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

Abstract

A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a lgt11 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.

Comparative Analysis of Mycobacterium tuberculosis pe and ppe Genes Reveals High Sequence Variation and an Apparent Absence of Selective Constraints

“…Similar results have also been found for ppe18 [21]. Less sensitive analysis based on the size of DNA repeats in the C-terminal region of ppe34 and ppe8 have also revealed a high frequency of polymorphism [12], [22]. In addition, some pe/ppe genes have been reported to display elevated levels of IS 6110 integration [23], [24], [25], [26], [27], [28] and homologous recombination [26], [29], [30], [31].…”

“…We hypothesise that if pe/ppe proteins are involved in immune evasion and antigenic variation their genes will have undergone rapid evolutionary change, as demonstrated by high levels of DNA sequence polymorphism and evidence for diversifying selection compared to other M. tuberculosis genes. Previous work on this theme [12], [18], [19], [20], [21], [22], [32] has produced conflicting results that may be due to the lack of sensitivity of the analysis technique, the decision to examine genes that belong to a particular pe or ppe sub-family that might show abnormal variation levels, or the decision to examine clinical isolates that are too closely related to reveal polymorphic differences. The resultant comparative gene analysis presented here provides new insights into the variation and evolution of these genes along with their potential role in providing the pathogen with a source of antigenic variation.…”

Diagnosis of extrapulmonary tuberculosis by PCR

“…The fatality rate for untreated TB meningitis is almost 100% and delay in treatment often leads to permanent neurological damage (Takahashi et al, 2008;Sharma et al, 2010a). The conventional microbiological tests to diagnose TB meningitis almost fail, and therefore, the detection of M. tuberculosis in CSF by PCR has been widely employed using IS6110, 65 kDa, 38 kDa, devR, MPB-64 or PPE gene target with varying sensitivities (Martins et al, 2000;Kulkarni et al, 2005;Quan et al, 2006;Srivastava et al, 2006;Rafi et al, 2007;Dora et al, 2008;Takahashi et al, 2008;Haldar et al, 2009; Table 1). The conventional microbiological tests to diagnose TB meningitis almost fail, and therefore, the detection of M. tuberculosis in CSF by PCR has been widely employed using IS6110, 65 kDa, 38 kDa, devR, MPB-64 or PPE gene target with varying sensitivities (Martins et al, 2000;Kulkarni et al, 2005;Quan et al, 2006;Srivastava et al, 2006;Rafi et al, 2007;Dora et al, 2008;Takahashi et al, 2008;Haldar et al, 2009; Table 1).…”

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“…Additionally, Srivastava et al . [33] showed in an analysis of more than 300 clinical isolates of M. tuberculosis that the MPTR domain of the PPE gene Rv0355c (PPE8) displayed several polymorphisms. There is also ample evidence available for differential expression of members of the PE/PE_PGRS family between different strains of M. tuberculosis [34] as well as under different environmental and experimental conditions.…”