García-Quintanilla A, Garcia L, Tudó G, Navarro M, González J, Jiménez de Anta M T
Departament de Microbiologia i Parasitologia Sanitàries, Institut d'Investigacions Biomèdiques Agustí Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Villarroel 170, 08036 Barcelona, Spain.
J Clin Microbiol. 2000 Mar;38(3):1166-9. doi: 10.1128/JCM.38.3.1166-1169.2000.
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.
为了在结核病快速诊断中获得更灵敏和特异的结果，我们开发了一种新方法，称为平衡半巢式PCR，该方法避免了不对称扩增的不便之处，并具有单管半巢式PCR的优点。这是通过用另一种在其5'端连接有内引物序列的引物取代标准半巢式技术中参与两轮扩增的外引物来实现的。当在80份涂片阴性培养阳性痰标本和60份对照标本中对结核分枝杆菌复合群的IS6110靶标进行这两种技术检测时，结果显示两种技术的特异性均为100%，半巢式PCR和平衡半巢式PCR的灵敏度分别为60%和75%（P = 0.02）。总之，平衡半巢式技术显示出比标准半巢式技术更高的灵敏度，并且通过将内引物连接到相对外引物的5'端，它可应用于任何PCR。因此，平衡半巢式技术为两条链中的内引物提供了靶标，避免了不对称扩增，从而实现了更有效的扩增，并且在实际应用中，在不损失特异性且交叉污染风险最小的情况下具有更高的灵敏度。
