title: Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection.
abstract: Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.
authors.affiliation: Centro de Desenvolvimento Científico e Tecnológico, Fundação Estadual de Produção e Pesquisa em Saúde, Porto Alegre, RS, Brasil.
authors.forename: Candice Tosi
authors.initials: CT
authors.lastname: Michelon
authors.forename: Franciele
authors.initials: F
authors.lastname: Rosso
authors.forename: Karen Barros
authors.initials: KB
authors.lastname: Schmid
authors.forename: Rosa Dea
authors.initials: RD
authors.lastname: Sperhacke
authors.forename: Martha Maria
authors.initials: MM
authors.lastname: Oliveira
authors.forename: Afrânio Lineu
authors.initials: AL
authors.lastname: Kritski
authors.forename: Leonides
authors.initials: L
authors.lastname: Rezende
authors.forename: Elis Regina Dalla
authors.initials: ER
authors.lastname: Costa
authors.forename: Andrezza Woloski
authors.initials: AW
authors.lastname: Ribeiro
authors.forename: Mirela
authors.initials: M
authors.lastname: Verza
authors.forename: Patrícia Izquierdo
authors.initials: PI
authors.lastname: Cafrune
authors.forename: Márcia Susana Nunes
authors.initials: MS
authors.lastname: Silva
authors.forename: Daniele
authors.initials: D
authors.lastname: Kuhleis
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Zaha
authors.forename: Maria Lucia Rosa
authors.initials: ML
authors.lastname: Rossetti
mesh_terms: D003124:Colorimetry; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*
chemical_list: D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D011933:Reagent Kits, Diagnostic