1887

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Volume 39, Issue 4

Use of PCR-mediated amplification of DNA in different types of clinical samples for the diagnosis of leprosy Free

Abstract

Summary

DNA of , obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an -specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, -specific amplification was obtained with as low as 100 ag (1 ag = 10 g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5 + to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.

  • Received:
  • Accepted:
  • Published Online:
© Society for General Microbiology, 1993
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1993-10-01
2024-04-28
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References

  1. Clark-Curtiss JE. Benefits of recombinant DNA technology for the study of Mycobacterium leprae. Curr Top Microbiol Immunol 1988; 138:61–79
     [Google Scholar]
  2. Woods SA, Cole ST. A rapid method for the detection of potentially viable Mycobacterium leprae in human biopsies: a novel application of PCR. FEMS Microbiol Lett 1989; 65:305–310
     [Google Scholar]
  3. Hartskeerl RA, De Wit MYL, Klatser PR. Polymerase chain reaction for the detection of Mycobacterium leprae. J Gen Microbiol 1989; 135:2357–2364
     [Google Scholar]
  4. Plikaytis BB, Gelber RH, Shinnick TM. Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay. J Clin Microbiol 1990; 28:1913–1917
     [Google Scholar]
  5. Williams DL, Gillis TP, Booth RJ, Looker D, Watson JD. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae. J Infect Dis 1990; 162:193–200
     [Google Scholar]
  6. Hermans PWM, Schuitema ARJ, van Soolingen D et al. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J Clin Microbiol 1990; 28:1204–1213
     [Google Scholar]
  7. De Wit MYL, Faber WR, Krieg SR et al. Application of a polymerase chain reaction for the detection of Mycobacterium leprae in skin tissues. J Clin Microbiol 1991; 29:906–910
     [Google Scholar]
  8. Wright PA, Wynford-Thomas D. The polymerase chain reaction : miracle or mirage? A critical review of its uses and limitations in diagnosis and research. J Pathol 1990; 162:99–117
     [Google Scholar]
  9. Brisson-Noel A, Lecossier D, Nassif X, Gicquel B, Levy-Frebault V, Hance AJ. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet 1989; 2:1069–1071
     [Google Scholar]
  10. Arnoldi J, Schliiter C, Duchrow M et al. Species-specific assessment of Mycobacterium leprae in skin biopsies by in situ hybridization and polymerase chain reaction. Lab Invest 1992; 66:618–623
     [Google Scholar]
  11. Draper P. Cell walls of Mycobacterium leprae. Int J Leprosy 1976; 44:95–98
     [Google Scholar]
  12. Santos AR, De-Miranda AB, Lima LM, Suffys PN, Degrave WM. Method for high yield preparation in large and small scale of nucleic acids from mycobacteria. J Microbiol Methods 1992; 15:83–94
     [Google Scholar]
  13. Mehra V, Sweetser D, Young RA. Efficient mapping of protein antigenic determinants. Proc Natl Acad Sci USA 1986; 83:7013–7017
     [Google Scholar]
  14. Sambrook J, Fritsch E, Maniatis T. Molecular cloning. A laboratory manual, Second edition. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989
     [Google Scholar]
  15. Leiker DL, McDougall AC. Technical guide for smear examination for leprosy by direct microscopy. Amsterdam: Leprosy Documentation Service; 1983
     [Google Scholar]
  16. Jopling WH, McDougall AC. Handbook of leprosy, 4th edn. Oxford: Heinemann Medical Books; 1988
     [Google Scholar]
  17. Sritharan V, Barker RH. A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR. Mol Cell Probes 1991; 5:385–395
     [Google Scholar]
  18. Clark-Curtiss JE, Jacobs WR, Docherty MA, Ritchie LR, Curtiss R. Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J Bacteriol 1985; 161:1093–1102
     [Google Scholar]
  19. Ridley DS, Jopling WH. Classification of leprosy according to immunity—a five group system. Int J Lepr 1966; 34:255–273
     [Google Scholar]
  20. Buck GE, O’Hara LC, Summersgill JT. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol 1992; 30:1331–1334
     [Google Scholar]
  21. Panaccio M, Lew A. PCR based diagnosis in the presence of 8% (v/v) blood. Nucleic Acids Res 1991; 19:1151
     [Google Scholar]
  22. Rao PS, Ekambaram V, Reddy BN, Krishnamoorthy P, Kumar SK, Dutta A. Is bacteriological examination by skin smear necessary in all paucibacillary leprosy patients in mass control programmes?. Lepr Rev 1991; 62:303–309
     [Google Scholar]
  23. Odinsen O, Nilson T, Humber DP. Viability of Mycobacterium leprae: a comparison of morphological index and fluorescent staining techniques in slit-skin smears and M. leprae suspensions. Int J Other Micobact Dis 1986; 54:403–408
     [Google Scholar]
  24. Gillis TP, Williams DL. Polymerase chain reaction and leprosy. Int J Lepr Other Mycobact Dis 1991; 59:311–316
     [Google Scholar]
  25. Shankara KM, Bedi BMS, Kasturi G, Kirchheimer WF, Balasubrahmanyan M. Demonstration of Mycobacterium leprae and its viability in the peripheral blood of leprosy patients. Lepr Rev 1972; 43:181–187
     [Google Scholar]
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Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy
J. Med. Microbiol. 39, 298 (1993); https://doi.org/10.1099/00222615-39-4-298
/content/journal/jmm/10.1099/00222615-39-4-298
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