Development and evaluation of a single-tube nested PCR with colorimetric assay for <i>Mycobacterium tuberculosis</i> detection

Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1 pg/μl. Its general sensitivity and specificity were 76.62 and 60.53%, respectively, with kappa index agreement of 0.166. A total of 318 biological samples (urine, plasma, peripheral blood mononuclear cells, pleural fluid and sputum) from pulmonary/extrapulmonary TB and non-TB patients were used in this study. The colorimetric STNPCR assay using IS6110 as the target gene was developed and optimized for Mtb detection based on similar validated systems. Cut-off values based on receiver operator characteristic curve analysis were defined to determine the sensitivity and specificity for each sample type. The technique's performance was assessed according to kappa index calculations and interpretation. To improve rapid molecular tuberculosis (TB) diagnosis, a novel molecular test was developed and evaluated in different biological samples. This test is based on single-tube nested PCR (STNPCR) with colorimetric assay using IS6110 as the target gene for Mycobacterium tuberculosis (Mtb) detection. A total of 318 biological samples from pulmonary/extrapulmonary TB and non-TB patients were used in this study. The colorimetric STNPCR assay used IS6110 as the molecular target optimized for Mtb detection based on validated similar systems. The results showed a detection limit of 1 pg/μl according to the absorbance analysis. The colorimetric STNPCR assay general sensitivity and specificity were 76.62% and 60.53%, respectively, with kappa index agreement of 0.166. Its sensitivity and specificity for the biological samples were: plasma (55.5% and 80%), PBMC (72.3% and 54.3%), urine (61.3% and 51.5%), pleural fluid (75% and 100%) and sputum (64.7% and 66.7%). The study demonstrated a quick and straightforward test with promising results, suggesting the automated colorimetric STNPCR assay as a potential addition to laboratory routines for both pulmonary and extrapulmonary TB diagnosis.