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Method for lysing Mycobacteria using achromopeptidase

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  • Publication Date:
    February 09, 1993
  • معلومة اضافية
    • Patent Number:
      5,185,242
    • Appl. No:
      07/720,076
    • Application Filed:
      June 24, 1991
    • نبذة مختصرة :
      The invention provides a rapid method for lysing Mycobacteria. In one embodiment is provided a method for lysing Mycobacteria which comprises exposing the bacteria to a lysis effective amount of the enzyme achromopeptidase.
      The method of the invention is particularly advantageous since only one step is involved, it is expedient compared to prior methods, and little instrumentation is necessary. By practicing the present invention it is possible to lyse Mycobacteria with minimal effort. In addition, practicing the invention results in liberating cellular components including deoxyribonucleic acid (DNA) from Mycobacteria. Not only is DNA liberated, but the DNA is suited for subsequent analysis by way of probe hybridization, restriction enzyme analysis, and the like.
    • Inventors:
      Keating, William E. (Durham, NC); Robson, Jillian A. (Pittsboro, NC)
    • Assignees:
      Becton Dickinson and Company (Franklin Lakes, NJ)
    • Claim:
      We claim
    • Claim:
      1. A method for lysing Mycobacteria which consists essentially of exposing the Mycobacteria to a lysis effective amount of the enzyme achromopeptidase for a time and under conditions for lysis.
    • Claim:
      2. The method of claim 1 in which the lysis effective amount of achromopeptidase is from about 50 to about 1000 units in about 25 to about 2500 microliters.
    • Claim:
      3. The method of claim 2 in which the lysis effective amount of achromopeptidase is from about 100 to about 300 units in about 100 to about 500 microliters.
    • Claim:
      4. The method of claim 1 in which the Mycobacteria is selected from the group consisting of Mycobacterium avium, Mycobacterium gordonae, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium bovis, Mycobacterium scrofulaceum, Mycobacterium paratuberculosis, Mycobacterium phlei, Mycobacterium marinum, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium intracellulare, Mycobacterium laprae, Mycobacterium xenopi, Mycobacterium ulcerans, Mycobacterium lepraemurium, Mycobacterium flavescens, Mycobacterium terrae, Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium asiaticum, Mycobacterium vaccae, Mycobacterium gastri, Mycobacterium triviale, Mycobacterium haemophicum, Mycobacterium africanum, Mycobacterium thermoresistable, and Mycobacterium smegmatis.
    • Claim:
      5. The method of claim 4 in which the Mycobacteria is Mycobacterium tuberculosis.
    • Claim:
      6. The method of claim 4 in which the Mycobacteria is M. bovis.
    • Claim:
      7. The method of claim 4 in which the Mycobacteria is Mycobacterium africanum.
    • Claim:
      8. The method of claim 4 in which the Mycobacteria is Mycobacterium intracellularae.
    • Claim:
      9. The method of claim 4 in which the Mycobacteria is Mycobacterium avium.
    • Claim:
      10. The method of claim 4 in which the Mycobacteria is Mycobacterium leprae.
    • Claim:
      11. The method of claim 4 in which the Mycobacteria is Mycobacterium chelonae.
    • Claim:
      12. The method of claim 4 in which the Mycobacteria is Mycobacterium paratuberculosis.
    • Claim:
      13. A method of isolating cellular components of Mycobacteria which consists essentially of exposing the Mycobacteria to a lysis effective amount of the enzyme achromopeptidase for a time and under conditions for lysis and isolating cellular components.
    • Claim:
      14. The method of claim 13 in which the cellular component isolated is DNA.
    • Claim:
      15. The method of claim 13 in which the cellular component isolated is RNA.
    • Claim:
      16. A method of amplifying Mycobacteria nucleic acid which consists essentially of exposing the Mycobacteria to a lysis effective amount of the enzyme achromopeptidase for a time and under conditions for lysis and amplifying Mycobacteria nucleic acid.
