype O FMDV-specific RT-LAMP assay did not detect 20 out of 22 strains of serotype O FMDVs, probably due to multiple mismatches between the primer and template sequences, showing that it is not suitable for detecting the serotype O FMDVs circulating in Pool 1 region countries, including Korea. In contrast, the developed sRT-LAMP assay with improved primers can rapidly and accurately diagnose serotype O FMDVs circulating in Pool 1 region countries and will be a useful alternative to RT-PCR and qRT-PCR.
一种基于敏感和特异的群体引物逆转录环介导等温扩增（sRT-LAMP）的方法被开发并评估，用于检测血清型 O 口蹄疫病毒（FMDV）。该方法特异性地扩增了血清型 O FMDV 的 VP3 基因，但不扩增其他血清型 FMDV 的 VP3 基因或任何其他病毒的 VP3 基因。该方法的检测限为 10 TCID/mL 或 10 RNA 拷贝/μL，比没有群体引物的 RT-LAMP 方法低 100 倍。该新方法比逆转录-聚合酶链反应（RT-PCR）灵敏 10 倍，与实时 RT-PCR（qRT-PCR）的灵敏度相当。使用不同血清型的 FMDV 株对该方法的评估表明，与 RT-PCR 结果完全一致。之前报道的血清型 O FMDV 特异性 RT-LAMP 方法未能检测到 22 株血清型 O FMDV 中的 20 株，可能是由于引物和模板序列之间存在多个错配，表明它不适合检测包括韩国在内的 1 区国家流行的血清型 O FMDV。相比之下，改进了引物的开发 sRT-LAMP 方法可以快速准确地诊断 1 区国家流行的血清型 O FMDV，将成为 RT-PCR 和 qRT-PCR 的有用替代方法。
