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

Abstract

Background

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 of B. pseudomallei is required for the appropriate disease management and prevention. The present study is designed to identify novel and unique sequences of B. pseudomallei and development of quantitative polymerase chain reaction (qPCR) assay.

Methods

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.

Results

In this study, an 80-nucleotide-long B. pseudomallei-specific region within the gene HrpB1 was identified by computational analysis. The developed HrpB1-based qPCR assay was highly specific for B. pseudomallei detection when evaluated with 65 different bacterial cultures. The sensitivity of the qPCR assay with the HrpB1-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.

Conclusion

The results of the study showed the applicability of a novel HrpB1-based qPCR assay for sensitive and specific detection of B. pseudomallei in diverse clinical and environmental samples.