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

Abstract

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 IS6110target 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.

Pulmonary tuberculosis in the West Bank, Palestinian Authority: molecular diagnostic approach

“…This phenomenon is not unique as it has been reported in several studies using similar methods (Clarridge et al 1993;Patnaik et al 2001;Lima et al 2008). To overcome the low sensitivity from sputum smear-negative samples, a nested PCR amplification has been used to enhance the DNA amplicon and found to be sufficient (Garcia-Quintanilla et al 2000). Detection of the bacteria among patients with a history of TB and patients having treatment because of active tuberculosis has been described previously (Rajalahti et al 1998).…”

“…2008). To overcome the low sensitivity from sputum smear‐negative samples, a nested PCR amplification has been used to enhance the DNA amplicon and found to be sufficient (Garcia‐Quintanilla et al. 2000).…”

A Genotyping Method for Detecting Foreign Buffalo Material in Mozzarella di Bufala Campana Cheese Using Allele-Specific- and Single-Tube Heminested-Polymerase Chain Reaction

“…To establish the presence/absence of amplified fragments with certainty and to directly determine the sample genotype, the AS-PCR analysis of DNA extracted from the somatic cells of milk samples (see Materials and Methods, Section 2.2 ) was performed using AS-FW and A1C-RV primers, of which the former was specific for the polymorphic variant. In parallel, the DNA extracted from cheese samples was analyzed using STHN-PCR [ 15 , 16 ] with a set of outer primers A1C-FW and A1C-RV, which were utilized in combination with the internal primer AS-FW. To increase the assay sensitivity and remove carry-over contamination, the subsequent opening of the tubes was avoided by adding reagents and oligonucleotides before the beginning of the amplification reaction [ 17 , 18 ].…”

Performance of the BD ProbeTec ET direct detection assay for the analysis of Mycobacterium tuberculosis in respiratory and non-respiratory clinical specimens

“…It is therefore imperative that accurate diagnosis of MTB and M. tuberculosis complex (MTBC) be sought because of the infectious nature of the organism. It was for this reason that a number of techniques based on polymerase chain reaction (PCR) amplification of Mycobacterium specific nucleic acids were introduced7, 8, 9, 10 for the direct detection of MTB; this includes the BD ProbeTec ET MBTC direct detection assay (DTB) 11 . The DTB assay is a semi-automated real-time molecular technique that targets MTB specific DNA (IS6110) using specific primers and fluorescently tagged probes.…”