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. 1991 Dec;29(12):2805–2808. doi: 10.1128/jcm.29.12.2805-2808.1991

Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine.

M J Gerritsen 1, T Olyhoek 1, M A Smits 1, B A Bokhout 1
PMCID: PMC270437  PMID: 1757552

Abstract

An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitive detection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction (PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows were used. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 10(7) to 10(8) Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelleted leptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation method enabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the need for dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by experimentally infected cows.

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Selected References

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

  1. Arthur R. R., Dagostin S., Shah K. V. Detection of BK virus and JC virus in urine and brain tissue by the polymerase chain reaction. J Clin Microbiol. 1989 Jun;27(6):1174–1179. doi: 10.1128/jcm.27.6.1174-1179.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bej A. K., Steffan R. J., DiCesare J., Haff L., Atlas R. M. Detection of coliform bacteria in water by polymerase chain reaction and gene probes. Appl Environ Microbiol. 1990 Feb;56(2):307–314. doi: 10.1128/aem.56.2.307-314.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bercovich Z., Taaijke R., Bokhout B. A. Evaluation of an ELISA for the diagnosis of experimentally induced and naturally occurring Leptospira hardjo infections in cattle. Vet Microbiol. 1990 Jan;21(3):255–262. doi: 10.1016/0378-1135(90)90036-u. [DOI] [PubMed] [Google Scholar]
  4. Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clewley J. P. Polymerase chain reaction assay of parvovirus B19 DNA in clinical specimens. J Clin Microbiol. 1989 Dec;27(12):2647–2651. doi: 10.1128/jcm.27.12.2647-2651.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellis W. A., Songer J. G., Montgomery J., Cassells J. A. Prevalence of Leptospira interrogans serovar hardjo in the genital and urinary tracts of non-pregnant cattle. Vet Rec. 1986 Jan 4;118(1):11–13. doi: 10.1136/vr.118.1.11. [DOI] [PubMed] [Google Scholar]
  7. Goodman J. L., Jurkovich P., Kramber J. M., Johnson R. C. Molecular detection of persistent Borrelia burgdorferi in the urine of patients with active Lyme disease. Infect Immun. 1991 Jan;59(1):269–278. doi: 10.1128/iai.59.1.269-278.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grover C. M., Thulliez P., Remington J. S., Boothroyd J. C. Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reaction and amniotic fluid. J Clin Microbiol. 1990 Oct;28(10):2297–2301. doi: 10.1128/jcm.28.10.2297-2301.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hsia K., Spector D. H., Lawrie J., Spector S. A. Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction. J Clin Microbiol. 1989 Aug;27(8):1802–1809. doi: 10.1128/jcm.27.8.1802-1809.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson R. C., Harris V. G. Differentiation of pathogenic and saprophytic letospires. I. Growth at low temperatures. J Bacteriol. 1967 Jul;94(1):27–31. doi: 10.1128/jb.94.1.27-31.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaneko S., Feinstone S. M., Miller R. H. Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J Clin Microbiol. 1989 Sep;27(9):1930–1933. doi: 10.1128/jcm.27.9.1930-1933.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuiken T., van Dijk J. E., Terpstra W. J., Bokhout B. A. The role of the common vole (Microtus arvalis) in the epidemiology of bovine infection with Leptospira interrogans serovar hardjo. Vet Microbiol. 1991 Aug 30;28(4):353–361. doi: 10.1016/0378-1135(91)90070-v. [DOI] [PubMed] [Google Scholar]
  13. Malloy D. C., Nauman R. K., Paxton H. Detection of Borrelia burgdorferi using the polymerase chain reaction. J Clin Microbiol. 1990 Jun;28(6):1089–1093. doi: 10.1128/jcm.28.6.1089-1093.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Olive D. M., al Mufti S., Simsek M., Fayez H., al Nakib W. Direct detection of human cytomegalovirus in urine specimens from renal transplant patients following polymerase chain reaction amplification. J Med Virol. 1989 Dec;29(4):232–237. doi: 10.1002/jmv.1890290403. [DOI] [PubMed] [Google Scholar]
  15. Sjöbring U., Mecklenburg M., Andersen A. B., Miörner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Oct;28(10):2200–2204. doi: 10.1128/jcm.28.10.2200-2204.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Telenti A., Marshall W. F., Smith T. F. Detection of Epstein-Barr virus by polymerase chain reaction. J Clin Microbiol. 1990 Oct;28(10):2187–2190. doi: 10.1128/jcm.28.10.2187-2190.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Van Eys G. J., Gravekamp C., Gerritsen M. J., Quint W., Cornelissen M. T., Schegget J. T., Terpstra W. J. Detection of leptospires in urine by polymerase chain reaction. J Clin Microbiol. 1989 Oct;27(10):2258–2262. doi: 10.1128/jcm.27.10.2258-2262.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Waitkins S. A. Leptospirosis as an occupational disease. Br J Ind Med. 1986 Nov;43(11):721–725. doi: 10.1136/oem.43.11.721. [DOI] [PMC free article] [PubMed] [Google Scholar]

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