Aonuma Hiroka, Iizuka-Shiota Itoe, Hoshina Tokio, Tajima Shigeru, Kato Fumihiro, Hori Seiji, Saijo Masayuki, Kanuka Hirotaka
Department of Tropical Medicine, The Jikei University School of Medicine, Tokyo, Japan.
Center for Medical Entomology, The Jikei University School of Medicine, Tokyo, Japan.
Trop Med Health. 2020 Oct 21;48:87. doi: 10.1186/s41182-020-00274-z. eCollection 2020.
Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries.
Common primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present.
The RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.
监测寨卡病毒病在无疫情国家的传入情况以及在流行国家的传播情况对于避免全球大流行至关重要。然而，由于寨卡病毒基因组序列存在大量突变，检测该病毒存在困难。为开发一种快速、简便且准确性高的方法，采用逆转录环介导等温扩增（RT-LAMP）技术检测来自多个国家的寨卡病毒株。
基于两种标准寨卡病毒株的基因组序列设计了RT-LAMP通用引物：非洲系MR-766和亚洲系PRVABC59。使用筛选出的靶向NS3区域的引物组进行RT-LAMP反应，可检测到两种寨卡病毒株。最低检测量为3×10 ng病毒RNA。观察到不同毒株之间反应时间存在可测量的延迟。RT-LAMP方法从一名有加勒比群岛旅行史患者的尿液和血清中扩增出目标病毒序列，还能预测出存在哪种寨卡病毒株系。
使用优化良好的引物组的RT-LAMP方法对检测基因组RNA序列多样的寨卡病毒株具有高特异性和敏感性。结合等温条件下LAMP反应的简便性，本研究建立的优化引物组可能有助于对寨卡热患者进行快速、准确诊断并提供病毒株信息。
