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

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 × 10 4 and 4.95 × 10 4 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 × 10 3 CFU/mL of B. pseudomallei . Detection limit in artificially spiked tap water and soil samples was determined to be 7.5 × 10 2 CFU/mL and 3.3 × 10 4 CFU per 5 g of B. pseudomallei , respectively.