Sharma Vijay K
Pre-Harvest Food Safety and Enteric Diseases Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, P O Box 70, Ames, IA 50010, USA.
Mol Cell Probes. 2006 Oct;20(5):298-306. doi: 10.1016/j.mcp.2006.03.001. Epub 2006 Mar 22.
A real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) was developed for detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to rfbE and eae, respectively, were targeted for reverse transcription, amplification, and real-time detection. A single-step RT-PCR kit containing a mixture of reverse transcriptases converted mRNA into cDNA, which was subsequently amplified by Taq polymerase included in the same kit. The real-time detection of amplification products was achieved by incorporating rfbE(O157)- and eae(O157:H7)-specific TaqMan probes in rRT-MPCR. The ability of two sets of primers and probes for specific detection of rfbE(O157) and eae(O157:H7) was initially verified by screening RNA of eight E. coli serotypes possessing different O antigens and eae alleles. These two sets of primers and probes were also tested in a standard real-time PCR (rPCR) using DNA prepared from several E. coli and non-E. coli strains to verify that only rfbE(O157)- and eae(O157:H7)-specific sequences were amplified and detected. The rRT-MPCR was then evaluated for detecting low-level contamination of EHEC O157:H7 in feces. When RNA prepared from bovine feces, which were artificially seeded with EHEC O157:H7 cells and cultured for five hours, was tested in rRT-MPCR as low as 1cfu/g of feces could be detected. The detection range for the two genes in fecal cultures was 5.1 x 10(-1)-5.1 x 10(4) cfu/g of feces. Thus, the described procedure could be applied to rapid detection of very low levels of EHEC O157:H7 using total RNA as a template. Since the presence of rfbE(O157)- and eae(O157:H7)-specific mRNA is dependent on replicating cells, rRT-MPCR could provide important information about the viability of EHEC O157:H7 in feces.
开发了一种实时逆转录多重聚合酶链反应（rRT-MPCR），用于检测肠出血性大肠杆菌（EHEC）O157:H7的rfbE和eae基因编码的mRNA。分别针对rfbE和eae的129bp和106bp序列特异性片段进行逆转录、扩增和实时检测。一种包含逆转录酶混合物的一步式RT-PCR试剂盒将mRNA转化为cDNA，随后由同一试剂盒中包含的Taq聚合酶进行扩增。通过在rRT-MPCR中加入rfbE（O157）和eae（O157:H7）特异性TaqMan探针实现对扩增产物的实时检测。通过筛选具有不同O抗原和eae等位基因的8种大肠杆菌血清型的RNA，初步验证了两组引物和探针特异性检测rfbE（O157）和eae（O157:H7）的能力。这两组引物和探针也在标准实时PCR（rPCR）中进行了测试，使用从几种大肠杆菌和非大肠杆菌菌株制备的DNA，以验证只有rfbE（O157）和eae（O157:H7）特异性序列被扩增和检测到。然后对rRT-MPCR检测粪便中EHEC O157:H7低水平污染的能力进行评估。当用EHEC O157:H7细胞人工接种并培养5小时的牛粪制备的RNA在rRT-MPCR中进行检测时，粪便中低至1cfu/g的含量都能被检测到。粪便培养物中这两个基因的检测范围为5.1×10（-1）-5.1×10（4）cfu/g粪便。因此，所述方法可用于以总RNA为模板快速检测极低水平的EHEC O157:H7。由于rfbE（O157）和eae（O157:H7）特异性mRNA的存在依赖于复制细胞，rRT-MPCR可以提供有关粪便中EHEC O157:H7活力的重要信息。
