Establishment And Preliminary Clinical Investigation Of A Highly Sensitive And Rapid Method For Detecting Mts90,a New Molecular Target Of Mycobacterium Tuberculosis

Background:Tuberculosis(TB)remains significant challenges of public health,especially in resource-limited countries with high TB burden.Failure in early diagnosis and lack of rapid and accurate diagnostic methods lead to ongoing prevalence and transmission of TB,coupled with continued emergence of multidrug resistant or extensively drug-resistant strains.Therefore,it is urgent to achieve rapid and accurate diagnosis for more TB suspected patients.There are disadvantages like poor sensitivity,time consumption and expensive equipment in existing methods for detecting mts90,a more specific target of Mycobacterium tuberculosis(MTB)identified in our previous research;so it is essential to develop a sensitive,rapid and low-cost method for detecting mts90.Objectives:We aim to establish a highly sensitive method targeting mts90 for rapidly detecting MTB,and this method is simple to operate and is not reliant on specialized equipment.Furthermore,investigate the diagnostic value and effectiveness of established method via test on clinical samples of suspected TB.Methods:Recently,recombinase polymerase amplification(RPA)technique makes it possible to rapidly amplify and detect nucleic acids without specialized devices;therefore we developed a RPA-based method for detecting mts90.Different screening methods were employed for selecting a preferred primer pair of amplification,including to design probe for specifically detecting mts90 in real-time.Sensitivity and specificity of this method was analyzed using mts90 recombinant plasmid and genomic DNA of related mycobacteria and common pathogens as templates.And clinical application performance was evaluated by means of Receiver Operating Characteristic(ROC)and by contrast with qPCR in detecting clinical specimens.Results:The preferred primer pair for amplification of mts90 by RPA was obtained by probe screening method,and a real-time RPA method for detecting mts90 was successfully established.The results showed that the real-time mts90 RPA assay was capable of detecting as sensitive as 6 copies of recombinant plasmid containing mts90 sequence per reaction.The assay was specific for detecting MTB,as it did not identify the genomic DNA from other mycobacteria and pathogens.When applied to analyze clinical samples,the Area Under the Cure(AUC)of real-time mts90 RPA assay was 0.876,and when the fluorescence detection threshold was 378 Relative Fluorescence Unit(RFU),sensitivity and specificity of the method were 96.4% and 76.5%,respectively,and Coincidence rate with clinical diagnosis was 88.9%(41/45).The sensitivity of mts90 RPA method was comparable to that of qPCR;however the average detection time of positive samples was 11.8 min,and the whole test procession could be completed within 20 min,which was faster than qPCR.Conclusion:The mts90 RPA assay can complete detection within 20 minutes at 39°C without thermal cycling,its simple operation and rapid detection suggest RPA-based MTB assays could be further developed for TB diagnosis in resource-poor settings.