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
Tuberculosis is one of the leading infectious diseases plaguing mankind and is mediated by the facultative pathogen, Mycobacterium tuberculosis(MTB). Once the pathogen enters the body, it subverts the host immune defe...Tuberculosis is one of the leading infectious diseases plaguing mankind and is mediated by the facultative pathogen, Mycobacterium tuberculosis(MTB). Once the pathogen enters the body, it subverts the host immune defenses and thrives for extended periods of time within the host macrophages in the lung granulomas, a condition called latent tuberculosis(LTB). Persons with LTB are prone to reactivation of the disease when the body's immunity is compromised. Currently there are no reliable and effective diagnosis and treatment options for LTB, which necessitates new research in this area. The mycobacterial proteins and genes mediating the adaptive responses inside the macrophage is largely yet to be determined. Recently, it has been shown that the mce operon genes are critical for host cell invasion by the mycobacterium and for establishing a persistent infection in both in vitro and in mouse models of tuberculosis. The Yrb E and Mce proteins which are encoded by the MTB mce operons display high degrees of homology to the permeases and the surface binding protein of the ABC transports, respectively. Similarities in structure and cell surface location impute a role in cell invasion at cholesterol rich regions and immunomodulation. The mce4 operon is also thought to encode a cholesterol transport system that enables the mycobacterium to derive both energy and carbon from the host membrane lipids and possibly generating virulence mediating metabolites, thus enabling the bacteria in its long term survival within the granuloma. Various deletion mutation studies involving individual or whole mce operon genes have shown to be conferring varying degrees of attenuation of infectivity or at times hypervirulence to the host MTB, with the deletion of mce4 A operon gene conferring the greatest degree of attenuation of virulence. Antisense technology using synthetic si RNAs has been used in knocking down genes in bacteria and over the years this has evolvedinto a powerful tool for elucidating the roles of various genes mediating infectivity and survival in mycobacteria. Molecular beacons are a newer class of antisense RNA tagged with a fluorophore/quencher pair and their use for in vivo detection and knockdown of mR NA is rapidly gaining popularity.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Tuberculosis is one of the leading infectious diseases plaguing mankind and is mediated by the facultative pathogen, Mycobacterium tuberculosis(MTB). Once the pathogen enters the body, it subverts the host immune defenses and thrives for extended periods of time within the host macrophages in the lung granulomas, a condition called latent tuberculosis(LTB). Persons with LTB are prone to reactivation of the disease when the body's immunity is compromised. Currently there are no reliable and effective diagnosis and treatment options for LTB, which necessitates new research in this area. The mycobacterial proteins and genes mediating the adaptive responses inside the macrophage is largely yet to be determined. Recently, it has been shown that the mce operon genes are critical for host cell invasion by the mycobacterium and for establishing a persistent infection in both in vitro and in mouse models of tuberculosis. The Yrb E and Mce proteins which are encoded by the MTB mce operons display high degrees of homology to the permeases and the surface binding protein of the ABC transports, respectively. Similarities in structure and cell surface location impute a role in cell invasion at cholesterol rich regions and immunomodulation. The mce4 operon is also thought to encode a cholesterol transport system that enables the mycobacterium to derive both energy and carbon from the host membrane lipids and possibly generating virulence mediating metabolites, thus enabling the bacteria in its long term survival within the granuloma. Various deletion mutation studies involving individual or whole mce operon genes have shown to be conferring varying degrees of attenuation of infectivity or at times hypervirulence to the host MTB, with the deletion of mce4 A operon gene conferring the greatest degree of attenuation of virulence. Antisense technology using synthetic si RNAs has been used in knocking down genes in bacteria and over the years this has evolvedinto a powerful tool for elucidating the roles of various genes mediating infectivity and survival in mycobacteria. Molecular beacons are a newer class of antisense RNA tagged with a fluorophore/quencher pair and their use for in vivo detection and knockdown of mR NA is rapidly gaining popularity.
