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A recombinase polymerase amplification lateral flow assay for rapid detection of Burkholderia pseudomallei, the causative agent of melioidosis

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Abstract

Burkholderia pseudomallei causes a fatal and infectious disease, melioidosis or Whitmore’s disease in humans and animals. Melioidosis is present in different parts of the world and is endemic in Southeast Asia and Northern Australia. Accurate diagnosis of melioidosis is difficult due to its common flu-like symptoms, potentially long incubation period and erroneous identification as culture contaminant. Early diagnosis of the disease is essentially required for administration of suitable antibiotics and disease containment. The present study reports a rapid, specific and sensitive recombinase polymerase amplification lateral flow assay for detection of B. pseudomallei. Specific primers and probe were designed and the assay was performed at 41 °C for 20 min in a portable incubator. End products were detected using ready-to-use lateral flow strips. RPA lateral flow assay could detect ≥ 250 fg genomic DNA of B. pseudomallei and ≥ 50 copies of recombinant plasmid harbouring the target DNA sequence. The assay was found to be highly specific and did not cross-react with other bacterial strains. In artificially spiked human blood and urine samples, the detection limit of the assay was 4.8 × 104 and 4.95 × 104 CFU/mL of B. pseudomallei, respectively. The detection limit of assay after 6 h of enrichment of artificially spiked urine samples was found to be 4.95 × 103 CFU/mL of B. pseudomallei. Detection limit in artificially spiked tap water and soil samples was determined to be 7.5 × 102 CFU/mL and 3.3 × 104 CFU per 5 g of B. pseudomallei, respectively.

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Acknowledgements

The authors thank Director, DRDE, Gwalior, for providing necessary research facilities and encouragement (DRDE/BPT/01/2021). AS is thankful to DRDO for providing Senior Research Fellowship.

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Authors and Affiliations

  1. Bioprocess Technology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior, 474002, India

    Apoorva Saxena, Vijai Pal, Nagesh Kumar Tripathi & Ajay Kumar Goel

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  1. Apoorva Saxena
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Correspondence to Vijai Pal.

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The experiments were performed according to the Institutional Animal Ethics Committee (IAEC) guidelines vide registration number 37/1999/CPCSEA and approved by Institutional Biosafety Committee vide protocol number IBSC/19/BPD/VP/2.

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Saxena, A., Pal, V., Tripathi, N. et al. A recombinase polymerase amplification lateral flow assay for rapid detection of Burkholderia pseudomallei, the causative agent of melioidosis. Braz J Microbiol 53, 185–193 (2022). https://doi.org/10.1007/s42770-021-00669-y

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