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

• Published in: Molecular diagnosis & therapy Vol. 28; no. 1; pp. 101 - 112
• Main Authors: Yadav, Pranjal Kumar, Paul, Moumita, Singh, Suchetna, Kumar, Sanjay, Ponmariappan, S., Thavaselvam, Duraipandian
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 2024; Springer Nature B.V
• Subjects: Assaying; Biomedical and Life Sciences; Biomedicine; Bioterrorism; Burkholderia pseudomallei; Burkholderia pseudomallei - genetics; Cancer Research; DNA, Bacterial - genetics; Evolution & development; Genes; Genetic diversity; Genomes; Human Genetics; Humans; Laboratories; Laboratory Medicine; Medical diagnosis; Melioidosis; Melioidosis - diagnosis; Melioidosis - microbiology; Molecular Medicine; Mortality; Nucleotides; Original Research Article; Pharmacotherapy; Polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Rivers; Sensitivity and Specificity; United States > US; India
• ISSN: 1177-1062; 1179-2000
• DOI: 10.1007/s40291-023-00686-7

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 Hrp B1 ( Hrp B1) 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  Hrp B1 was identified by computational analysis. The developed  Hrp B1-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  Hrp B1-recombinant plasmid was found to be five copies per qPCR reaction. The assay’s detection limit was found to be 5 × 10 2 CFU/mL for human blood and urine, 5 × 10 1 CFU/mL in river water, and 2 × 10 3 CFU/gm in paddy field soil. Conclusion The results of the study showed the applicability of a novel  Hrp B1-based qPCR assay for sensitive and specific detection of  B. pseudomallei  in diverse clinical and environmental samples.