specific and sensitive detection of h. pylori in biological specimens by real-time rt-pcr and in situ hybridization特异、敏感检测幽门螺杆菌在生物标本的实时rt - pcr和原位杂交.pdf

Specific and Sensitive Detection of H. pylori in Biological Specimens by Real-Time RT-PCR and In Situ Hybridization Hui Liu, Arifur Rahman, Cristina Semino-Mora, Sonia Q. Doi, Andre Dubois* Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America Abstract PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this b