مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Development of loop-mediated isothermal amplification targeting mpt64 for rapid and reliable detection of Mycobacterium tuberculosis
Development of loop-mediated isothermal amplification targeting mpt64 for rapid and reliable detection of Mycobacterium tuberculosis
Ehsan Aryan,1,*Seyedeh-Zohreh Mirbagheri,2Zahra Meshkat,3Amir-Hooshang Alvandi,4Hadi Safdari,5
1. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences 2. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences 3. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences 4. Department of Medical Microbiology, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Islamic Republic of Iran 5. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences
Introduction: Conventional methods remain as the cornerstone of laboratory diagnosis of tuberculosis (TB). However, certain drawbacks of these approaches have attracted attentions to the development of newer molecular methods for detection of Mycobacterium tuberculosis (Mtb). To reliably detect Mtb in clinical specimens, we developed a loop-mediated isothermal amplification (LAMP) reaction targeting mpt64, a gene presents in all strains of Mtb, and the results were compared with mpt64-nested PCR (nPCR).
Methods: Six specific primers were designed to target mpt64 genomic sequence of Mycobacterium tuberculosis complex. The analytical specificity of the primers was examined using 20 mycobacterial and 6 non mycobacterial species. The optimal temperature and time of the reaction were 64˚C and 90min, respectively. The mpt64-LAMP was compared with mpt64- nested PCR using 189 clinical specimens collected during a cross-sectional study.
Results: Eighty five out of 189 clinical specimens evaluated in this cross-section study were rule out to have TB and they were used to calculate the specificity of the reactions that was 89.4% (76/85, 95% confidence interval [CI], 81-95%) and 84.7% (72/85, CI, 75.3-92%) respectively for nPCR and LAMP. Although the analytical sensitivity of nPCR was 1000-fold greater than the one obtained by the LAMP, the overall diagnostic sensitivity of LAMP (84.6% [88/104], CI, 76.2-91%) was higher than the one of nPCR (71.2% [74/104], CI, 62-80%). Interestingly, further analysis showed that 90% (27/30) of false-negative nPCR results were due to the presence of inhibitors in clinical specimens, while 77.8% (21/27) of these specimens were positive by the LAMP.
Conclusion: Accordingly, significant higher tolerance of LAMP to inhibitors than that of nPCR (p<0.0001) leads to improved detection of Mtb using this new generation of gene amplification method. Moreover, lack of popular genes such as IS6110 in some strains of Mtb poses mpt64 as a more reliable target.