title: Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine.
abstract: BACKGROUND
  Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples.
  
  
  METHODS
  Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested).
  
  
  RESULTS
  STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary).
  
  
  CONCLUSIONS
  STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Juliana Figueirêdo da Costa
authors.initials: JF
authors.lastname: Lima
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Gabriela de Moraes Rêgo
authors.initials: Gde M
authors.lastname: Guedes
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Juliana Falcão de Araújo
authors.initials: JF
authors.lastname: Lima
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Laís Ariane de Siqueira
authors.initials: LA
authors.lastname: Lira
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Fabiana Cristina Fulco
authors.initials: FC
authors.lastname: Santos
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Mercia Eliane de
authors.initials: ME
authors.lastname: Arruda
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Lílian Maria Lapa
authors.initials: LM
authors.lastname: Montenegro
authors.affiliation: Laboratório de Imunoepidemiologia, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, Brazil.
authors.forename: Haiana Charifker
authors.initials: HC
authors.lastname: Schindler
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D004269:DNA, Bacterial / Q000097:blood / Q000652:urine; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis*; D055815:Young Adult
chemical_list: D004269:DNA, Bacterial