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Development and validation of signature sequence-based PCR for improved molecular diagnosis of tuberculosis.
Journal of Molecular Diagnostics : JMD 2021 June 9
Reliable, fast, and affordable diagnosis for TB remains a challenge to reduce disease incidence in resource-poor countries. Tests based on nucleotide sequences that are 'signature' to M. tuberculosis (M. tb) have the potential to make a positive impact on case detection rates, which can eventually help control TB. Using extensive comparative bioinformatics approach, we mined the genome for M. tb-specific genes and identified four genes so-called "Signature sequence (SS)". With <25% homology with other known genes/proteins of mycobacterial/non-mycobacterial origin in various databases, these SS-genes are ideal targets for species-specific identification. Sputum from suspected patients was liquefied using novel "Complete Liquefying Reagent" (CLR), and DNA was isolated. Samples from patients (n=417), reporting to TB clinics at two different hospitals, which met our inclusion criteria, were collected for this study. A small number (n=143) was used for initial standardization and the remaining patient samples (n=274) were evaluated by SS and compared with smear microscopy, GeneXpert, culture, and clinical outcome. An overwhelming sensitivity of 97.0%, significantly higher than GeneXpert (95.0%), was seen. SS could pick all smear-negative, but culture-positive samples, along with other culture-negative samples, some of the latter were declared clinically positive. Our results yielded superior sensitivity and specificity through conventional PCR.
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