Abstract
A PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid identification of mycobacterial DNA coding for 16S rRNA was developed. The PCR selectively targeted and amplified part of the 16S rRNA gene from all mycobacteria while simultaneously labelling one strand of the amplified product with a 5' fluorescein-labelled primer. The identity of the labelled strand was subsequently determined by hybridization to a panel of mycobacterial species-specific capture probes, which were immobilized via their 5' biotin ends to a streptavidin-coated microtiter plate. Specific hybridization of a 5' fluorescein-labelled strand to a species probe was detected colorimetrically with an anti-fluorescein enzyme conjugate. The assay was able to identify 10 Mycobacterium spp. A probe able to hybridize to all Mycobacterium species (All1) was also included. By a heminested PCR, the assay was sensitive enough to detect as little as 10 fg of DNA, which is equivalent to approximately three bacilli. The assay was able to detect and identify mycobacteria directly from sputa. The specificities of the capture probes were assessed by analysis of 60 mycobacterial strains corresponding to 18 species. Probes Avi1, Int1, Kan1, Xen1, Che1, For1, Mal1, Ter1, and Gor1 were specific. The probe Tbc1 cross-hybridized with the Mycobacterium terrae amplicon. Analysis of 35 strains tested blind resulted in 34 strains being correctly identified. This method could be used for rapid identification of early cultures and may be suitable for the detection and concurrent identification of mycobacteria within clinical specimens.
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- Böddinghaus B., Rogall T., Flohr T., Blöcker H., Böttger E. C. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol. 1990 Aug;28(8):1751–1759. doi: 10.1128/jcm.28.8.1751-1759.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Böddinghaus B., Wolters J., Heikens W., Böttger E. C. Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level. FEMS Microbiol Lett. 1990 Jul;58(2):197–203. doi: 10.1111/j.1574-6968.1990.tb13978.x. [DOI] [PubMed] [Google Scholar]
- Citron D. M., Baron E. J., Finegold S. M., Goldstein E. J. Short prereduced anaerobically sterilized (PRAS) biochemical scheme for identification of clinical isolates of bile-resistant Bacteroides species. J Clin Microbiol. 1990 Oct;28(10):2220–2223. doi: 10.1128/jcm.28.10.2220-2223.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cormican M., Glennon M., Riain U. N., Flynn J. Multiplex PCR for identifying mycobacterial isolates. J Clin Pathol. 1995 Mar;48(3):203–205. doi: 10.1136/jcp.48.3.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Beenhouwer H., Liang Z., De Rijk P., Van Eekeren C., Portaels F. Detection and identification of mycobacteria by DNA amplification and oligonucleotide-specific capture plate hybridization. J Clin Microbiol. 1995 Nov;33(11):2994–2998. doi: 10.1128/jcm.33.11.2994-2998.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Debrunner M., Salfinger M., Brändli O., von Graevenitz A. Epidemiology and clinical significance of nontuberculous mycobacteria in patients negative for human immunodeficiency virus in Switzerland. Clin Infect Dis. 1992 Aug;15(2):330–345. doi: 10.1093/clinids/15.2.330. [DOI] [PubMed] [Google Scholar]
- Eisenach K. D., Cave M. D., Bates J. H., Crawford J. T. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis. 1990 May;161(5):977–981. doi: 10.1093/infdis/161.5.977. [DOI] [PubMed] [Google Scholar]
- Falkinham J. O., 3rd Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev. 1996 Apr;9(2):177–215. doi: 10.1128/cmr.9.2.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fiss E. H., Chehab F. F., Brooks G. F. DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory. J Clin Microbiol. 1992 May;30(5):1220–1224. doi: 10.1128/jcm.30.5.1220-1224.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ford E. G., Snead S. J., Todd J., Warren N. G. Strains of Mycobacterium terrae complex which react with DNA probes for M. tuberculosis complex. J Clin Microbiol. 1993 Oct;31(10):2805–2806. doi: 10.1128/jcm.31.10.2805-2806.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hance A. J., Grandchamp B., Lévy-Frébault V., Lecossier D., Rauzier J., Bocart D., Gicquel B. Detection and identification of mycobacteria by amplification of mycobacterial DNA. Mol Microbiol. 1989 Jul;3(7):843–849. doi: 10.1111/j.1365-2958.1989.tb00233.x. [DOI] [PubMed] [Google Scholar]
- Higgins D. G., Bleasby A. J., Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992 Apr;8(2):189–191. doi: 10.1093/bioinformatics/8.2.189. [DOI] [PubMed] [Google Scholar]
