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Indian journal of microbiology2010; 50(2); 172-178; doi: 10.1007/s12088-010-0003-3

Evaluation of PCR, DNA hybridization and immunomagnetic separation – PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.

Abstract: Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1-2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.
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Publication Date: 2010-03-05 PubMed ID: 23100824PubMed Central: PMC3450331DOI: 10.1007/s12088-010-0003-3Google Scholar: Lookup
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Summary

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This research article outlines the development of a rapid detection strategy for Burkholderia mallei, a bacteria that causes glanders in equines and can be transmitted to humans, in environmental samples. The researchers found that polymerase chain reaction (PCR) is the most effective method, particularly when using a specific 823 bp DNA sequence of B. mallei.

Selection of Media for Bacterial Growth

  • The researchers began by selecting a suitable medium to support the growth of the B. mallei bacteria. They evaluated several different bacteriological media.
  • They found that the brain heart infusion broth medium with selective supplements (BHIB-SS) successfully supported the maximum growth of B. mallei. This was true even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus.

Standardization of PCR and DNA Hybridization Method

  • The team then worked on developing a method to identify and isolate the bacteria. They standardized a Polymerase Chain Reaction (PCR) and a DNA hybridization method for a specific 823 base pair DNA sequence of B. mallei.
  • The PCR method was deemed superior as it provided a lower detection limit, meaning the method could detect the presence of B. mallei even in lower concentrations.

Implementation of Immunomagnetic Separation Method

  • To directly and quickly isolate B. mallei from environmental samples, an immunomagnetic separation (IMS) method was also standardized by the researchers.
  • The rationale behind using IMS was to bring down the detection limit still further, allowing for quicker and more efficient detection of the bacteria.

Testing of Detection Methods on Artificially Contaminated Samples

  • The researchers then contaminated water, husk, grass, and gram samples with B. mallei bacteria for testing.
  • After letting the bacteria grow in BHIB-SS medium, they then observed and analyzed the samples using the PCR and DNA hybridization methods.
  • The researchers found that PCR was superior to DNA hybridization, with an ability to detect concentrations of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices.

Conclusion and Recommendations

  • The research concluded that PCR is the most effective method for detecting B. mallei in environmental samples, either following enrichment in BHIB-SS or after IMS.
  • They recommended the IMS-PCR method in situations where high numbers of B. mallei bacteria are expected and results need to be obtained quickly.

Cite This Article

APA
Merwyn S, Kumar S, Agarwal GS, Rai GP. (2010). Evaluation of PCR, DNA hybridization and immunomagnetic separation – PCR for detection of Burkholderia mallei in artificially inoculated environmental samples. Indian J Microbiol, 50(2), 172-178. https://doi.org/10.1007/s12088-010-0003-3

Publication

Indian journal of microbiology
ISSN: 0046-8991
NlmUniqueID: 0374703
Country: India
Language: English
Volume: 50
Issue: 2
Pages: 172-178

Researcher Affiliations

Merwyn, S
  • Division of High Containment Facility, Defence Research and Development Establishment, Gwalior, 474 002 India.
Kumar, S
    Agarwal, G S
      Rai, G P

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        Citations

        This article has been cited 3 times.
        1. Gaspar EB, Santos LRD, Egito AAD, Santos MGD, Mantovani C, Rieger JDSG, Abrantes GAS, Suniga PAP, Favacho JM, Pinto IB, Nassar AFC, Santos FLD, Araújo FR. Assessment of the Virulence of the Burkholderia mallei Strain BAC 86/19 in BALB/c Mice. Microorganisms 2023 Oct 20;11(10).
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        2. Knox A, Zerna G, Beddoe T. Current and Future Advances in the Detection and Surveillance of Biosecurity-Relevant Equine Bacterial Diseases Using Loop-Mediated Isothermal Amplification (LAMP). Animals (Basel) 2023 Aug 18;13(16).
          doi: 10.3390/ani13162663pubmed: 37627456google scholar: lookup
        3. Abreu DC, Gomes AS, Tessler DK, Chiebao DP, Fava CD, Romaldini AHCN, Araujo MC, Pompei J, Marques GF, Harakava R, Pituco EM, Nassar AFC. Systematic monitoring of glanders-infected horses by complement fixation test, bacterial isolation, and PCR. Vet Anim Sci 2020 Dec;10:100147.
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