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. 1992 Dec;30(12):3095–3098. doi: 10.1128/jcm.30.12.3095-3098.1992

Effects of fixation on polymerase chain reaction detection of Mycobacterium leprae.

P Fiallo 1, D L Williams 1, G P Chan 1, T P Gillis 1
PMCID: PMC270594  PMID: 1452690

Abstract

The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Dubeau L., Chandler L. A., Gralow J. R., Nichols P. W., Jones P. A. Southern blot analysis of DNA extracted from formalin-fixed pathology specimens. Cancer Res. 1986 Jun;46(6):2964–2969. [PubMed] [Google Scholar]
  2. FRAENKEL-CONRAT H. Reaction of nucleic acid with formaldehyde. Biochim Biophys Acta. 1954 Oct;15(2):307–309. doi: 10.1016/0006-3002(54)90083-9. [DOI] [PubMed] [Google Scholar]
  3. Franzblau S. G., White K. E. Comparative in vitro activities of 20 fluoroquinolones against Mycobacterium leprae. Antimicrob Agents Chemother. 1990 Feb;34(2):229–231. doi: 10.1128/aac.34.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Goelz S. E., Hamilton S. R., Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun. 1985 Jul 16;130(1):118–126. doi: 10.1016/0006-291x(85)90390-0. [DOI] [PubMed] [Google Scholar]
  5. HASELKORN R., DOTY P. The reaction of formaldehyde with polynucleotides. J Biol Chem. 1961 Oct;236:2738–2745. [PubMed] [Google Scholar]
  6. Hartskeerl R. A., de Wit M. Y., Klatser P. R. Polymerase chain reaction for the detection of Mycobacterium leprae. J Gen Microbiol. 1989 Sep;135(9):2357–2364. doi: 10.1099/00221287-135-9-2357. [DOI] [PubMed] [Google Scholar]
  7. Impraim C. C., Saiki R. K., Erlich H. A., Teplitz R. L. Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun. 1987 Feb 13;142(3):710–716. doi: 10.1016/0006-291x(87)91472-0. [DOI] [PubMed] [Google Scholar]
  8. Olive D. M. Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. J Clin Microbiol. 1989 Feb;27(2):261–265. doi: 10.1128/jcm.27.2.261-265.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. RIDLEY D. S., JOPLING W. H. A classification of leprosy for research purposes. Lepr Rev. 1962 Apr;33:119–128. doi: 10.5935/0305-7518.19620014. [DOI] [PubMed] [Google Scholar]
  10. Shibata D. K., Arnheim N., Martin W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med. 1988 Jan 1;167(1):225–230. doi: 10.1084/jem.167.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Starnbach M. N., Falkow S., Tompkins L. S. Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization. J Clin Microbiol. 1989 Jun;27(6):1257–1261. doi: 10.1128/jcm.27.6.1257-1261.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Williams D. L., Gillis T. P., Booth R. J., Looker D., Watson J. D. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. J Infect Dis. 1990 Jul;162(1):193–200. doi: 10.1093/infdis/162.1.193. [DOI] [PubMed] [Google Scholar]
  13. de Wit M. Y., Faber W. R., Krieg S. R., Douglas J. T., Lucas S. B., Montreewasuwat N., Pattyn S. R., Hussain R., Ponnighaus J. M., Hartskeerl R. A. Application of a polymerase chain reaction for the detection of Mycobacterium leprae in skin tissues. J Clin Microbiol. 1991 May;29(5):906–910. doi: 10.1128/jcm.29.5.906-910.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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