Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Aug;33(8):2131–2135. doi: 10.1128/jcm.33.8.2131-2135.1995

Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization.

M Kunakorn 1, R B Markham 1
PMCID: PMC228348  PMID: 7559961

Abstract

Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin-labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic.

Full Text

The Full Text of this article is available as a PDF (264.1 KB).

Selected References

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

  1. Cimino G. D., Metchette K. C., Tessman J. W., Hearst J. E., Isaacs S. T. Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res. 1991 Jan 11;19(1):99–107. doi: 10.1093/nar/19.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Kemp D. J., Churchill M. J., Smith D. B., Biggs B. A., Foote S. J., Peterson M. G., Samaras N., Deacon N. J., Doherty R. Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients. Gene. 1990 Oct 15;94(2):223–228. doi: 10.1016/0378-1119(90)90391-4. [DOI] [PubMed] [Google Scholar]
  3. Kox L. F., Rhienthong D., Miranda A. M., Udomsantisuk N., Ellis K., van Leeuwen J., van Heusden S., Kuijper S., Kolk A. H. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol. 1994 Mar;32(3):672–678. doi: 10.1128/jcm.32.3.672-678.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Kunakorn M., Boonma P., Khupulsup K., Petchclai B. Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis. J Clin Microbiol. 1990 Jun;28(6):1249–1253. doi: 10.1128/jcm.28.6.1249-1253.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kunakorn M., Wichukchinda N., Raksakait K., Petchclai B., Jutavijittum P., Mandee Y., Kamthon N., Kurimura T. Screening of HIV-1 proviral DNA using simple protocol polymerase chain reaction in Thai blood donors. AIDS. 1993 Dec;7(12):1681–1682. doi: 10.1097/00002030-199312000-00025. [DOI] [PubMed] [Google Scholar]
  6. Lazar J. G. Advanced methods in PCR product detection. PCR Methods Appl. 1994 Aug;4(1):S1–14. doi: 10.1101/gr.4.1.s1. [DOI] [PubMed] [Google Scholar]
  7. Leelarasamee A., Bovornkitti S. Melioidosis: review and update. Rev Infect Dis. 1989 May-Jun;11(3):413–425. doi: 10.1093/clinids/11.3.413. [DOI] [PubMed] [Google Scholar]
  8. Lew A. E., Desmarchelier P. M. Detection of Pseudomonas pseudomallei by PCR and hybridization. J Clin Microbiol. 1994 May;32(5):1326–1332. doi: 10.1128/jcm.32.5.1326-1332.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Longo M. C., Berninger M. S., Hartley J. L. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990 Sep 1;93(1):125–128. doi: 10.1016/0378-1119(90)90145-h. [DOI] [PubMed] [Google Scholar]
  10. Niederhauser C., Höfelein C., Wegmüller B., Lüthy J., Candrian U. Reliability of PCR decontamination systems. PCR Methods Appl. 1994 Oct;4(2):117–123. doi: 10.1101/gr.4.2.117. [DOI] [PubMed] [Google Scholar]
  11. Pang J., Modlin J., Yolken R. Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA. Mol Cell Probes. 1992 Jun;6(3):251–256. doi: 10.1016/0890-8508(92)90024-r. [DOI] [PubMed] [Google Scholar]
  12. Pruksachartvuthi S., Aswapokee N., Thankerngpol K. Survival of Pseudomonas pseudomallei in human phagocytes. J Med Microbiol. 1990 Feb;31(2):109–114. doi: 10.1099/00222615-31-2-109. [DOI] [PubMed] [Google Scholar]
  13. Rys P. N., Persing D. H. Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products. J Clin Microbiol. 1993 Sep;31(9):2356–2360. doi: 10.1128/jcm.31.9.2356-2360.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Thornton C. G., Hartley J. L., Rashtchian A. Utilizing uracil DNA glycosylase to control carryover contamination in PCR: characterization of residual UDG activity following thermal cycling. Biotechniques. 1992 Aug;13(2):180–184. [PubMed] [Google Scholar]
  15. Tyler S. D., Strathdee C. A., Rozee K. R., Johnson W. M. Oligonucleotide primers designed to differentiate pathogenic pseudomonads on the basis of the sequencing of genes coding for 16S-23S rRNA internal transcribed spacers. Clin Diagn Lab Immunol. 1995 Jul;2(4):448–453. doi: 10.1128/cdli.2.4.448-453.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ulrich P. P., Romeo J. M., Daniel L. J., Vyas G. N. An improved method for the detection of hepatitis C virus RNA in plasma utilizing heminested primers and internal control RNA. PCR Methods Appl. 1993 Feb;2(3):241–249. doi: 10.1101/gr.2.3.241. [DOI] [PubMed] [Google Scholar]
  17. Wagner R. The regulation of ribosomal RNA synthesis and bacterial cell growth. Arch Microbiol. 1994;161(2):100–109. doi: 10.1007/BF00276469. [DOI] [PubMed] [Google Scholar]
  18. Wilson S. M., McNerney R., Nye P. M., Godfrey-Faussett P. D., Stoker N. G., Voller A. Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis. J Clin Microbiol. 1993 Apr;31(4):776–782. doi: 10.1128/jcm.31.4.776-782.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yabuuchi E., Kosako Y., Oyaizu H., Yano I., Hotta H., Hashimoto Y., Ezaki T., Arakawa M. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol. 1992;36(12):1251–1275. doi: 10.1111/j.1348-0421.1992.tb02129.x. [DOI] [PubMed] [Google Scholar]
  20. de Lamballerie X., Zandotti C., Vignoli C., Bollet C., de Micco P. A one-step microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res Microbiol. 1992 Oct;143(8):785–790. doi: 10.1016/0923-2508(92)90107-y. [DOI] [PubMed] [Google Scholar]
  21. van Nues R. W., Rientjes J. M., van der Sande C. A., Zerp S. F., Sluiter C., Venema J., Planta R. J., Raué H. A. Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA. Nucleic Acids Res. 1994 Mar 25;22(6):912–919. doi: 10.1093/nar/22.6.912. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES