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Journal of Clinical Microbiology, March 2000, p. 1166-1169, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Single-Tube Balanced Heminested PCR for Detecting Mycobacterium tuberculosis in Smear-Negative Samples

Albert García-Quintanilla,1 Lourdes Garcia,2 Griselda Tudó,1 Maria Navarro,1 Julià González,3,* and Maria T. Jiménez de Anta3

Departament de Microbiologia i Parasitologia Sanitàries, Institut d'Investigacions Biomèdiques Agustí Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona,1 and Servei de Microbiologia, Departament de Microbiologia i Parasitologia Sanitàries, IDIBAPS, Hospital Clínic,3 Villarroel 170, 08036 Barcelona, and Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Campus Universitari, 08193 Bellaterra,2 Spain

Received 6 July 1999/Returned for modification 9 September 1999/Accepted 6 December 1999

In order to achieve more sensitive and specific results for the rapid diagnosis of tuberculosis, we have developed a new method, named balanced heminested PCR, which avoids the inconvenience of asymmetric amplification and has the advantages of single-tube heminested PCR. This was achieved by replacing the outer primer that participates in both rounds of amplification in the standard heminested technique by another primer containing the sequence of the inner primer attached at its 5' end. When both techniques were tested for the IS6110 target of Mycobacterium tuberculosis complex in 80 smear-negative culture-positive sputum samples and 60 control samples, the results showed 100% specificity for both techniques and sensitivities of 60 and 75% for heminested PCR and balanced heminested PCR, respectively (P = 0.02). In conclusion, the balanced heminested technique shows a higher sensitivity than that of the standard heminested, and it could be applied to any PCR by attaching the inner primer at the 5' end of the opposite outer primer. Thus, the balanced heminested technique provides a target for the inner primer in both strands, avoiding asymmetric amplification and thereby resulting in a more efficient amplification, and, in practice, a higher sensitivity without loss of specificity and with a minimum risk of cross-contamination.

* Corresponding author. Mailing address: Servei de Microbiologia, Hospital Clínic, c/Villarroel 170, Barcelona 08036, Spain. Phone: 34-932275522. Fax: 34-932275454. E-mail: jgm{at}medicina.ub.es.

Journal of Clinical Microbiology, March 2000, p. 1166-1169, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Garcia-Quintanilla, A., Gonzalez-Martin, J., Tudo, G., Espasa, M., Jimenez de Anta, M. T. (2002). Simultaneous Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex in Clinical Samples by 5'-Exonuclease Fluorogenic PCR. J. Clin. Microbiol. 40: 4646-4651 [Abstract] [Full Text]  
  • Patnaik, M., Liegmann, K., Peter, J. B. (2001). Rapid Detection of Smear-Negative Mycobacterium tuberculosis by PCR and Sequencing for Rifampin Resistance with DNA Extracted Directly from Slides. J. Clin. Microbiol. 39: 51-52 [Abstract] [Full Text]  


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