Fliss I, Blais B W, Holley R, Simard R E
Département de Sciences et Technologie des Aliments, Université Laval, Québec, Canada.
J Appl Bacteriol. 1995 Aug;79(2):195-202. doi: 10.1111/j.1365-2672.1995.tb00935.x.
A simple multiplex riboprobe system for detection of virulent Yersinia enterocolitica was developed using a pool of RNA probes specific for various chromosomal and plasmid-borne virulence gene sequences (yadA, virC, ail and yst). Riboprobes were synthesized by a rapid, simple and efficient technique involving in vitro transcription of polymerase chain reaction-generated templates incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the priming oligonucleotides. After dot blotting target DNA samples on nitrocellulose and hybridization with the riboprobes, the RNA: DNA hybrids formed were detected by a simple immunoenzymic assay involving sequential reactions with an anti-RNA : DNA hybrid monoclonal antibody, anti-mouse Ig-peroxidase conjugate and chromogenic or chemiluminescent enzyme substrate solution. This multiplex riboprobe system targeting both chromosomal and plasma-borne sequences permitted detection of virulent Y. enterocolitica, regardless of plasmid loss during handling of cultures, and was unreactive with a virulent Y. enterocolitica, other Yersinia and other bacteria. This system resulted in a significant improvement in the limit of detection in comparison to that obtained with individual probes.
利用一组针对各种染色体和质粒携带的毒力基因序列（yadA、virC、ail和yst）的RNA探针，开发了一种用于检测致病性小肠结肠炎耶尔森菌的简单多重核糖探针系统。核糖探针通过一种快速、简单且高效的技术合成，该技术涉及对聚合酶链反应生成的模板进行体外转录，其中一个引物寡核苷酸中包含噬菌体T7 RNA聚合酶启动子序列。将目标DNA样品点样到硝酸纤维素膜上并与核糖探针杂交后，通过一种简单的免疫酶测定法检测形成的RNA:DNA杂交体，该测定法包括与抗RNA:DNA杂交单克隆抗体、抗小鼠Ig-过氧化物酶偶联物和显色或化学发光酶底物溶液依次反应。这种针对染色体和质粒携带序列的多重核糖探针系统能够检测致病性小肠结肠炎耶尔森菌，而不管培养物处理过程中质粒是否丢失，并且与致病性小肠结肠炎耶尔森菌、其他耶尔森菌和其他细菌无反应。与使用单个探针相比，该系统显著提高了检测限。
