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 × 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.

A real-time loop mediated isothermal amplification assay for molecular detection of Burkholderia mallei, the aetiological agent of a zoonotic and re-emerging disease glanders.

Burkholderia mallei, a potential biological warfare agent, is the causative agent of an infectious, fatal, and zoonotic disease, called glanders. Accurate and early diagnosis of glanders is important to control the disease lethality and infection spread. Molecular detection of B. mallei is considered strenuous because B. mallei is a subtractive genomic clone of B. pseudomallei. The present study was aimed at development of a real-time LAMP assay for detection of B. mallei. The LAMP assay was highly sensitive and could detect ≥250 fg of genomic DNA of B. mallei and ≥100 copies of recombinant plasmid containing target DNA sequence. In artificially spiked blood and water samples, it could detect ≥2.1 × 103 and ≥4.73 × 102 CFU/mL of B. mallei, respectively. The assay was highly specific for B. mallei as none of the other bacteria used in the study tested positive. The reported LAMP assay being simple and rapid can be a viable alternative to PCR-based glanders diagnostic assays in glanders endemic regions with resource-limited settings.

Development of a rapid and sensitive recombinase polymerase amplification-lateral flow assay for detection of Burkholderia mallei.

Burkholderia mallei, a potential biothreat agent is the aetiological agent of glanders, a zoonotic disease primarily affecting equines. B. mallei shares close genetic proximity with B. pseudomallei, the aetiological agent of melioidosis. Hence, molecular detection of B. mallei and its differentiation from B. pseudomallei has always been challenging. Early diagnosis of glanders is critical for timely treatment in humans and disease containment in animals. In this study a recombinase polymerase amplification-lateral flow (RPA-LF) assay has been developed for early and accurate detection of B. mallei. RPA-LF assay was found to be highly sensitive and detected as low as 10 fg genomic DNA of B. mallei. The assay was highly specific and could differentiate B. mallei and B. pseudomallei. The assay also detected B. mallei in artificially spiked blood, tap water and garden soil. The established assay is simple, rapid and does not require complex instrumentation. The field deployable assay can have better implications in rapid glanders diagnosis and environmental detection of B. mallei over PCR-based detection tools in glanders endemic areas with limited laboratory resources.