Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Jul;63(7):2702–2707. doi: 10.1128/aem.63.7.2702-2707.1997

Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

K Erlandson 1, C A Batt 1
PMCID: PMC168565  PMID: 9212417

Abstract

A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Akopyanz N., Bukanov N. O., Westblom T. U., Berg D. E. PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori. Nucleic Acids Res. 1992 Dec 11;20(23):6221–6225. doi: 10.1093/nar/20.23.6221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akopyanz N., Bukanov N. O., Westblom T. U., Kresovich S., Berg D. E. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res. 1992 Oct 11;20(19):5137–5142. doi: 10.1093/nar/20.19.5137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Betzl D., Ludwig W., Schleifer K. H. Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes. Appl Environ Microbiol. 1990 Sep;56(9):2927–2929. doi: 10.1128/aem.56.9.2927-2929.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bidochka M. J., Walsh S. R., Ramos M. E., Leger R. J., Silver J. C., Roberts D. W. Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes. Appl Environ Microbiol. 1995 Feb;61(2):556–560. doi: 10.1128/aem.61.2.556-560.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bringel F., Curk M. C., Hubert J. C. Characterization of lactobacilli by Southern-type hybridization with a Lactobacillus plantarum pyrDFE probe. Int J Syst Bacteriol. 1996 Apr;46(2):588–594. doi: 10.1099/00207713-46-2-588. [DOI] [PubMed] [Google Scholar]
  7. Bruce J. L., Hubner R. J., Cole E. M., McDowell C. I., Webster J. A. Sets of EcoRI fragments containing ribosomal RNA sequences are conserved among different strains of Listeria monocytogenes. Proc Natl Acad Sci U S A. 1995 May 23;92(11):5229–5233. doi: 10.1073/pnas.92.11.5229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cancilla M. R., Powell I. B., Hillier A. J., Davidson B. E. Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels. Appl Environ Microbiol. 1992 May;58(5):1772–1775. doi: 10.1128/aem.58.5.1772-1775.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crowhurst R. N., Hawthorne B. T., Rikkerink E. H., Templeton M. D. Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA. Curr Genet. 1991 Nov;20(5):391–396. doi: 10.1007/BF00317067. [DOI] [PubMed] [Google Scholar]
  10. Czajka J., Batt C. A. Verification of causal relationships between Listeria monocytogenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified polymorphic DNA patterns. J Clin Microbiol. 1994 May;32(5):1280–1287. doi: 10.1128/jcm.32.5.1280-1287.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Czajka J., Bsat N., Piani M., Russ W., Sultana K., Wiedmann M., Whitaker R., Batt C. A. Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms. Appl Environ Microbiol. 1993 Jan;59(1):304–308. doi: 10.1128/aem.59.1.304-308.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davin-Regli A., Abed Y., Charrel R. N., Bollet C., de Micco P. Variations in DNA concentrations significantly affect the reproducibility of RAPD fingerprint patterns. Res Microbiol. 1995 Sep;146(7):561–568. doi: 10.1016/0923-2508(96)80562-6. [DOI] [PubMed] [Google Scholar]
  13. Hahn D., Amann R. I., Ludwig W., Akkermans A. D., Schleifer K. H. Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides. J Gen Microbiol. 1992 May;138(5):879–887. doi: 10.1099/00221287-138-5-879. [DOI] [PubMed] [Google Scholar]
  14. Klijn N., Weerkamp A. H., de Vos W. M. Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems. Appl Environ Microbiol. 1995 Feb;61(2):788–792. doi: 10.1128/aem.61.2.788-792.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Köhler G., Ludwig W., Schleifer K. H. Differentiation of lactococci by rRNA gene restriction analysis. FEMS Microbiol Lett. 1991 Dec 1;68(3):307–312. doi: 10.1111/j.1574-6968.1991.tb04615.x. [DOI] [PubMed] [Google Scholar]
  16. Mazurier S. I., Audurier A., Marquet-Van der Mee N., Notermans S., Wernars K. A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates. Res Microbiol. 1992 Jun;143(5):507–512. doi: 10.1016/0923-2508(92)90097-8. [DOI] [PubMed] [Google Scholar]
  17. Rodtong S., Tannock G. W. Differentiation of Lactobacillus strains by ribotyping. Appl Environ Microbiol. 1993 Oct;59(10):3480–3484. doi: 10.1128/aem.59.10.3480-3484.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Salama M., Sandine W., Giovannoni S. Development and application of oligonucleotide probes for identification of Lactococcus lactis subsp. cremoris. Appl Environ Microbiol. 1991 May;57(5):1313–1318. doi: 10.1128/aem.57.5.1313-1318.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sharpe A. N., Michaud G. L. Hydrophobic grid-membrane filters: new approach to microbiological enumeration. Appl Microbiol. 1974 Aug;28(2):223–225. doi: 10.1128/am.28.2.223-225.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Terzaghi B. E., Sandine W. E. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975 Jun;29(6):807–813. doi: 10.1128/am.29.6.807-813.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Veiga da Cunha M., Foster M. A. Sugar-glycerol cofermentations in lactobacilli: the fate of lactate. J Bacteriol. 1992 Feb;174(3):1013–1019. doi: 10.1128/jb.174.3.1013-1019.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Voigt K., Wöstemeyer J. The combination of Gilbert/Maxam chemical sequencing and the dideoxynucleotide chain termination approach facilitates the construction of species specific PCR-primers based on diagnostic RAPD bands. Microbiol Res. 1995 Nov;150(4):373–377. doi: 10.1016/S0944-5013(11)80019-4. [DOI] [PubMed] [Google Scholar]
  23. Welsh J., McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990 Dec 25;18(24):7213–7218. doi: 10.1093/nar/18.24.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wiedmann M., Bruce J. L., Knorr R., Bodis M., Cole E. M., McDowell C. I., McDonough P. L., Batt C. A. Ribotype diversity of Listeria monocytogenes strains associated with outbreaks of listeriosis in ruminants. J Clin Microbiol. 1996 May;34(5):1086–1090. doi: 10.1128/jcm.34.5.1086-1090.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Williams J. G., Kubelik A. R., Livak K. J., Rafalski J. A., Tingey S. V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Xia X., Bollinger J., Ogram A. Molecular genetic analysis of the response of three soil microbial communities to the application of 2,4-D. Mol Ecol. 1995 Feb;4(1):17–28. doi: 10.1111/j.1365-294x.1995.tb00188.x. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES