Single-tube balanced heminested PCR for detecting Mycobacterium tuberculosis in smear-negative samples

In order to achieve more sensitive and specific results for the rapid diagn osis of tuberculosis, we have developed a new method, named balanced hemine sted PCR, which avoids the inconvenience of asymmetric amplification and ha s the advantages of single-tube heminested PCR, This was achieved by replac ing the outer primer that participates in both rounds of amplification in t he standard heminested technique by another primer containing the sequence of the inner primer attached at its 5' end, When both techniques were teste d for the IS6110 target of Mycobacterium tuberculosis complex in 80 smear-n egative 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 b y attaching the inner primer at the 5' end of the opposite outer primer. Th us, the balanced heminested technique provides a target for the inner prime r in both strands, avoiding asymmetric amplification and thereby resulting in a more efficient amplification, and, in practice, a higher sensitivity w ithout loss of specificity and with a minimum risk of cross-contamination.