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J Med Microbiol 55 (2006), 1071-1077; DOI: 10.1099/jmm.0.46379-0
© 2006 Society for General Microbiology
ISSN 1473-5644

Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis

Ranjana Srivastava1, D. Kumar1, M. N. Waskar1, Meera Sharma2, V. M. Katoch3 and Brahm S. Srivastava1

1 Microbiology Division, Central Drug Research Institute, Lucknow 226001, India

2 Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

3 Central JALMA Institute for Leprosy, Agra, India

Correspondence
Ranjana Srivastava
drranjana{at}yahoo.com

Received 18 October 2005
Accepted 28 April 2006

A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a {lambda}gt11 library of M. tuberculosis by DNA–DNA hybridization using genomic DNA of M. tuberculosis as probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment of M. tuberculosis containing repetitive sequences, which produced positive hybridization signals with M. tuberculosis DNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) of M. tuberculosis. The use of CD192 as a DNA probe for the identification of M. tuberculosis in culture and clinical samples was investigated. The 1291 bp sequence was present in M. tuberculosis, Mycobacterium bovis and M. bovis BCG, but was not present in many of the other mycobacterial strains tested, including M. tuberculosis H37Ra. More than 300 clinical isolates of M. tuberculosis were probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detected M. tuberculosis with 100 % specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis.

Abbreviations: ATT, antitubercular treatment; CSF, cerebrospinal fluid; DR, direct repeat; IAC, internal amplification control; MPTR, major polymorphic tandem repeat; NPV, negative predictive value; PGRS, polymorphic GC-rich repetitive sequence; PPV, positive predictive value; TBM, tuberculous meningitis.

The GenBank/EMBL/DDBJ accession number for the CD192 sequence of M. tuberculosis is 810514.






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