Direct Identification of Mycobacterium Tuberculosis from
Sputum on Ziehl-Neelsen Acid Fast Stained Slides by Use
of Silica-Based Filter Combined with Polymerase Chain
Reaction Assay
UNCHALEE T ANSUPHASIRI, MSc*,
PREYANUCH BOONRAT, MSc**,
SOMSAK RIENTHONG, MSc***
Affiliation : * Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok 10400,
** Paolo Memorial Hospital, Bangkok 10400,
*** Tuberculosis Division, Department of Communicable Disease Control, Ministry of Public Health, Nonthaburi 11000,
Thailand.
AbstractThis paper describes a method for isolation of deoxyribonucleic acid (DNA) from Ziehl
Neelsen stained sputum smears on glass slides; and isolated DNA was used for the IS6J 10 polymerase
chain reaction (PCR)-based identification of M. tuberculosis. A total of 221 samples from newly diag
nosed suspected tuberculosis cases were first examined by microscopic examination. For DNA extrac
tion by silica-based filter, a home-made modified spin column gave the efficacy as did the nucleospin
tissue reagent kit and therefore was selected for PCR template preparation. The extracted DNA was
amplified by the IS6J 10 PCR using a primer pair that amplifies a 377-bp target, and the product was
analyzed by agarose gel electrophoresis with confirmation by Southern blot hybridization. In compa
rison with culture, PCR with template prepared by the silica based filter showed overall sensitivity and
specificity of 91.7 and 100 per cent, respectively. This study used the over one year and less than one
year slides samples to study the effect of storage time. In the more than one year storage group, PCR
assay gave a sensitivity and specificity of 83.3 and 100 per cent, respectively. In conclusion, the appli
cability of the PCR directly to DNA extracted from Ziehi-Neelsen stained smears could become a
valuable alternative approach for rapid identification of M. tuberculosis, and could be used to evaluate
quality of the control of local laboratories in tuberculosis (TB) screening and solve the problem of
specimen transportation. In addition, the method could be used in retrospective studies involving a wide
range of PCR-based analyses, such as detection of rifampicin resistant gene in multidrug-resistant
tuberculosis (MDR-TB) study.
Keywords : Mycobacterium Tuberculosis, PCR, Sputum, Ziehi-Neelsen Acid Fast Stained Slides, DNA
Isolation, Silica-Based Filter
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