Reports of Biochemistry and Molecular Biology
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Basic Sciences
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2322-3480
2322-3480
10.61186/rbmb
en
jalali
1398
10
1
gregorian
2020
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1
8
4
online
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Development of a Cost-Effective Line Probe Assay for Rapid Detection and Differentiation of Mycobacterium Species: A Pilot Study
میکروب شناسی
Microbiology
مقالات اصلی
Original Article
<div style="text-align: justify;"><strong><em>Background:</em></strong> The line probe assay (LPA) is one of the most accurate diagnostic tools for detection of different <em>Mycobacterium</em> species. Several commercial kits based on the LPA for detection of <em>Mycobacterium</em> 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.<br>
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<strong><em>Methods:</em></strong> 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.<br>
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<strong><em>Results:</em></strong> 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. <em>Mycobacterium</em> species of the nine clinical specimens and their cultivated isolates were correctly identified by PCR-LPA and confirmed by sequencing.<br>
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<strong><em>Conclusions:</em></strong> 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.<br>
</div>
Diagnosis, Line Probe Assay (LPA), Mycobacterium Infection, Tuberculosis.
383
393
http://rbmb.net/browse.php?a_code=A-10-334-2&slc_lang=en&sid=1
Reza
kamali Kakhki
reza.kamali99@yahoo.com
10031947532846006770
10031947532846006770
No
Antimicrobial Resistance Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
Ehsan
Aryan
aryanE@mums.ac.ir
10031947532846006771
10031947532846006771
No
Antimicrobial Resistance Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
Zahra
Meshkat
meshkatz@mums.ac.ir
10031947532846006772
10031947532846006772
No
Antimicrobial Resistance Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.
Mojtaba
Sankian
sankianm@mums.ac.ir
10031947532846006773
10031947532846006773
Yes
Immunobiochemistry Laboratory, Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran.