Detection of uncommon enteric bacterial pathogens from acute diarrheal specimens using sybr-green real time pcr

Abstract

SUMMARY: Acute diarrheal disease is a major health problem, and the second most common cause of death in children under 5 years of age. Conventional diagnostic methods are laborious, time consuming, and occasionally inaccurate. We used SYBR-Green real-time PCR for the detection of 10 uncommon bacterial pathogens using fecal specimens from acute diarrheal patients. In the SYBR-Green real-time PCR assay, the products formed were identified based on a melting point temperature curve analysis, and the assay was validated with the respective reference strain. In a retrospective study, we tested 1,184 stool specimens previously examined using conventional culture methods. Enterotoxigenic Bacteriodes fragilis was detected in 6.7% of the samples followed by enterotoxigenic Bacillus cereus (5.1%), Clostridium perfringens (3.9%), and Aeromonas hydrophila (3.8%). In the prospective study, A. hydrophila, Staphylococcus aureus, and C. perfringens were predominantly detected in 11 > 5 years of age, using real-time PCR. The real-time PCR assay is comprehensive, rapid, accurate, and well suited for surveillance or diagnostic purposes to detect uncommon bacterial pathogens, and should be useful in initiating appropriate care and thereby reducing patient risk.