sybr-green-i and Bacterial-Infections

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.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bacteria; Bacterial Infections; Benzothiazoles; Child; Child, Preschool; Diamines; Diarrhea; Feces; Female; Humans; Infant; Infant, Newborn; Male; Middle Aged; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Retrospective Studies; Staining and Labeling; Transition Temperature; Young Adult

In this study, we report a novel isothermal nucleic acid amplification method only requires one pair of primers and one enzyme, termed Polymerase Spiral Reaction (PSR) with high specificity, efficiency, and rapidity under isothermal condition. The recombinant plasmid of blaNDM-1 was imported to Escherichia coli BL21, and selected as the microbial target. PSR method employs a Bst DNA polymerase and a pair of primers designed targeting the blaNDM-1 gene sequence. The forward and reverse Tab primer sequences are reverse to each other at their 5' end (Nr and N), whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The PSR method was performed at a constant temperature 61 °C-65 °C, yielding a complicated spiral structure. PSR assay was monitored continuously in a real-time turbidimeter instrument or visually detected with the aid of a fluorescent dye (SYBR Greenı), and could be finished within 1 h with a high accumulation of 10(9) copies of the target and a fine sensitivity of 6 CFU per reaction. Clinical evaluation was also conducted using PSR, showing high specificity of this method. The PSR technique provides a convenient and cost-effective alternative for clinical screening, on-site diagnosis and primary quarantine purposes.

Topics: Bacteria; Bacterial Infections; Base Sequence; Benzothiazoles; beta-Lactamases; Diamines; DNA; DNA Polymerase I; DNA Primers; Enzyme Stability; Escherichia coli; Geobacillus stearothermophilus; Humans; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Organic Chemicals; Plasmids; Quinolines; Reproducibility of Results; Species Specificity; Temperature