Molecular Diagnostics

Eisenach, Kathleen D.

doi:10.1002/9781444311433.ch9

Cited by 5 publications

(4 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The high-copy number of IS6110 is thought to result in increased sensitivity of a PCR assay [18], although a potentially more serious problem is the existence of mycobacterial strains that lack IS6110 [19,20]. However, there have been few reports citing the presence of such strains and it is unlikely that false-negative PCR results are attributable to strains lacking this element [18]. Moreover, preliminary data obtained from CNS and extra-CNS isolates available from our centre has shown that none of the strains were lacking the insertion element and majority of them were high-copy numbered isolates (our unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Rapid diagnosis of tuberculous meningitis: A comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen

Rafi

Venkataswamy

Ravi

et al. 2007

Journal of the Neurological Sciences

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

Rapid diagnosis of tuberculous meningitis: A comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen

Rafi

Venkataswamy

Ravi

et al. 2007

Journal of the Neurological Sciences

View full text Add to dashboard Cite

“…However, a more sensitive test is required to detect samples with a low bacterial load. Since the advent of PCR, there has been an explosion in its use for TB diagnosis owing to its speed and sensitivity (Eisenach, 1998). However, these advantages are partially offset by economic and technical factors and simplification is required before PCR can be widely employed.…”

Section: Introductionmentioning

confidence: 99%

Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons

Haldar

Chakravorty

Bhalla

et al. 2007

Journal of Medical Microbiology

View full text Add to dashboard Cite

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the 'gold' standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a costeffective manner.

show abstract

“…On the other hand, nucleic acid probes coupled with amplification allow rapid and specific identification of M. tuberculosis in clinical samples (Pfyfer et al, 1996). Some of the probes include insertion elements (IS6110, IS1081), genes for immunodominant antigens (38 kDa, 85 protein complex, 30/32 kDa, MPB64) and ribosomal sequences (16S and 23S rRNA) (Eisenach, 1999). These DNA probes are genus and species specific and utilize a wide array of sequences from a single-copy sequence to repetitive DNA elements.…”

Section: Introductionmentioning

confidence: 99%

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

Srivastava

Kumar

Waskar

et al. 2006

Journal of Medical Microbiology

View full text Add to dashboard Cite

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.

show abstract

Molecular Diagnostics

Cited by 5 publications

References 26 publications

Rapid diagnosis of tuberculous meningitis: A comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen

Rapid diagnosis of tuberculous meningitis: A comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen

Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons

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

Contact Info

Product

Resources

About