The Establishment And Primary Application Of Ag85B-based Genetic Method For Rapidly Detecting Mycobacterium Tuberculosis Via RPA Technology

Part I The establishment and verification of Ag85B-based genetic method for rapidly detecting Mycobacterium tuberculosis via RPA technologyObjective:Tuberculosis(TB),as a disease resulted from Mycobacterium tuberculosis(MTB),has become one of the top ten causes of death and a severe threat to people's health.The course of treatment needs to be monitored due to the long period of therapy.Thus,the rapid diagnosis and rational treatment is an important measurement for controlling the epidemic of tuberculosis.Nevertheless,the sensitivity of traditional sputum smear is low and DNA detection of nucleic acid cannot reveal the viable bacteria.The method of Xpert MTB/RIF recommended by WHO is expensive.Therefore,it is promising to provide multiple choices and promote utilization via developing a precise,rapid and economic alternative method.Recombinase polymerase amplification(RPA)is an in vitro isothermal amplification technology of nucleic acid,which can detect single molecule within 20 min and has widely applied to medical diagnosis,agriculture,food and biosafety due to its characters of speediness,convenience and high sensitivity and specificity.The previous study deemed that the mRNA of prokaryote can serve as the symbol of viable bacteria.Thus,we detected DNA and mRNA of typical Ag85B gene form MTB via RPA to confirm and monitor therapy efficiency in real time,which is conducive to judging the therapy efficiency and sensitivity of drugs.Based on above features of RPA,this study was to build and verify a novel RPA technology for detecting MTB and provides a promising strategy for the rapid diagnosis and point of care test(POCT)of TB.Methods:Typical Ag85B gene of MTB was selected to serve as the targeted gene,probe and primers were designed following the guidelines of Twist Dx according to the conserved region of Ag85B.The optimization of reaction temperature and primers were then conducted to screen the optimal primers combination and reaction temperature.The standard plasmid was prepared and then serially diluted to serve as reaction template to ascertain the lowest limit of detection.The sensitivity of RPA were analyzed and the standard curve and regression equation were established.The DNA of standard strain M.tuberculosis(M.:Mycobacterium)H37Rv and BCG vaccine,non-tuberculous mycobacteria(NTM)and control pathogens was extracted and then tested via RPA approach to investigate the specificity of RPA method.The reliability of RPA technology was evaluated by detecting the DNA extracted from 20 cases of MTB and20 cases of NTM strains.Results:1.According to the gene sequence of targeted gene,four pairs of primers and one probe were designed.Three different concentrations of standard plasmid were selected as template,screened optimal primers combination was F4-R4 and optimal reaction temperature was 39 ^oC.2.The standard plasmid was prepared and sequenced,the concentration of standard plasmid was 4.0×10¹⁰ copies/?L.3.The detection limit of the real-time RPA could reach 4.0 copies/?L within 20 min.The regression equation:Y=14.22-3.33X(R²=0.9420).4.When testing DNA samples,only H37Rv and BCG showed the positive fluorescence signals in the real-time RPA tests,others including NTM(M.kansasii and M.intracellulare)and respiratory pathogens(Klebsiella pneumonia and Acinetobacter baumannii)showed the negative signals,which indicated a 100%of specificity.5.The DNA samples of MTB(20)and NTM strains(20)were determined via RPA and all the DNA samples from MTB displayed the positive signals.Meanwhile,no signals were noticed for DNA samples of NTM strains,informing a satisfactory reliability.Conclusions:In this study,we designed and established an Ag85B-based genetic method for rapidly detecting Mycobacterium tuberculosis via RPA technology.The detection of M.tuberculosis could be completed within 20 min at 39°C via an accessible RPA assay.The detection limit RPA technology could reach 4.0 copies/?L.Moreover,the specificity of Ag85B-based RPA was 100%and the detection result was reliable.Taken together,the Ag85B-based RPA provides a rapid determination approach of nucleic acid for diagnosis of MTB and a potential for POCT.Part II The primary clinical application of RPA to rapidly determine Mycobacterium tuberculosisObjective:More than 30% of cases were unrecorded and missed due to the diagnosis difficulty of TB,which is adverse to control the TB.RPA technology can serve as one of fast measured