Comparison of DNA Extraction Protocols and Molecular Targets to Diagnose Tuberculous Meningitis

Abstract

Tuberculous meningitis (TBM) is a severe form of extrapulmonary tuberculosis. The aims of this study were to evaluate in-house molecular diagnostic protocols of DNA extraction directly from CSF samples and the targets amplified by qPCR as an accurate and fast diagnosis of TBM. One hundred CSF samples from 68 patients suspected of TBM were studied. Four DNA extraction techniques (phenol-chloroform-thiocyanate guanidine, silica thiocyanate guanidine, resin, and resin with ethanol) were compared and CSF samples were used to determine the best target (IS6110, MPB64, and hsp65 KDa) by qPCR. The extraction protocol using the phenol-chloroform-thiocyanate guanidine showed the best results in terms of quantification and sensitivity of PCR amplification, presenting up to 10 times more DNA than the second best protocol, the silica guanidine thiocyanate. The target that showed the best result for TBM diagnosis was the IS6110. This target showed 91% sensitivity and 97% specificity when we analyzed the results by sample and showed 100% sensitivity and 98% specificity when we analyzed the results by patient. The DNA extraction with phenol-chloroform-thiocyanate guanidine followed by IS6110 target amplification has been shown to be suitable for diagnosis of TBM in our clinical setting.

Evolution of Nanotech Assisted PCR Diagnosis of Mycobacterium Tuberculosis and Its Assessment With Conventional Methods

“…The disadvantage of PCR is its high cost, molecular lab infrastructure and skilled technologist. At present-day, in India real-time PCR is suitable as a public tool for rapid and early detection of TB [21]. Nanotech assisted PCR with higher efficiencies has been evolved [22][23][24].…”

“…These results are found superior than conventional methods. The magnetic nanoparticle (MNP) assisted DNA extraction protocol showed better results in terms of quantification and sensitivity of PCR diagnosis of Mycobacterium tuberculosis, evolving nanotech assisted innovative method [12].…”

An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

“…Another study by Tang et al reports an integrated device employing HDA for detection of S. typhimurium in various spiked samples 23 . The residual lysis and the precipitation buffer components used in these studies are likely to have reduced the sensitivity of nucleic acid amplification 24,25 . While there are various reports of integrated assays on paper (Table 1), due to extensive washing steps to filter amplification inhibitors there is an increase in the total assay time and complexity, which limits the translation of these assays.…”

An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min fromE. coliandM. smegmatiscells on a paper substrate

“…Secondly, the background signal from lysates should be much lower compared to the signal 23 . The residual lysis and the precipitation buffer components used in these studies are likely to have reduced the sensitivity of nucleic acid amplification 24,25 . While there are various reports of integrated assays on paper (Table 1), due to extensive washing steps to filter amplification inhibitors there is an increase in the total assay time and complexity, which limits the translation of these assays.…”