title: Loop-mediated isothermal amplification assay for rapid and sensitive diagnosis of tuberculosis.
abstract: OBJECTIVE
  Loop-mediated isothermal amplification (LAMP) is a newly developed molecular method that can be performed isothermally. We developed and evaluated a LAMP assay using novel primers to diagnose tuberculosis directly from clinical samples.
  
  
  METHODS
  Primers were designed to amplify the specific novel esat-6 gene target of Mycobacterium tuberculosis (MTB). Quantitated DNA was used to determine analytical sensitivity and specificity was evaluated by testing 29 NTM and 37 other bacterial species. After standardization, its sensitivity and specificity were evaluated on samples from 118 TB suspected and 31 non-TB patients and compared it with smear, culture and mPCR methods.
  
  
  RESULTS
  LAMP was able to detect 5 fg DNA (one MTB) within 21 min and found to be 10 times more sensitive than mPCR and showed 100% specificity against NTM and other bacterial species. In clinical samples, LAMP showed highest MTB detection rate (52.5%) as compared to mPCR (44%) and culture (30.5%). On culture positive and mPCR positive samples, the sensitivity of LAMP was found to be 100% (95% CI 90.2-100) and 96.1% (95% CI 86.7-99.5) respectively with 93.5% (95% CI 78.5-99.2) of overall specificity.
  
  
  CONCLUSIONS
  LAMP was found to be more sensitive than culture and mPCR for the detection of MTB. It showed specificity comparable to mPCR but was rapid and cost effective.
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, All India Institute of Medical Sciences, New Delhi 110029, India.
authors.forename: Parveen
authors.initials: P
authors.lastname: Kumar
authors.affiliation: AmpliGene India Biotech Pvt. Ltd., Ahmedabad, Gujarat, India.
authors.forename: Deepal
authors.initials: D
authors.lastname: Pandya
authors.affiliation: National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India.
authors.forename: Niti
authors.initials: N
authors.lastname: Singh
authors.affiliation: National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India.
authors.forename: Digambar
authors.initials: D
authors.lastname: Behera
authors.affiliation: Department of Emergency Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Praveen
authors.initials: P
authors.lastname: Aggarwal
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, All India Institute of Medical Sciences, New Delhi 110029, India. Electronic address: sarman_singh@yahoo.com.
authors.forename: Sarman
authors.initials: S
authors.lastname: Singh
mesh_terms: D000328:Adult; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*; D055815:Young Adult
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: LAMP; Multiplex PCR; Mycobacterium tuberculosis; esat-6