Frontiers in microbiology · 2026
Analytical validation of a multiplex PCR assay for differentiation of five clinically significant nontuberculous mycobacterial species.
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Gaikwad Shaina, Tiwari Antisha, Agrawal Falguni, Singh Jitendra, Khadanga Sagar, Khurana Alkesh, Purwar Shashank, Maurya Anand Kumar
Аннотация
BACKGROUND: In TB endemic areas, accurate identification of non-tuberculous mycobacteria (NTM) from Mycobacterium tuberculosis complex remains challenging, which frequently results in incorrect diagnosis and ineffective treatment. There is a vital need for quick and affordable molecular methods to identify therapeutically relevant NTM at the species level.
OBJECTIVE: To develop and analytically validate an in-house multiplex PCR (mPCR) assay for simultaneous differentiation of five clinically relevant NTM species.
METHODS: The assay was developed using reference strains of M. abscessus, M. avium complex, M. chelonae, M. fortuitum, and M. kansasii. Before multiplex integration, species-specific primers were designed and verified with simplex PCR. Analytical sensitivity was determined using serial dilutions of purified genomic DNA tested in replicate reactions. Standardized bacterial suspensions were added to culture-negative samples to further assess assay performance. M. tuberculosis (H37Rv) and other non-target bacterial and mycobacterial species were used to evaluate analytical specificity.
RESULTS: The in-house mPCR technique successfully detected all five target species simultaneously and showed clear species-specific amplification, with distinct amplicon patterns (178-392 bp). The analytical limit of detection was established at 1 ng/μL using purified DNA, with reproducible amplification across replicate reactions. Spiked sample evaluation confirmed detection capability in complex biological matrices, though reduced band clarity was observed at lower concentrations. High analytical specificity was confirmed by the absence of cross-reactivity with non-target species, such as M. tuberculosis.
CONCLUSION: The developed in-house mPCR assay offers a rapid, economical molecular method for differentiating clinically important NTM species and has strong analytical performance. To justify its use in standard diagnostic labs, further extensive clinical validation is necessary.
OBJECTIVE: To develop and analytically validate an in-house multiplex PCR (mPCR) assay for simultaneous differentiation of five clinically relevant NTM species.
METHODS: The assay was developed using reference strains of M. abscessus, M. avium complex, M. chelonae, M. fortuitum, and M. kansasii. Before multiplex integration, species-specific primers were designed and verified with simplex PCR. Analytical sensitivity was determined using serial dilutions of purified genomic DNA tested in replicate reactions. Standardized bacterial suspensions were added to culture-negative samples to further assess assay performance. M. tuberculosis (H37Rv) and other non-target bacterial and mycobacterial species were used to evaluate analytical specificity.
RESULTS: The in-house mPCR technique successfully detected all five target species simultaneously and showed clear species-specific amplification, with distinct amplicon patterns (178-392 bp). The analytical limit of detection was established at 1 ng/μL using purified DNA, with reproducible amplification across replicate reactions. Spiked sample evaluation confirmed detection capability in complex biological matrices, though reduced band clarity was observed at lower concentrations. High analytical specificity was confirmed by the absence of cross-reactivity with non-target species, such as M. tuberculosis.
CONCLUSION: The developed in-house mPCR assay offers a rapid, economical molecular method for differentiating clinically important NTM species and has strong analytical performance. To justify its use in standard diagnostic labs, further extensive clinical validation is necessary.