A simple but high effective specimen preparation for DNA extraction of Mycobacterium tuberculosis and its clinical application in quantitative-PCR determination of tuberculosis infection

Abstract Background： In the recent few years, there is an ongoing outbreak of tuberculosis worldwide. Molecular diagnosis based on genomic amplification is a rapid and reliable method for early detection of tuberculosis infection. However, the quality and quantity of nucleic acid of Mycobacterium tuberculosis ( MTB ) extracted from different human specimens is still a bottleneck for molecular analysis because of the difficulty associated with breaking cell walls of MTB . Methods： In this study, new specimens handling methods including Dounce homogenizer with a loose pestle and a tight pestle, compared with a conventional procedure consisted of sodium hydroxide, were applied for DNA isolation from human samples (sputum and pleural effusion) with or without MTB , and real time quantitative PCR (RT-qPCR) were evaluated DNA recovery, sensitivity and specificity for early and accurate diagnosis of tuberculosis infection. Results： 45 patients infected with tuberculosis and 56 normal controls were enrolled. With specimens handling with both the tight pestle and loose pestle of Dounce homogenizer, MTB DNA was recovered about 300 -10 4 times than traditional specimen pretreated method and 83.67%-100% tuberculosis infections could be better detected. The new DNA extraction methods were also performed excellent in plural effusion with detection rate in the range of 85.71%-90.47%. The relative lower specificity (80.55%-92.31%) was also well explained by follow-up. RT-qPCR handling with D-Tight data expanding with another 209 suspicious TB individuals (164 diagnosed with TB infection and 45 diagnosed with non-TB infection) showed a high sensitivity (97.47%, taken clinical diagnosis as golden standard) with no significant diverse between TB treated group and TB non-treated group (96.30% VS 98.08%). Conclusions： The results demonstrates that Dounce homogenizer could offer high quality and quantity MTB DNA to RT-qPCR determination of MTB infection.