Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei

Abstract

BackgroundBurkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance.MethodsSignature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated.ResultsA single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction.ConclusionsThe new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

Recent Advances in Burkholderia mallei and B. pseudomallei Research

“…Although the assay detected psu in all 19 B. pseudomallei samples, it failed to detect BuMC in three of the B. pseudomallei isolates. Although no sensitivity or specificity data was provided, the limit of detection of the assay was estimated to be <1 genome equivalent per reaction [41]. …”

Gene Amplification and Sequencing for Bacterial Identification

“…Using DNA probes generated from subtractive genome comparison, a number of assays have been designed, with applications ranging from environmental microbiology (Price et al, 2013), industrial quality control (Yu et al, 2010), clinical diagnostics (Ho et al, 2012;Ho, Yuen, et al, 2011;Wang, Sun, & Lu, 2012) and rapid detection and identification of bacterial pathogens with bioterrorism potential Janse, Hamidjaja, Hendriks, & van Rotterdam, 2013;. As the probes generated from the in silico analysis can be selected based on their genomic characteristics, e.g., number of copies, highly sensitive assays and quantitative assays may be designed using multi-copy and single-copy probes, respectively.…”

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues

“…In recent years, multiplexed TaqMan qPCR has become a useful tool for the identification and quantification of pathogens in different areas such as food safety (Köppel et al, 2019;Wei et al, 2019), medical environment (Janse et al, 2013;Kamau et al, 2013), agronomics (Wei et al, 2008;Zitnick-Anderson et al, 2018), GMO detection (Choi et al, 2018;Wang et al, 2018), and the environment (Hulley et al, 2019). For plant pathogens, these methods have been tested on samples of naturally infected plants, spiked samples (Li et al, 2009;Willsey et al, 2018), and on mixtures of plant and pathogen DNAs (Abraham et al, 2018).…”