- Kirschner P., Böttger E. C. Microheterogeneity within rRNA of Mycobacterium gordonae. J Clin Microbiol. 1992 Apr;30(4):1049–1050. doi: 10.1128/jcm.30.4.1049-1050.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirschner P., Kiekenbeck M., Meissner D., Wolters J., Böttger E. C. Genetic heterogeneity within Mycobacterium fortuitum complex species: genotypic criteria for identification. J Clin Microbiol. 1992 Nov;30(11):2772–2775. doi: 10.1128/jcm.30.11.2772-2775.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirschner P., Rosenau J., Springer B., Teschner K., Feldmann K., Böttger E. C. Diagnosis of mycobacterial infections by nucleic acid amplification: 18-month prospective study. J Clin Microbiol. 1996 Feb;34(2):304–312. doi: 10.1128/jcm.34.2.304-312.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirschner P., Springer B., Vogel U., Meier A., Wrede A., Kiekenbeck M., Bange F. C., Böttger E. C. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol. 1993 Nov;31(11):2882–2889. doi: 10.1128/jcm.31.11.2882-2889.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kox L. F., van Leeuwen J., Knijper S., Jansen H. M., Kolk A. H. PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples. J Clin Microbiol. 1995 Dec;33(12):3225–3233. doi: 10.1128/jcm.33.12.3225-3233.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kusunoki S., Ezaki T. Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov. Int J Syst Bacteriol. 1992 Apr;42(2):240–245. doi: 10.1099/00207713-42-2-240. [DOI] [PubMed] [Google Scholar]
- Martin C., Lévy-Frébault V. V., Cattier B., Legras A., Goudeau A. False positive result of Mycobacterium tuberculosis complex DNA probe hybridization with a Mycobacterium terrae isolate. Eur J Clin Microbiol Infect Dis. 1993 Apr;12(4):309–310. doi: 10.1007/BF01967270. [DOI] [PubMed] [Google Scholar]
- Patel S., Wall S., Saunders N. A. Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains. J Clin Microbiol. 1996 Jul;34(7):1686–1690. doi: 10.1128/jcm.34.7.1686-1690.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pierre C., Lecossier D., Boussougant Y., Bocart D., Joly V., Yeni P., Hance A. J. Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol. 1991 Apr;29(4):712–717. doi: 10.1128/jcm.29.4.712-717.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plikaytis B. B., Plikaytis B. D., Yakrus M. A., Butler W. R., Woodley C. L., Silcox V. A., Shinnick T. M. Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis. J Clin Microbiol. 1992 Jul;30(7):1815–1822. doi: 10.1128/jcm.30.7.1815-1822.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rogall T., Flohr T., Böttger E. C. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol. 1990 Sep;136(9):1915–1920. doi: 10.1099/00221287-136-9-1915. [DOI] [PubMed] [Google Scholar]
- Rogall T., Wolters J., Flohr T., Böttger E. C. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol. 1990 Oct;40(4):323–330. doi: 10.1099/00207713-40-4-323. [DOI] [PubMed] [Google Scholar]
- Shinnick T. M., Good R. C. Mycobacterial taxonomy. Eur J Clin Microbiol Infect Dis. 1994 Nov;13(11):884–901. doi: 10.1007/BF02111489. [DOI] [PubMed] [Google Scholar]
- Springer B., Böttger E. C., Kirschner P., Wallace R. J., Jr Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S rRNA gene and proposal of Mycobacterium mucogenicum sp. nov. Int J Syst Bacteriol. 1995 Apr;45(2):262–267. doi: 10.1099/00207713-45-2-262. [DOI] [PubMed] [Google Scholar]
- Springer B., Stockman L., Teschner K., Roberts G. D., Böttger E. C. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol. 1996 Feb;34(2):296–303. doi: 10.1128/jcm.34.2.296-303.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Telenti A., Marchesi F., Balz M., Bally F., Böttger E. C., Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 1993 Feb;31(2):175–178. doi: 10.1128/jcm.31.2.175-178.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vaneechoutte M., De Beenhouwer H., Claeys G., Verschraegen G., De Rouck A., Paepe N., Elaichouni A., Portaels F. Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J Clin Microbiol. 1993 Aug;31(8):2061–2065. doi: 10.1128/jcm.31.8.2061-2065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Victor T., du Toit R., van Helden P. D. Purification of sputum samples through sucrose improves detection of Mycobacterium tuberculosis by polymerase chain reaction. J Clin Microbiol. 1992 Jun;30(6):1514–1517. doi: 10.1128/jcm.30.6.1514-1517.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wayne L. G., Sramek H. A. Agents of newly recognized or infrequently encountered mycobacterial diseases. Clin Microbiol Rev. 1992 Jan;5(1):1–25. doi: 10.1128/cmr.5.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson P. A., Phipps J., Samuel D., Saunders N. A. Development of a simplified polymerase chain reaction-enzyme immunoassay for the detection of Chlamydia pneumoniae. J Appl Bacteriol. 1996 Apr;80(4):431–438. doi: 10.1111/j.1365-2672.1996.tb03239.x. [DOI] [PubMed] [Google Scholar]
- Wilton S., Cousins D. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl. 1992 May;1(4):269–273. doi: 10.1101/gr.1.4.269. [DOI] [PubMed] [Google Scholar]
- Woods G. L., Washington J. A., 2nd Mycobacteria other than Mycobacterium tuberculosis: review of microbiologic and clinical aspects. Rev Infect Dis. 1987 Mar-Apr;9(2):275–294. doi: 10.1093/clinids/9.2.275. [DOI] [PubMed] [Google Scholar]