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EVALUATION OF HSP 65,
TB ,SP REGIONS IN
IDENTIFYING MYCOBACTERIUM OTHER
THANTUBERCULOSIS( MOTT);USING PCR-RFLP
Noorolhoda Saadaee Jahromi ,Shima Seif, Parissa Farnia, Mehdi Kazempour,Mohammad Kargar, JamilehNowroozi, Mehdi
Kazempour, Mohammadreza Masjedi,Aliakbar Velayati Mycobacteriology Research Center (MRC),National Research
Institute Of Tuberculosis and Lung Disease(NRITLD),Shahid Beheshti University Medical Campus.Tehran,Iran.
Background
Mycobacteria Other Than Tuberculosis (MOTT) are frequent causes of pulmonary infections resembling TB, but differ
from MTB complex by being opportunistic pathogens and are acquired mainly from the environment. Recent investigators
reported an increasing cause of pulmonary infection by MOTT species, in Iran. Identification of MOTT by conventional
biochemical methods is cumbersome and time-consuming. Therefore in the present study,the capabilities of 3
different regions (Tb,Sp,Hsp 65
) were examined using three primers. We demonstrated that Hsp 65
genotyping would be
very helpful to identify mycobacteria at the species level.
Material & Method
DNA were extracted from 121 culture positive specimens during the year 2007-2009.The amplification carried out
using following primers ( Tb ; Tb 11
5’-ACCAACGATGGTGTGTCCAT-3’ Tb 5’-CTTGTCGAACCGCATACCCT) ,(Sp
, 12
; Sp 1
5’-ACCTCCTTTCTAAGGAGCACC-3’ ,
Sp 2
5’- GATGCTCGCAACCACTATCCA-3’), ( Hsp ; HSP F3 5’-ATCGC-
CAAGGAGATCGAGCT-3’ ,
HSP R4 5’-AAGGTGCCGCGGATCTTGTT-3’)
The PCR products (Tb: 439bp ,Sp: 250-330 bp ,HSP: 644 bp) were digested using restriction enzymes with BsteII and
HaeIII for Tb ,HaeIII for Sp and AvaII ,HphI,HpaII for HSP regions.The digested patterns were analyzed on 2% agarose gel.
Result
The results demonstrated different sensitivity ratio for various Mycobacterium species by Tb ,Sp and Hsp regions. For
RGM , Rapid Growing Mycobacteria , group(e.g., M. furtitium and M. chelonei ) the sensitivity of Tb primer was the highest
among the other two regions(92%). Although, for slow growing mycobacterium(nonphotochromgen & phtochromgen)
the combination of two primers(i.e., Tb+Sp) was required . Thereafter ,the sensitivity for identification of such species
reached upto 94%. Incontrast , scotochromoghen(e.g M. gordonae and M. scrofalceum) were differentiated more precisely
using Sp primer(74.3% ).
Conclusion
The recent increase in MOTT species within the country , underline the need to rapidly distinguish such Mycobacteria
from tuberculosis complex .We demonstrate the use of combined primers for accurate identification of mycobacterium
up to species level.
Key Word: Identification,Atypic Mycobacteria,PCR-RFLP,RGM
European Society of Mycobacteriology | 30 th Annual Congress | July 2009 | Porto - Portugal
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