Study On The Detection Of Mycobacterium Tuberculosis 16S RRNA By Reverse Transcription Loop-mediated Isothermal Amplification

Mycobacterium tuberculosis(MTB)is a pathogen causing tuberculosis.Early diagnosis plays an important role in the rational treatment and the termination of infection.The currently methods like PCR,Fluorescence Quantitive PCR,loop-mediated isothermal amplification and other molecular biological assays are based on MTB DNA as the target sequence for primer designing.However,they have positive results for dead or viable bacteria,so it is impossible to evaluate the effectiveness of anti-tuberculosis treatment in the clinic.The sensitivity and specificity of detection are low,and can't meet the needs of clinical diagnosis.Bacterial DNA is usually a single or a few copies,and 16S rRNA can be up to 10~4 copies in a live MTB.Therefore,the detection of 16S rRNA should increase the detection sensitivity.At the same time,MTB 16S rRNA is only present in metabolically viable live bacteria,so the detection of 16S rRNA can improve the diagnostic specificity.In addition,the LAMP technology is cheap and easy to operate;the product does not require any instrument and is visible by naked eye after being dyed with calcein or hydroxynaphthol blue.LAMP is rapid and efficient amplification,which is suitable for detection under various conditions,especially for the grass-roots and on-site use.In this study,reverse transcription LAMP(RT-LAMP)was used for detection of MTB.Part ?.Detection of Mycobacterium tuberculosis 16S rRNA in Sputum by RT-LAMP.Objective:To provide a high sensitivity,specific,can detect viable live bacteria and simple method for rapid detection of tuberculosis by establishing a new method of RT-LAMP priming 16S rRNA.Methods:After comparing the 16S rRNA complete sequences of MTB with non MTB,the primers were designed by Primer Explorer V5 software.The sensitivity was determined by comparison the results of RT-LAMP and LAMP after serial 10-fold dilution of MTB primary bacterial solution.The specificity was determined by observing the detection results of NTM and other known respiratory bacteria(Staphylococcus aureus,Pseudomonas aeruginosa,Klebsiella pneumoniae).RT-LAMP,LAMP and L-J were used to detect sputum specimens from 131 patients with tuberculosis and 24 cases of control definitely without tuberculosis.Results:The sensitivity of RT-LAMP,LAMP methods were determined.Among which,the most sensitive one,RT-LAMP,was 10 times higher than LAMP method.RT-LAMP can detect a single copy of the MTB repeatedly.RT-LAMP method was used to detect 1 common MTB,8 NTM and 3common respiratory bacteria,and only MTB positive was found.The results showed that the positive rate of MTB in clinical sputum specimens detected by RT-LAMP,LAMP and L-J methods were 97.7%,78.6%,73.2%respectively,There was only one false positive case in the negative control group by L-J method.The positive rate by RT-LAMP method was higher than that of LAMP and L-J(P<0.01,P<0.01).Eighteen cases of positive sputum tuberculosis,sputum specimens have a higher detection rate by RT-LAMP and LAMP detection before the treatment of tuberculosis.However,with the extension of treatment time,the positive rate of RT-LAMP detection decreased rapidly,while the positive rate of LAMP detection decreased slowly.Conclusions:1.According to the characteristic of multiple copies and only exist in live bacteria of MTB,we creatively established the 16S rRNA RT-LAMP technology and used it to detect viable bacteria.It not only proved its high sensitivity and specificity,but also is easy to operate,time-saving,suitable for grass-roots and low-resource hospitals.This technology is expected to become an effective means for rapid detection of MTB.2.16S rRNA of MTB was used to design specific primers for RT-LAMP technique to detect MTB in sputum.RT-LAMP was Compared with LAMP technology and L-J method,proved that it has higher sensitivity and specificity.Part ?.Detection of Mycobacterium tuberculosis 16S rRNA in cerebrospinal fluid by RT-LAMP.Objective:To provide a new method for rapid diagnosis of tuberculous meningitis(TBM)by using the previously established 16S rRNA RT-LAMP technology to detect MTB in cerebrospinal fluid.Methods:We selected 105 Cerebrospinal Fluid(CSF)samples from patients,of which TBM has 52 cases,suspected TBM has 33 cases and control group has20 cases.Using the 16S rRNA RT-LAMP method of MTB established earlier with RT-PCR as a control,the sensitivity,specificity and positive rate of actual sample were tested by comparison of the two detection methods,In clinical sample detection,FQ-PCR was used as the control to compare the detection effect of the two methods.Results:The sensitivity of RT-LAMP is higher than that of RT-PCR,in which the lowest detection value was 1 CFU/ml and 10~2 CFU/ml respectively.In the 5 common cerebrospinal fluid specimens of pathogens of the central nervous system,no specific bands were amplified by two methods.The positive rate of clinical cerebrospinal fluid by RT-LAMP method was 96.1%(50/52),72.3%(24/33)in TBM,suspected TBM,respectively,while by RT-PCR method was 71.1%(37/52),33.3%(11/33),The detection rates of FQ-PCR in the two group were 65.3%(34/52),24.2%(8/33),respectively.None of the three methods showed positive amplification in the control group,and the detection rate of RT-LAMP was higher than that of FQ-PCR.There has significant difference between the two methods(P<0.01),The detection rate of RT-PCR in TBM group and suspected TBM group was higher than that of FQ-PCR.There was no significant difference between the two methods(P>0.05).Conclusions:Detection of MTB in cerebrospinal fluid by RT-LAMP was compared with RT-PCR,which also proved RT-LAMP high sensitivity and specificity.