    • Claim:
      17. The method of claim 16 in which the nucleic acid is DNA.
    • Claim:
      18. The method of claim 16 in which the nucleic acid is RNA.
    • Claim:
      19. The method of claim 5 which further comprises the isolation of DNA.
    • Claim:
      20. The method of claim 6 which further comprises the isolation of DNA.
    • Claim:
      21. The method of claim 7 which further comprises the isolation of DNA.
    • Claim:
      22. The method of claim 8 which further comprises the isolation of DNA.
    • Claim:
      23. The method of claim 9 which further comprises the isolation of DNA.
    • Claim:
      24. A method for identifying Mycobacteria which consists essentially of exposing the Mycobacteria to a lysis effective amount of the enzyme achromopeptidase for a time and under conditions for lysis and adding a Mycobacteria identifying agent.
    • Claim:
      25. The method of claim 24 in which the Mycobacteria identifying agent is a nucleic acid probe.
    • Claim:
      26. The method of claim 25 in which the nucleic acid probe is deoxyribonucleic acid.
    • Claim:
      27. The method of claim 25 in which the nucleic acid probe is ribonucleic acid.
    • Claim:
      28. The method of claim 24 in which the Mycobacteria is obtained from the source selected from the group consisting of feces, sputum, blood, tissue, urine, and other body fluids.
    • Claim:
      29. A kit comprising a Mycobacteria identifying agent and a lysis effective amount of achromopeptidase.
    • Claim:
      30. The kit of claim 29 in which the Mycobacteria identifying agent is a nucleic acid probe.
    • Claim:
      31. The kit of claim 30 in which the nucleic acid probe is deoxyribonucleic acid.
    • Claim:
      32. The kit of claim 29 in which the nucleic acid probe is ribonucleic acid.
    • Claim:
      33. A method for lysing Mycobacteria which consists essentially of isolating Mycobacteria from a sample and exposing the Mycobacteria to a lysis effective amount of achromopeptidase for a time and under conditions for lysis.
    • Claim:
      34. The method of claim 4 in which the lysis effective amount of achromopeptidase is from about 50 to about 1000 units in about 25 to about 2500 microliters.
    • Claim:
      35. The method of claim 4 in which the lysis effective amount of achromopeptidase in from about 100 to about 300 units in about 100 to 500 microliters.
    • Claim:
      36. The method of claim 34 in which the Mycobacteria is selected from the group consisting of Mycobacterium avium, Mycobacterium gordonae, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium bovis, Mycobacterium scrofulaceum, Mycobacterium paratuberculosis, Mycobacterium phlei, Mycobacterium marinum, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium intracellulare, Mycobacterium laprae, Mycobacterium xenopi, Mycobacterium ulcerans, Mycobacterium lepraemurium, Mycobacterium flavescens, Mycobacterium terrae, Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium asiaticum, Mycobacterium vaccae, Mycobacterium gastri, Mycobacterium triviale, Mycobacterium haemophicum, Mycobacterium africanum, Mycobacterium thermoresistable, and Mycobacterium smegmatis.
    • Claim:
      37. The method of claim 33 in which the Mycobacteria is Mycobacterium tuberculosis.
    • Claim:
      38. The method of claim 33 in which the Mycobacteria is Mycobacterium intracellulare.
    • Claim:
      39. The method of claim 33 which further comprises isolation of cellular components.
    • Claim:
      40. The method of claim 4 which further comprises amplification of nucleic acid.
    • Claim:
      41. The method of claim 4 which further comprises the addition of a Mycobacteria identifying agent.
    • Claim:
      42. The method of claim 38 in which the identifying agent is a nucleic acid probe.
    • Current U.S. Class:
      435/6; 435/220; 4352/531; 435/270; 435/91; 536/231
    • Current International Class:
      C12Q 168; C12N 952; C12P 1934; C07H 2100
    • Patent References Cited:
      4900677 February 1990 Hewitt
    • Other References:
      Zinsser Microbiology, 1984, Joklik et al., eds. Appleton-Century-Crofts, Norwalk, Conn., p. 12.
      D. L. Whipple et al., J. Clin. Microbiol. 25:1511 (1987).
      R. N. Picken et al., Molecular and Cellular Probes 2:289 (1988).
      S. S. Hurley et al., J. Clin. Microbiol. 25:2227 (1987).
      T. Ezaki et al., J. Clin. Microbiol. 16:844 (1982).
    • Primary Examiner:
      Yarbrough, Amelia B.
    • Assistant Examiner:
      Bennett, Lisa
    • Attorney, Agent or Firm:
      Stierwalt, Brian K.
    • الرقم المعرف:
      edspgr.05185242