Kongkasuriyachai Darin, Yongkiettrakul Suganya, Kiatpathomchai Wansika, Arunrut Narong
National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
Methods Mol Biol. 2017;1572:431-443. doi: 10.1007/978-1-4939-6911-1_28.
Loop-mediated isothermal amplification (LAMP) has been used to detect several pathogens including malaria parasites from field and clinical samples. In this protocol, the malaria LAMP technology is developed to differentiate between Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) species by targeting the dihydrofolate reductase thymidylate synthase (dhfr-ts) gene, a known target for the antifolate class of drugs such as Pyrimethamine. LAMP primer sets are designed and validated for species specific amplification. Additionally, specific probes help improve detection and visualization of the products when combined with lateral flow dipstick-based (LFD) detection. The protocols are further simplified to eliminate tedious sample preparation steps, such that crude lysis prepared simply by diluting few microliter (μL) of blood sample with distilled water is sufficient. The LAMP-LFD malaria dhfr-ts protocols are sensitive and can detect as little as 1 picogram (pg) of PfDNA and 1 nanogram (ng) of PvDNA, or a few microliters of crude lysate from infected blood samples (Yongkiettrakul et al., Parasitol Int 63: 777-784, 2014). These simplified steps not only reduce cost but also increase the potential for large application in the fields and clinical settings.
环介导等温扩增技术（LAMP）已被用于从野外和临床样本中检测包括疟原虫在内的多种病原体。在此方案中，开发了疟疾LAMP技术，通过靶向二氢叶酸还原酶胸苷酸合成酶（dhfr-ts）基因来区分恶性疟原虫（Pf）和间日疟原虫（Pv），该基因是乙胺嘧啶等抗叶酸类药物的已知靶点。设计并验证了用于物种特异性扩增的LAMP引物组。此外，当与基于侧向流动试纸条（LFD）的检测相结合时，特异性探针有助于提高产物的检测和可视化。该方案进一步简化以消除繁琐的样品制备步骤，以至于仅用蒸馏水稀释几微升血液样本制备的粗裂解物就足够了。LAMP-LFD疟疾dhfr-ts方案灵敏，能够检测低至1皮克（pg）的PfDNA和1纳克（ng）的PvDNA，或来自感染血液样本的几微升粗裂解物（Yongkiettrakul等人，《寄生虫学国际》63：777 - 784，2014）。这些简化步骤不仅降低了成本，还增加了在野外和临床环境中大规模应用的潜力。
