Reza kamali Kakhki - Antimicrobial Resistance Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
Ehsan Aryan - Antimicrobial Resistance Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
Zahra Meshkat - Antimicrobial Resistance Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
Mojtaba Sankian - Immunobiochemistry Laboratory, Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: The line probe assay (LPA) is one of the most accurate diagnostic tools for detection of different Mycobacterium species. Several commercial kits based on the LPA for detection of Mycobacterium species are currently available. Because of their high cost, especially for underdeveloped and developing countries, and the discrepancy of non-tuberculous mycobacteria (NTM) prevalence across geographic regions, it would be reasonable to consider the development of an in-house LPA. The aim of this study was to develop an LPA to detect and differentiate mycobacterial species and to evaluate the usefulness of PCR-LPA for direct application on clinical samples. Methods: One pair of biotinylated primers and 15 designed DNA oligonucleotide probes were used based on multiple aligned internal transcribed spacer (ITS) sequences. Specific binding of the PCR-amplified products to the probes immobilized on nitrocellulose membrane strips was evaluated by the hybridization method. Experiments were performed three times on separate days to evaluate the assay’s repeatability. The PCR-LPA was evaluated directly on nine clinical samples and their cultivated isolates. Results: All 15 probes used in this study hybridized specifically to ITS sequences of the corresponding standard species. Results were reproducible for all the strains on different days. Mycobacterium species of the nine clinical specimens and their cultivated isolates were correctly identified by PCR-LPA and confirmed by sequencing. Conclusions: In this study, we describe a PCR-LPA that is readily applicable in the clinical laboratory. The assay is fast, cost-effective, highly specific, and requires no radioactive materials.  

Diagnosis, Line Probe Assay (LPA), Mycobacterium Infection, Tuberculosis.