Development of a Novel Internally Controlled HrpB1 Gene-Based Real-Time qPCR Assay for Detection of Burkholderia pseudomallei.

Abstract

Melioidosis, caused by category B bioterrorism agent Burkholderia pseudomallei, is a seasonal disease of tropical and subtropical regions with a high mortality rate. An early and culture-independent detection ofB. pseudomalleiis required for the appropriate disease management and prevention. The present study is designed to identify novel and unique sequences ofB. pseudomalleiand development of quantitative polymerase chain reaction (qPCR) assay. A novel B. pseudomallei-specific target sequence was identified by in silico analysis for the qPCR assay development. The specificity of the developed assay was assessed using purified DNA of 65 different bacterial cultures, and the sensitivity was estimated using a cloned target gene. Further, a type III secretion protein HrpB1 (HrpB1) gene-based duplex qPCR assay incorporating suitable extraction and amplification control was developed, and its viability was assessed in different clinical and environmental matrices for the detection of B. pseudomallei. In this study, an 80-nucleotide-longB. pseudomallei-specific region within the geneHrpB1was identified by computational analysis. The developedHrpB1-based qPCR assay was highly specific forB. pseudomalleidetection when evaluated with 65 different bacterial cultures. The sensitivity of the qPCR assay with theHrpB1-recombinant plasmid was found to be five copies per qPCR reaction. The assay's detection limit was found to be 5 × 102 CFU/mL for human blood and urine, 5 × 101 CFU/mL in river water, and 2 × 103 CFU/gm in paddy field soil. The results of the study showed the applicability of a novelHrpB1-based qPCR assay for sensitive and specific detection ofB. pseudomalleiin diverse clinical and environmental samples.