Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA

Abstract

The detection of amplified products resulting from polymerase chain reactions (PCRs) remains a complicated process. To simplify the detection procedures, we developed a colorimetric microtiter plate hybridization assay for the specific detection of 5'-biotinylated PCR fragments of Mycobacterium leprae DNA. For this assay, an M. leprae DNA capture probe was made and immobilized on the wells of a microtiter plate. Hybridization of the biotin-labeled PCR fragments was detected through enzymatic color development. The resulting optical densities showed a logarithm-linear relationship with the amount of template DNA and corresponded to the intensity of the bands obtained through gel analysis and Southern blotting of the PCR products. The sensitivity of the assay was found to be 125 fg of genomic M. leprae DNA, or 20 lysed bacilli, revealing a detection limit similar to that of agarose gel analysis. The efficient coamplification of human DNA was used as a positive control for the presence of inhibitory substances in clinical material. For detection of human PCR products, a human DNA capture probe was also constructed for the colorimetric assay. This dual setup for hybridization, which thus detected both M. leprae and human DNA PCR products, was useful for ascertaining the presence of inhibiting substances in clinical specimens. All biopsy specimens (n = 10) from untreated patients with leprosy were positive. Apparently, this assay is more sensitive than microscopy, because biopsy specimens from half of the patients were negative upon histopathological examination. Biopsy specimens from three treated patients were negative, as were those from the three patients who did not have leprosy. We conclude that this colorimetric assay can replace agarose gel analysis and Southern hybridization, because it is as sensitive as those methods. Its advantages over conventional gel analysis and Southern hybridization are that it is less cumbersome and more rapid.

Real-time PCR in the microbiology laboratory

“…Existing combinations of PCR and amplicon detection assays will be called 'conventional PCR' throughout this review. The detection components include agarose gel electrophoresis [19], Southern blot [20] and ELISA-like systems [21]. Conventional PCR has been used to obtain quantitative data, with promising results [22].…”

Colorimetric microwell plate hybridization assay for detection of amplified Mycobacterium tuberculosis DNA from sputum samples

“…An objective quantitation of the amplified products would make it possible to determine a significant level of amplified DNA which is clearly relevant for active tuberculosis. Recently, it was shown that the PCR in combination with the colorimetric microwell plate hybridization assay (CoMPHA) could be used for semiquantitative analysis of the level of Mycobacterium leprae template DNA in biopsy specimens (14). Here, we show that 5Ј-biotinylated PCR products of M. tuberculosis can be detected by an M. tuberculosis capture probe in the CoMPHA, allowing a clear distinction between PCR-positive and PCRnegative samples.…”

“…Amplification with this primer set resulted in a 188-bp fragment with restriction sites at both ends. This fragment was digested and cloned into M13BM20 and subsequently transfected in Escherichia coli as described previously (14). Single-stranded DNA was isolated from the recombinant M13 phages and served as a capture probe for identification of the M. tuberculosis PCR products.…”

Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

“…Whilst Southern blot detection of amplicon by hybridisation with a labelled oligonucleotide probe (oligoprobe) increases the specificity of amplicon detection, it is time consuming, frequently uses radioactive labels and requires multiple PCR product handling steps, increasing the risk of spreading amplicon throughout the laboratory (Holland et al, 1991). Alternatively, PCR -ELISA has been used to capture amplicon onto a solid phase via biotin or digoxigenin-labelled primers, oligoprobes or by direct capture after incorporation of the biotin or digoxigenin into the amplicon (van der Vliet et al, 1993;Keller et al, 1990;Kemp et al, 1990;Kox et al, 1996;Dekonenko et al, 1997;Watzinger et al, 2001). Once captured, amplicon is detected using an enzyme-labelled avidin or anti-digoxigenin reporter molecule in a manner similar to a standard ELISA format.…”