Ram Siya, Shanker Rishi
Environmental Microbiology Division (Gheru Campus), Industrial Toxicology Research Center, P.O. Box 80, Mahatma Gandhi Marg, Lucknow-226001, India.
In Silico Biol. 2005;5(5-6):499-504.
Diarrheagenic E. coli strains contribute to water related diseases in urban and rural environment in developing and developed world. E. coli pathotype and pathogenicity varies due to complex multifactorial mechanism involving a large number of virulence factors. Rapid assessment of the virulence pattern of E. coli isolates is possible by Real-Time PCR probes like TaqMan. For designing TaqMan probes and primers for multiplex PCR selected E. coli gene sequences: stx1, stx2, hlyA, chuA, eae, lacZ, lamB and fimA were retrieved from NCBI's GenBank database. The alignment of the multiple sequences and analysis of conserved sequences was carried out using ClustalW and BLAST programs. The primers and Taqmen probes were designed using Beacon Designer software version 2.1 for two multiplexed PCR assays. In silico PCR simulation of these assays showed PCR products for stx2 (248bp) stx1 (102 bp), lacZ (228bp) and lamB (86 bp) in multiplex #1 and eae (200bp), chuA (147 bp), hlyA (141bp) and fimA (79 bp) in multiplex #2, respectively. These multiplexed PCR amplification products and probes can be used to identify and confirm presence of O157:H7/ H7-, O157:H43/45 and O26:H-/H11 serotypes. In conclusion, multiplex Real-Time Polymerase Chain Reaction oligomers and TaqMan probes designed and validated in silico will be helpful in management of water quality and outbreaks, by improving specificity and minimizing time needed for in vitro verification work.
致泻性大肠杆菌菌株在发展中国家和发达国家的城乡环境中导致与水相关的疾病。由于涉及大量毒力因子的复杂多因素机制，大肠杆菌的致病型和致病性各不相同。通过TaqMan等实时荧光定量PCR探针可以快速评估大肠杆菌分离株的毒力模式。为设计用于多重PCR的TaqMan探针和引物，从NCBI的GenBank数据库中检索了选定的大肠杆菌基因序列：stx1、stx2、hlyA、chuA、eae、lacZ、lamB和fimA。使用ClustalW和BLAST程序对多个序列进行比对并分析保守序列。使用Beacon Designer软件2.1版设计了用于两种多重PCR检测的引物和TaqMan探针。这些检测的计算机模拟PCR显示，多重检测1中stx2（248bp）、stx1（102bp）、lacZ（228bp）和lamB（86bp）以及多重检测2中eae（200bp）、chuA（147bp）、hlyA（141bp）和fimA（79bp）分别有PCR产物。这些多重PCR扩增产物和探针可用于鉴定和确认O157:H7/H7-、O157:H43/45和O26:H-/H11血清型的存在。总之，通过计算机设计和验证的多重实时聚合酶链反应寡聚物和TaqMan探针，将有助于水质管理和疫情爆发应对，提高特异性并减少体外验证工作所需的时间。
