Ou Hong-Yu, Ju Cindy Teh Shuan, Thong Kwai-Lin, Ahmad Norazah, Deng Zixin, Barer Michael R, Rajakumar Kumar
Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai, China.
J Mol Diagn. 2007 Nov;9(5):624-30. doi: 10.2353/jmoldx.2007.070064.
The use of pathogen genome sequence data for the control and management of infections remains an ongoing challenge. We describe a broadly applicable, web-enabled approach that can be used to develop bacteria-specific polymerase chain reaction (PCR) assays. Salmonella enterica Paratyphi A has emerged as a major cause of enteric fever in Asia. Culture-based diagnosis is slow and frequently negative in patients with suspected typhoid and paratyphoid fever, potentially compromising patient management and public health. We used the MobilomeFINDER web-server to perform in silico subtractive hybridization, thus identifying 43 protein-coding sequences (CDSs) that were present in two Paratyphi A strains but not in other sequenced Salmonella genomes. After exclusion of 29 CDSs found to be variably present in Paratyphi A strains by microarray hybridization and grouping of remaining CDSs by genomic location, four dispersed targets (stkF, spa2473, spa2539, hsdM) were used to develop a highly discriminatory multiplex PCR assay. All 52 Paratyphi A strains within the diverse panel investigated produced one of two pathognomonic four-band signatures. Given rapid and ongoing expansion of DNA and comparative genomics databases, our universally accessible web-server-supported do-it-yourself approach offers the potential to contribute significantly to the rapid development of species-, serovar-, or pathotype-specific PCR assays targeting pre-existing and emerging bacterial pathogens.
利用病原体基因组序列数据进行感染的控制和管理仍然是一项持续存在的挑战。我们描述了一种广泛适用的、基于网络的方法，可用于开发针对细菌的聚合酶链反应（PCR）检测方法。甲型副伤寒沙门氏菌已成为亚洲肠热症的主要病因。基于培养的诊断方法速度慢，对于疑似伤寒和副伤寒热的患者检测结果常常为阴性，这可能会影响患者的治疗和公共卫生。我们使用MobilomeFINDER网络服务器进行电子减法杂交，从而鉴定出43个蛋白质编码序列（CDS），这些序列存在于两株甲型副伤寒沙门氏菌菌株中，但在其他已测序的沙门氏菌基因组中不存在。通过微阵列杂交排除在甲型副伤寒沙门氏菌菌株中发现的29个可变存在的CDS，并根据基因组位置对其余CDS进行分组后，使用四个分散的靶点（stkF、spa2473、spa2539、hsdM）开发了一种高分辨率多重PCR检测方法。在所研究的不同菌株组中的所有52株甲型副伤寒沙门氏菌菌株都产生了两种特征性四带信号之一。鉴于DNA和比较基因组学数据库的快速持续扩展，我们这种普遍可访问的、由网络服务器支持的自助式方法有潜力为针对现有和新出现的细菌病原体的物种、血清型或致病型特异性PCR检测方法的快速开发做出重大贡献。
