Background:Tuberculosis(TB)remains a leading cause of mortality worldwide,particularly in developing nations.Currently,available diagnostic methods are often too costly or insufficiently sensitive for effective use in...Background:Tuberculosis(TB)remains a leading cause of mortality worldwide,particularly in developing nations.Currently,available diagnostic methods are often too costly or insufficiently sensitive for effective use in low-and middle-income countries.Developing a rapid,convenient,and accurate method for detecting the Mycobacterium tuberculosis complex(MTBC)is crucial to curtail the spread of TB.Methods:Primers and probes targeting conserved regions of IS1081 were designed,and the RNase P gene was introduced as an internal control to prevent false-negative results.M.tuberculosis control was used to optimize the reaction temperature.Additionally,we calculated and compared the limit of detection,specificity,and coincidence rate between this platform and the TaqMan real-time fluorescence quantification method(RT-qPCR)using two sets of national reference panels and 10 strains of MTBC.Results:An on-site MTBC-multiplex recombinase polymerase amplification(MTBC-mRPA)platform was established,with detection within 30 min over a broad temperature range(25℃-45℃).Probit analysis estimated a 95% limit of detection of 557.16(95% confidence interval:406.76-1062.67)bacteria/mL(p<0.0001),close to the limit of detection of 461.84(95% confidence interval:342.55-881.57)bacteria/mL(p<0.0001)of qPCR.The platform differentiated between non-tuberculous mycobacteria and other common respiratory bacteria,showing 100% specificity.The coincidence rate between multiplex real-time recombinase polymerase amplification(RT-mRPA)and RT-qPCR was 100%,indicating substantial similarity.Conclusion:A simple,rapid,and visual MTBC-mRPA platform coupled with rapid DNA extraction was developed for sensitive and specific detection of MTBC,especially suitable for on-site screening of TB in low-resource settings.展开更多
基金supported by the Natural Science Foundation of Jiangsu Province(Grant No.BK20200682).
文摘Background:Tuberculosis(TB)remains a leading cause of mortality worldwide,particularly in developing nations.Currently,available diagnostic methods are often too costly or insufficiently sensitive for effective use in low-and middle-income countries.Developing a rapid,convenient,and accurate method for detecting the Mycobacterium tuberculosis complex(MTBC)is crucial to curtail the spread of TB.Methods:Primers and probes targeting conserved regions of IS1081 were designed,and the RNase P gene was introduced as an internal control to prevent false-negative results.M.tuberculosis control was used to optimize the reaction temperature.Additionally,we calculated and compared the limit of detection,specificity,and coincidence rate between this platform and the TaqMan real-time fluorescence quantification method(RT-qPCR)using two sets of national reference panels and 10 strains of MTBC.Results:An on-site MTBC-multiplex recombinase polymerase amplification(MTBC-mRPA)platform was established,with detection within 30 min over a broad temperature range(25℃-45℃).Probit analysis estimated a 95% limit of detection of 557.16(95% confidence interval:406.76-1062.67)bacteria/mL(p<0.0001),close to the limit of detection of 461.84(95% confidence interval:342.55-881.57)bacteria/mL(p<0.0001)of qPCR.The platform differentiated between non-tuberculous mycobacteria and other common respiratory bacteria,showing 100% specificity.The coincidence rate between multiplex real-time recombinase polymerase amplification(RT-mRPA)and RT-qPCR was 100%,indicating substantial similarity.Conclusion:A simple,rapid,and visual MTBC-mRPA platform coupled with rapid DNA extraction was developed for sensitive and specific detection of MTBC,especially suitable for on-site screening of TB in low-resource settings.