method of MTB nucleic acid.Meanwhile,we have built a detected method of Ag85 B via in first part.In this part,we collected sputum samples from TB patients diagnosed via clinic.The samples were then tested via real-time RPA and new molecular method built in the first part for rapid measurement of TB was investigated by comparing with traditional sputum bacteriologic method and Xpert MTB/RIF recommended by WHO.Methods:Recruiting 70 cases of patients diagnosed with TB and other 50 cases of patients with respiratory diseases during the same period were used as the controls.The demographic and clinical data were collected.Subsequently,morning and spot sputum was collected and stored in sterile sealed plastic box with screw top at 4 oC.Extracting and purifying DNA and RNA from sputum.The 85 BDNA from sputum was determined via RPA,the results were compared with that tested using Xpert MTB/RIF.The detection method of 85 BmRNA via RPA was built.The TB patients were monitor to observe the changes before and after treatment,the results were compared with that tested by sputum smear and RPA aiming at 85 BDNA.Results:1.Among 70 cases of TB patients,48 patients(68.6%)were male and 22 patients(31.4%)were female,with an average age of 59.57±17.8 years old.The occupation was dominated by farmer and retiree and 30 patients(43%)had visible pulmonary cavity observed by chest CT,25 patients(35.7%)had a history of smoking and 45 patients(64.3%)have a history of chronic diseases such as: hypertension,diabetes,chronic obstructive pulmonary disease,bronchiectasis,bronchial asthma,tumor,cerebral infarction,chronic kidney disease,chronic liver disease,rheumatoid arthritis,dermatomyositis and psychosis.All patients were negative for HIV testing.21 patients(30%)had a history of tuberculosis or extrapulmonary tuberculosis and 2 patients had tuberculosis relatives.The control group had 50 cases of patients.Among them,30 patients(60%)were male and 20 patients(40%)were female,with an average age of 63.24±19.5 years old.23 patients(46%)had chronic obstructive pulmonary disease,16 patients(32%)had pneumonia,6 patients(12%)were diagnosed with bronchiectasis and 5 patients(10%)were tested with tumor.2.Among 70 cases,the 85 BDNA of sputum was evaluated by RPA method,63 patients(90.0%)were positive,7 patients(10%)were negative and patients in the control group,1 patient(2%)was positive,49 patients(98%)were negative.In 70 cases,the samples were detected by Xpert MTB/RIF,65 patients(92.9%)were positive and 5 patients(7.1%)were negative.In 50 control cases,Xpert MTB/RIF was used to detect sputum samples,all detected by Xpert MTB/RIF were negative.The detection sensitivity,specificity,positive predictive value and negative predictive value of the RPA method were 90%(80-96),98%(89-100),98%(92-100)and 88%(76-95),respectively.Meanwhile,the index above determined employing Xpert MTB/RIF was 93%(84-98),100%(93-100),100%(94-100)and 91%(80-97),respectively.The Kappa values were 0.865 and 0.915,respectively,and the results showed a perfect consistency.3.85 BmRNA was determined via RT-RPA,the results showed that standard strain H37 Rv and BCG displayed the positive signals,but M.kansasii,M.intracellulare,klebsiella pneumonia and acinetobacter baumannii did not have the fluorescence signals.4.26 cases were observed subsequently,after treatment with standard antituberculosis(2HRZE/4HR),the positive number of patients detected by RPA-85 BDNA,RT-RPA 85 B mRNA and sputum smear were all reduced at 2,4 and 8 week,of which the positive number of patients evaluated by RPA-85 BDNA were highest.The positive number of patients tested by RT-RPA 85 BDNA were comparable to that detected by at 2 week,but the positive number detected by sputum smear were lower than that tested by RT-RPA 85 BDNA at 0,4 and 8 week,which can be attributed to the low sensitivity of sputum smear.Comparing the three methods,RT-RPA 85 BmRNA can preferably reveal the condition of live bacteria and monitored therapy efficiency in time.Conclusions:We previously established a rapid detection method of nucleic acid via Ag85B-based RPA and then applied it to the determination of clinical samples.This approach has relatively high sensitivity and specificity,which is similar with Xpert MTB/RI recommended by WHO but superior to traditional smear.Subsequently,85 BmRNA was measured employing RT-RPA because the level of 85 BmRNA can preferably reflect the condition of viable bacteria,which can serve as an indication of therapy efficiency during the treatment and assist clinicist to find drug-resistant patients timely.