פיתוח תבחינים מולקולתיים מהירים לזיהוי מחוללי תחלואת מעיים

תקציר

Development of Molecular Tests for Rapid Detection of Enteropathogens 

Miri Yavzori, Nir Uriel, Nurith Porat, Ron Dagan, Ruhama Ambar, Ofer Shpilberg, Dani Cohen

Army Health Branch Research Unit, Medical Corps, Israel Defense Forces; Pediatric Infectious Disease Unit, Soroka University Medical Center and Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheba; and Sackler Faculty of Medicine, Tel Aviv University

Amplification of specific DNA sequences by polymerase chain reaction (PCR), enables rapid, sensitive and direct, specific identification of pathogens at very low concentrations in clinical samples. Studies in recent years have reported identification of several enteropathogens directly from stool samples by PCR. The amplification process includes the use of primers complementary to the DNA sequences specific to the pathogen, thus relying on the pathogen's genotype, rather than its phenotype on which identification by the methods of classical microbiology were based.

We have developed PCR protocols for the differential identification of enteropathogens resembling the normal flora (enterotoxigenic E. coli (ETEC), E. coli O-157), Shigella spp, and the detection of enteropathogens that can not be grown on classic growth media (Norwalk virus). The amplification process is inhibited by several substrates present in fecal material (phenol, hemoglobin), limiting DNA extraction by phenol. The protocols we have developed for direct detection of Shigella spp and ETEC in stools circumvent inhibition of PCR by the use of a 4-hour pre-enrichment step in brain-heart infusion broth.

Rapid and accurate identification of enteropathogens is important for prompt and focused intervention to stop the chain of transmission in outbreaks of gastroenteritis in military and civilian populations.