Journal of Pure and Applied MicrobiologyVol. 6 No. 1

The Use of DNA Extracts from Ziehl-Neelsen Stained Slides for Spoligotyping in National Reference TB Laboratory of Iran

Mohadese Mozafari, Parissa Farnia and Ali Akbar Velayati

Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), WHO & UNION Collaborating Centre for TB & Lung Diseases , Shahid Beheshti University (Medical Campus), Darabad, Tehran,19556, P.O: 19575/154, Iran.

Received on 02 August 2011 and accepted on 14 September 2011



Spoligotyping is a molecular typing method that is used to differentiate M.tuberculosis complex. The method is based on polymorphism of the chromosomal DR locus consisting of identical 36-bp DRs alternating with 35-41 unique spacers. In this study, the spoligotyping was applied to Ziehl-Neelsen stained smears and its utility in smears with different positivity rate was assessed. DNA were extracted from smears (n=115) with different positivity rate [3+ (39; 33%), 2+ (28; 24), 1+ (33; 28%) and Scanty (15; 13%)]. Thirty negative smear slides were also used as a control. PCR was performed with primers specific for direct repeats and the product was detected by ECL hybridization kit. The obtained data entered the X-file and were analyzed with previously published database (spolDB4). The utility rate in 3+, 2+ and 1+ smears were 84.6%, 71.4%, 36.4% respectively (p<0.01). The smears with scanty results could not produce any patterns. In overall, the spoligopatterns was observed in 56.5% of samples. The predominant spoligopatterns identified as Haarlem (47.6%), CAS (26%) and T (15.3%). Spoligotyping can be reliable in smears with high positivity rate. Therefore, the method may be the best option to identify the circulating strains within limited resources laboratories.

Keywords : Ziehl-Neelsen slides, Spoligotyping, Molecular epidemiolog.