Development of a duplex-polymerase chain reaction for rapid detection of pathogenic Leptospira.

A duplex-polymerase chain reaction (PCR) for the rapid detection of pathogenic leptospires was developed by using two sets of newly designed primers which amplified in the same reaction two different DNA fragments simultaneously: 279-bp of LipL32 and 430-bp of 16S rRNA. For DNA extraction from bacterial cultures, the silica-based spin column method was found to be more suitable and was selected for the extraction of DNAs from all 92 bacterial strains including 56 strains of pathogenic Leptospira, 15 strains of non-pathogenic Leptospira and 21 other strains of bacteria. The PCR products were analyzed by agarose gel-electrophoresis with confirmation by Southern and dot hybridization using synthetic DNA probe prepared from LipL32 gene of a pathogenic reference strain, L.

Duplex PCR-hybridization based detection of pathogenic Leptospira in environmental water samples obtained from endemic areas in northeast region of Thailand.

Leptospirosis, a major health problem worldwide, is known to be endemic in the northeastern part of Thailand with the risk of infection by exposure to pathogenic Leptospira in contaminated aquatic environment. A method based on PCR-hybridization detection of pathogenic Leptospira in water was established. The method included filtration of water sample through membrane filters of two pore sizes, DNA extraction from filters using a guanidine thiocyanate extraction method, a duplex-PCR assay with two primer pairs, and hybridization with a synthetic LipL32 DNA probe.

Antibiotic resistance of enterococci isolated from frozen foods and environmental water.

We evaluated 239 isolates of enterococci (113 from frozen foods and 126 from environmental water) for their resistance to 8 antibiotics by agar disk diffusion method. Most isolates from both sources were resistant to tetracycline (64.1% food strains; 46.

An enrichment broth culture-duplex PCR combination assay for the rapid detection of enterotoxigenic Clostridium perfringens in fecal specimens.

An enrichment broth culture-duplex PCR combination assay was devised to identify Clostridium perfringens directly from fecal samples. The method consists of a combination of short enrichment of samples in selective media, DNA isolation, and performing duplex PCR using two pairs of primers which identify C. perfringens strains that harbor the virulence enterotoxin gene.

Antimicrobial resistance among Clostridium perfringens isolated from various sources in Thailand.

Antimicrobial resistance among Clostnridium perfringens isolated from feces of humans and pigs, food and other environmental sources was examined by testing of 201 PCR-confirmed strains for resistance to 7 antimicrobial agents. The minimal inhibitory concentrations (MICs) were determined by the agar dilution method. Overall, C.

Evaluation of synthetic DNA probes for confirmation of Clostridium perfringens enterotoxin gene PCR products.

A new diagnostic reagent was developed that is capable of detecting the presence of Clostridium perfringens rapidly and accurately compared to the conventional methods. C. perfringens enterotoxin (cpe) gene is the gene of interest since it encodes the enterotoxin responsible for food poisoning.

A test strip IgM dot-ELISA assay using leptospiral antigen of endemic strains for serodiagnosis of acute leptospirosis.

A test strip IgM dot-ELISA assay for the detection of leptospire-specific IgM antibodies in human sera was developed. Antigen dotted on a nitrocellulose paper strip was the pool sonicated antigen prepared from three predominant reactive Leptospira serovars currently in endemic area, i.e.

Two simple immunoassays using endemic leptospiral antigens for serodiagnosis of human leptospirosis.

Two simple enzyme immunoassays, a conventional microplate and dot-ELISA, were developed to detect specific IgM antibodies using pool sonicated antigen prepared from three of the most reactive serovars of Leptospira associated with disease in Thailand. Both assays were evaluated and compared with the standard microscopic agglutination test (MAT) performed with 343 serum samples. A battery of 16 pathogenic serovars of L.

Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water.

Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C.

Direct identification of Mycobacterium tuberculosis from sputum on Ziehl-Neelsen acid fast stained slides by use of silica-based filter combined with polymerase chain reaction assay.

This paper describes a method for isolation of deoxyribonucleic acid (DNA) from Ziehl-Neelsen stained sputum smears on glass slides; and isolated DNA was used for the IS6110 polymerase chain reaction (PCR)-based identification of M. tuberculosis. A total of 221 samples from newly diagnosed suspected tuberculosis cases were first examined by microscopic examination.

Drug-resistant tuberculosis among prisoners of three prisons in Bangkok and the vicinity.

The purpose of this study was to determine the prevalence of drug-resistant tuberculosis and some factors associated with drug resistance among prisoners of three prisons in Bangkok and the vicinity. Susceptibility testing to four first-line antituberculous drugs was performed on 165 M. tuberculosis strains isolated from prisoners of three prisons including Klongprem Central (KC) prison, Bangkwang Central (BC) prison and the Correctional Institution (CI) for Male Drug Addicts.

PCR detection and prevalence of enterotoxin (cpe) gene in Clostridium perfringens isolated from diarrhea patients.

Unlabelled: Clostridium perfringens isolated from patients with diarrhea (n=233) were analysed by a duplex PCR assay, in order to determine the prevalence of enterotoxin (cpe) gene and various factors involved in patients with cpe-positive isolates. This duplex PCR uses two sets of primers which amplify in the same reaction two different gene fragments: the phospholipase C (plc, alpha-toxin) and the enterotoxin (cpe) genes in C. perfringens.

Comparison of microplate hybridization with gel electrophoresis and dot blot hybridization for the rapid detection of Mycobacterium tuberculosis PCR products.

A microplate ELISA hybridization assay has been developed for the detection of the IS6110 PCR products of M. tuberculosis from sputum specimens. In this study, its efficacy was evaluated by comparison with agarose gel electrophoresis (AGE) and dot blot hybridization (DBH), with culture results as the 'gold standard'.

Evaluation of sputum staining by modified cold method and comparison with Ziehl-Neelsen and fluorochrome methods for the primary diagnosis of tuberculosis.

An improved acid-fast staining technique for sputum examination for the primary diagnosis of tuberculosis is described. The technique was modified and simplified by the elimination of heating and by combining the stages of counterstaining: making the technique easier and safer, with less risk of phenol aerosols. The efficiency of this method was evaluated by comparison with two conventional methods, Ziehl-Neelsen (ZN) staining and fluorochrome staining; culture was deemed the gold standard for tuberculosis diagnosis.