Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Mahidol Oxford Tropical Medicine Research Unit (MORU), Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
PLoS Negl Trop Dis. 2022 Aug 29;16(8):e0010659. doi: 10.1371/journal.pntd.0010659. eCollection 2022 Aug.
Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.
检测导致类鼻疽病的病原菌伯克霍尔德菌仍然是一项具有挑战性的任务，因为检测时间长、实验室要求高，而且许多当前检测方法的特异性和灵敏度都不高。在这项研究中，我们提出了一种新方法，通过利用 CRISPR-Cas12a 结合等温扩增技术，从临床分离物中识别伯克霍尔德菌 DNA，从而规避了这些问题。通过对保守的 CRISPR-Cas12a 靶位点进行计算机搜索，我们设计了 CRISPR-Cas12a 来包含一个针对伯克霍尔德菌的高度特异性间隔序列，命名为 crBP34。基于 crBP34 的检测方法可以检测到少至 40 个拷贝的伯克霍尔德菌基因组 DNA，同时能够区分其他测试的常见病原体。当与侧流纸条结合使用时，检测结果可以简单地读取，而不会降低灵敏度，并且不需要昂贵的设备。这种基于 crBP34 的检测方法为伯克霍尔德菌 DNA 提供了高灵敏度、特异性和简单的检测方法。直接在临床样本上使用该检测方法可能需要进一步优化，因为这些样本与高水平的人 DNA 复杂在一起。
