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

Cho, Sung Rae; Vliet, G. M. E. Van Der; Park, Sun; Baik, S Hyuk; Kim, Sung Kyu; Chong, Yunsop; Kolk, A. H. J.; Klatser, P. R.; Kim, Joo-Deuk

doi:10.1128/jcm.33.3.752-754.1995

Cited by 18 publications

(6 citation statements)

References 14 publications

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“…This technique proved to be as sensitive for the detection of amplified products as traditional Southern hybridization but faster and more convenient, because it does not require radioisotopes, as demonstrated for Mycobacterium leprae (33), hepatitis B virus (17), and human immunodeficiency virus type 1 (24). For bacteria, similar techniques have been used to detect different species of Mycobacterium (M. tuberculosis [8,23,37], M. leprae [33], and M. xenopi [13]), as well as Bordetella pertussis (5), Salmonella species (6), C. trachomatis (3), and enterotoxigenic Escherichia coli (28). These studies have shown that colorimetric hybridization is an effective method for routine diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, several investigators have presented sensitive assays for the diagnosis of various viral (16,17,24,30,35), bacterial (3,5,7,8,20,22,23,28,33,37), fungal (14), and protozoal (2) infections on the basis of nonradioactive (colorimetric or chemiluminescent) nucleic acid hybridization. These kinds of techniques are simple, and colorimetry requires only a conventional microtiter spectrophotometric reader and can be readily done on a large scale.…”

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confidence: 99%

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Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens

1997

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PCR is recognized as a promising method for the detection of Helicobacter pylori in gastric biopsy specimens. However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical laboratories. The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products. The three assays were based on the amplification of a fragment of the ureC gene of H. pylori and a colorimetric hybridization assay. The first assay (GEN-ETI-K DNA enzyme immunoassay; Sorin, Sallugia, Italy) was based on the hybridization of amplified DNA with a probe bound in microtiter wells and detected with labelled anti-DNA antibody. The second assay (Pylori-prob; Biocode, Sclessin, Belgium) comprised a solid-phase sandwich hybridization system with a specific biotinylated probe being used for detection. Finally, the third assay (PCR enzyme-linked immunosorbent assay; Boehringer, Mannheim, Germany) was based on the hybridization of amplified DNA labelled with digoxigenin as a probe (used as a coating in microtiter wells) and detected with antidigoxigenin-peroxidase as conjugate. The sensitivity of the colorimetric assay was evaluated by using amplification products from PCR assays performed on several 10-fold dilutions of DNA from H. pylori CIP 101260, and the specificity was assessed with different urease-positive bacteria. Biopsy specimens from 199 patients were tested; 106 were classified as H. pylori positive, and 93 were classified as H. pylori negative by culture and/or histological examination as the "gold standard." The receiving operating characteristic curve was used to determine the best cutoff point for each assay. The detection of PCR products by colorimetric hybridization increases the sensitivity up to 100-fold compared to that with gel electrophoresis. The results are rapid (4 h) and easy to interpret and can be automated.

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confidence: 99%

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Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens

1997

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“…The use of PCR to detect the presence of Mycobacterium tuberculosis in clinical specimens has been widely reported (3,6,7,9,15). Although PCR can be specific and sensitive, there are difficulties associated with the technique.…”

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Comparative evaluation of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex in respiratory specimens

et al. 1997

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Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor.

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“…These products are captured in a microtiter plate with streptavidincoated wells and detected by hybridization with DIG-labeled oligonucleotide probes. Others have described similar capture assays for the detection of amplicons (3,15,17,18), but these authors did not use a built-in control for inhibition of the PCRs. In our assay, two DIG-labeled oligonucleotide probes are used, permitting detection of amplicons from the M. tuberculosis complex and amplicons from recombinant M. smegmatis 1008 (containing a modified IS6110).…”

Section: Discussionmentioning

confidence: 99%

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control

et al. 1996

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A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization.

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

Cited by 18 publications

References 14 publications

Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens

Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens

Comparative evaluation of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex in respiratory specimens

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control

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