Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods:One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis we...Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods:One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl-Neelsen staining,by cultivation on solid medium and by a balanced hemincsted fluorometric PCR system(Orange C3TB) that could preserve worker safety and produce a rather pure material free of potential inhibitors. DNA amplification was performed in a low cost tuberculosis termocycler-fluorotneter.Produced double stranded DNA was flurometrically detected.The whole reaction was conducted in one single tube which would not be opened after adding the processed sample in order to minimize the risk of cross contamination with amplicons.Results:The assay was able to delect 30 bacillus per sample mL with 99.8%interassay variation coefficient.PCR was positive in 23(21.9%) tested samples(21 of them were smear negative).In our study it showed a preliminary sensitivity of 94.5%for sputum and an overall specificity of 98.7%.Conclusions:Total run time of the test is 4 h with 2.5 real working time.All PCR positive samples are also positive by microbiological culture and clinical criteria.Results show that it could be a very useful tool to increase detection efficiency of tuberculosis disease in low bacilus load samples.Furthermore,its low cost and friendly using make it feasible to run in poor regions.展开更多
This study presents a fast, accurate and sensitive technique using gas chromatography-mass spectrometry (GC-MS) for the identification and quantification of N-acyl homoserine lactones (AHLs) in the extracts of bacteri...This study presents a fast, accurate and sensitive technique using gas chromatography-mass spectrometry (GC-MS) for the identification and quantification of N-acyl homoserine lactones (AHLs) in the extracts of bacterial strain of Pseudomonas aeruginosa and sputum sample of a cystic fibrosis patient. This method involves direct separation and determination of AHLs by using GC-MS as simultaneous separation and characterization of AHLs were possible without any prior derivatiza-tion. Electron ionization resulted in a common fragmentation pattern with the most common fragment ion at m/z 143 and other minor peaks at 73, 57 and 43. The limit of detection for N-butanoyl, N-hexanoyl, N-octanoyl, N-decanoyl, N-dodecanoyl and N-tetradecanoyl homoserine lactones was 2.14, 3.59, 2.71, 2.10, 2.45 and 2.34 μg/L, respectively. The presence of AHLs in the culture of P. aeruginosa strain and spu-tum of a cystic fibrosis patient was achieved in selected ion monitoring (SIM) mode by using the prominent fragment at m/z 143.展开更多
文摘Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods:One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl-Neelsen staining,by cultivation on solid medium and by a balanced hemincsted fluorometric PCR system(Orange C3TB) that could preserve worker safety and produce a rather pure material free of potential inhibitors. DNA amplification was performed in a low cost tuberculosis termocycler-fluorotneter.Produced double stranded DNA was flurometrically detected.The whole reaction was conducted in one single tube which would not be opened after adding the processed sample in order to minimize the risk of cross contamination with amplicons.Results:The assay was able to delect 30 bacillus per sample mL with 99.8%interassay variation coefficient.PCR was positive in 23(21.9%) tested samples(21 of them were smear negative).In our study it showed a preliminary sensitivity of 94.5%for sputum and an overall specificity of 98.7%.Conclusions:Total run time of the test is 4 h with 2.5 real working time.All PCR positive samples are also positive by microbiological culture and clinical criteria.Results show that it could be a very useful tool to increase detection efficiency of tuberculosis disease in low bacilus load samples.Furthermore,its low cost and friendly using make it feasible to run in poor regions.
文摘This study presents a fast, accurate and sensitive technique using gas chromatography-mass spectrometry (GC-MS) for the identification and quantification of N-acyl homoserine lactones (AHLs) in the extracts of bacterial strain of Pseudomonas aeruginosa and sputum sample of a cystic fibrosis patient. This method involves direct separation and determination of AHLs by using GC-MS as simultaneous separation and characterization of AHLs were possible without any prior derivatiza-tion. Electron ionization resulted in a common fragmentation pattern with the most common fragment ion at m/z 143 and other minor peaks at 73, 57 and 43. The limit of detection for N-butanoyl, N-hexanoyl, N-octanoyl, N-decanoyl, N-dodecanoyl and N-tetradecanoyl homoserine lactones was 2.14, 3.59, 2.71, 2.10, 2.45 and 2.34 μg/L, respectively. The presence of AHLs in the culture of P. aeruginosa strain and spu-tum of a cystic fibrosis patient was achieved in selected ion monitoring (SIM) mode by using the prominent fragment at m/z 143.
