Published online before print August 6, 2009

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Journal of Molecular Diagnostics 2009, Vol. 11, No. 5
Copyright © 2009 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2009.080135

Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons

Parameet Kumar^*, Kapili Nath^*, Bimba Rath, Manas K. Sen, Potharuju Vishalakshi, Devender S. Chauhan^¶, Vishwa M. Katoch^||, Sarman Singh^**, Sanjay Tyagi, Vishnubhatla Sreenivas and Hanumanthappa K. Prasad^*

From the Departments of Biotechnology, * Division of Clinical Microbiology, Laboratory Medicine, ** and Biostatistics, All India Institute of Medical Sciences, New Delhi, India; the Department of Pediatrics, Kalawati Saran Children’s Hospital, New Delhi, India; the Department of Pulmonary Critical Care and Sleep Medicine, VMMC and Safdarjung Hospital, New Delhi, India; the LRS Institute of Tuberculosis and Respiratory Diseases, New Delhi, India; the National JALMA Institute of Leprosy and other Mycobacterial Diseases, ^¶ Tajganj, Agra, India; the Department of Health Research, Ministry of Health and Family Welfare, ^|| Government of India and Indian Council of Medial Research, New Delhi, India; and the Department of Molecular Genetics, Public Health Research Institute, New York, New York

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.