基于 SYBR Green I 的闭管环介导等温扩增（LAMP）检测方法用于已知利什曼原虫疟疾诊断的验证。
Validation of SYBR green I based closed-tube loop-mediated isothermal amplification (LAMP) assay for diagnosis of knowlesi malaria.
机构信息
Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia.
Sarawak State Health Department, Jalan Diplomatik, Off Jalan Bako, Kuching, Sarawak, 93050, Malaysia.
出版信息
Malar J. 2021 Mar 25;20(1):166. doi: 10.1186/s12936-021-03707-0.
BACKGROUND
As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi.
METHODS
A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes.
RESULTS
LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2-99.7%) and 100% specific (95% CI 83.2-100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange.
CONCLUSIONS
These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.
背景
作为 PCR 方法的替代方法，LAMP 在分子诊断领域的应用越来越多。在 65°C 的等温条件下，整个过程大约需要 30 分钟完成。在本研究中，我们建立了一种在封闭管系统中用于检测疟原虫 knowlesi 的敏感可视化 LAMP 方法。
方法
从砂拉越州卫生部获得了 71 份采集于血斑的疟疾显微镜阳性血液样本。使用 18s rRNA 作为靶基因，在提取 DNA 后进行巢式 PCR 和 SYBR 绿 I LAMP 检测。通过肉眼观察 LAMP 终产物的颜色变化。
结果
与 100 拷贝/µL 的巢式 PCR 相比，LAMP 检测的下限为 10 拷贝/µL。在采集的 71 份疟原虫 knowlesi 血液样本中，LAMP 检测到 69 份显微镜阳性样本。LAMP 比巢式 PCR 检测具有更高的灵敏度。SYBR 绿 I LAMP 检测的灵敏度为 97.1%（95%CI 90.2-99.7%），特异性为 100%（95%CI 83.2-100%）。在不打开盖子的情况下，将 SYBR 绿 I 掺入管内帽中，可在扩增完成后直接进行结果的可视化。阳性样本立即变为绿色，而阴性样本仍为橙色。
结论
这些结果表明，SYBR 绿 I LAMP 检测法是一种在偏远地区检测疟原虫 knowlesi 的便捷诊断工具。
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