Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Michelon, Candice Tosi; Rosso, Franciele; Schmid, Karen Barros; Sperhacke, Rosa Dea; Oliveira, Martha Maria de; Kritski, Afrânio Lineu; Rezende, Lilian Cristina; Costa, Elis Regina Dalla; Ribeiro, Andrezza; Verza, Mirela; Cafrune, Patrícia Izquierdo; Silva, Márcia Susana Nunes; Kuhleis, Daniele; Zaha, Arnaldo; Rossetti, Maria Lúcia Rosa

doi:10.1590/s0074-02762011000200013

Cited by 17 publications

(30 citation statements)

References 28 publications

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“…All of the 108 isolates analysed by LINE-TB/MDR showed specific hybridisation to the IS 6110 probe, which was used for M. tuberculosis identification (amongst other members of the MTBC), indicating that this method can be a reliable tool for TB diagnosis. This result is similar to that found previously by our research group when testing a method to detect TB (DETEC-TB) (Michelon et al 2011). In addition, the 55 DNA samples that did not contain mutations in the genes associated with resistance and were drug susceptible according to the DST, were correctly identified by the LINE-TB/MDR method.…”

Section: Discussionsupporting

confidence: 92%

“…Reverse-line blot hybridisation and colorimetric detection - Reverse hybridisation and colorimetric detection were performed based on the previously described protocol (Maschmann et al 2011, Michelon et al 2011), with some modifications. The membranes were washed in 2x SSC/0.1% sodium dodecyl sulfate (SDS) for 5 min at 50ºC and were incubated in 2x SSC with 5% bovine serum albumin (BSA) at 50ºC for 15 min.…”

Section: Methodsmentioning

confidence: 99%

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In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Ferreira

Costa

Santos

et al. 2014

Mem. Inst. Oswaldo Cruz

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Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

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Section: Discussionsupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Ferreira

Costa

Santos

et al. 2014

Mem. Inst. Oswaldo Cruz

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“…A non-organic method developed in our laboratory was used to extract the DNA from the clinical specimens (Michelon et al , 2011). Briefly, 500 μL of each sample was concentrated by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

Association between human papillomavirus (HPV) DNA and micronuclei in normal cervical cytology

et al. 2014

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The aim of the present study was to investigate the association between HPV-DNA and micronucleus (MN) frequency in women with normal cervical cytology. A total of 158 normal cervical smears were analyzed cytologically. The HPV genome was amplified using the GP5+/bioGP6+ consensus primers. HPV-DNA of high-risk types 16, 18, 31, 33, 39, 45 and 59 were also investigated. Of the 158 samples, 20 (12.7%) and 47 (29.7%) were positive for HPV-DNA and MN, respectively. Evidence for MN was found in 11 out of 20 (55%) HPV-DNA positive samples and in 36 out of 138 (26.1%) HPV-DNA negative ones. MN presence was significantly higher in HPV-DNA positive samples (p = 0.016). On the other hand, the absence of MN observed in a considerable number of HPV-DNA negative samples (102) may be of great value in predicting the absence of HPV. The mean age of HPV-DNA positive women (34.2 ± 12.6) was significantly lower than the mean age of HPV-DNA negative women (43.9 ± 13.7) (p = 0.003). Infection by one or multiple HPV types was found in 11 out of 20 (55.0%) and 9 out of 20 (45.0%) samples, respectively. The evaluation of MN using cervical smears collected for cytology tests could, thus, be used as additional information to monitor a population’s exposure to HPV.

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“…Detect‐TB (Labtest, MG, Brazil) is a new molecular commercial kit that has been developed in Brazil, based on the colorimetric detection of amplified product of the IS6110 region hybridized with specific probe fixed on microplate. DNA extraction, purification and amplification were performed as described previously . To prevent DNA contamination, strict room separation was used, including a workflow from initiation of the PCR to hybridization.…”

Section: Methodsmentioning

confidence: 99%

Polymerase chain reaction test in induced sputum of patients with pulmonary tuberculosis

Paiva

Staub

Valentini

et al. 2018

Clinical Respiratory J

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Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Cited by 17 publications

References 28 publications

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Association between human papillomavirus (HPV) DNA and micronuclei in normal cervical cytology

Polymerase chain reaction test in induced sputum of patients with pulmonary tuberculosis

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