Improvement of Mycobacterium tuberculosis detection in clinical samples using DNA purified by glass matrix

Rossetti, Maria L.R.; Jardim, Susana; Rodrigues, Vivian; Moura, Andréia R. de; Oliveira, Hugo Goulart de; Zaha, Arnaldo

doi:10.1016/s0167-7012(97)00978-0

Cited by 18 publications

(17 citation statements)

References 16 publications

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“…Direct submission of the biological sample to amplification by PCR was shown to be better than a protocol that used DNA extraction and purification by glass matrix. We chose the GM-PCR protocol because our group already uses this method to detect M. tuberculosis DNA and it showed the best results with CSF samples of patients with tuberculous meningitis (Rossetti et al, 1997). It is possible that some samples presented low concentrations of bacteria, that DNA losses could have occurred during the wash steps when the purification procedure with the glass matrix was applied or that some Taq DNA polymerase inhibitor(s) remained in the preparation.…”

Section: Discussionmentioning

confidence: 99%

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Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory

Baethgen¹,

Moraes²,

Weidlich³

et al. 2003

Journal of Medical Microbiology

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A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88·5 % and, using dot-blot DNA detection, the sensitivity increased to 96·4 %. The detection limit for meningococcus in cerebrospinal fluid was 2310 2 c.f.u. ml À1 .Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96·4 % using dot-blot DNA detection.

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Section: Discussionmentioning

confidence: 99%

“…Ninety-one DNA samples were prepared from 500 ìl CSF or serum using a glass matrix (Sephaglas; Pharmacia Biotech) and eluted in a final volume of 30 ìl, as described by Rossetti et al (1997). Aliquots (10 ìl) of the extracted and purified DNA were submitted to PCR amplification.…”

Section: Methodsmentioning

confidence: 99%

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory

Baethgen¹,

Moraes²,

Weidlich³

et al. 2003

Journal of Medical Microbiology

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“…Nevertheless, many problems have been described during the use of in-house methodologies, including low reproducibility and subjective readings of the results (Kox et al 1996). Our group has developed and evaluated an in-house polymerase chain reaction (PCR) using IS6110 as a target and two differ-ent procedures to detect M. tuberculosis complex DNA: agarose gel electrophoresis and membrane hybridization (Rossetti et al 1997, Sperhacke et al 2004, Verza et al 2009). Because the sensitivity of PCR is dependent on the efficient preparation of clinical specimens before amplification and the detection method, this study describes a pre-commercial molecular test, Detect-TB (Ampligenix Biotech, Brazil), that includes an improved purification method for M. tuberculosis complex DNA and the development of a colorimetric method for its detection, in which an oligonucleotide probe is fixed into the wells of microwell plates and hybridized with biotinylated PCR amplification products derived from clinical samples.…”

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confidence: 99%

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Michelon

Rosso

Schmid

et al. 2011

Mem. Inst. Oswaldo Cruz

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“…This technique was performed using a modifi ed protocol, as fi rst utilized by Rossetti et al 14,21 . A water-containing negative control was included to assess any cross-contamination between the biological specimens.…”

Section: Mycobacterium Tuberculosis Dna Extraction and Purifi Cationmentioning

confidence: 99%

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Montenegro

Silva

Lima

et al. 2013

Rev. Soc. Bras. Med. Trop.

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Introduction: This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fl uid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil. Methods: A prospective double-blind study with 37 hospitalized patients of both sexes, aged over 15, was used to investigate the diagnosis of pleural effusion. The criteria used to defi ne the cases included the demonstration of bacillus in biological samples by smear or culture or by a granulomatous fi nding in the histopathological examination, associated with an evident response to specifi c treatments to each clinical situation. Pleural fl uid, blood and urine samples were collected and subjected to routine tests and the nested PCR technique to assess for M. tuberculosis amplifi cation. Results: In total, 37 pleural effusion patients took part in the study, of whom 19 (51.3%) had tubercular etiologies and 18 (48.7%) had etiologies from other causes. When the pleural fl uid, blood and/or urine sample in-house nested-PCR sensitivities were evaluated simultaneously, the results were positive regardless of the biological specimen (the sensitivity was 84.2%); however, when the blood and/or urine samples were analyzed together, the sensitivity was 72.2%. When the pleural fl uid samples were evaluated alone, the sensitivity was only 33.3%. Conclusions: The performance of the diagnostic pleural tuberculosis nested-PCR was directly related to the diversity of the samples collected from the same patient. Additionally, this study may identify a need to prioritize non-invasive blood and urine collection for this diagnosis.

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Improvement of Mycobacterium tuberculosis detection in clinical samples using DNA purified by glass matrix

Cited by 18 publications

References 16 publications

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

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