Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications

Abstract

The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

Loss of Methyltransferase Function and Increased Efflux Activity Leads to Doxycycline Resistance in Burkholderia pseudomallei

“…Preamplification was performed on the cDNA to enhance the sensitivity of the qPCR assays due to the low expression levels of amrB in some isolates. Six B. pseudomallei strains were subjected to preamplification using conventional PCR for amrB (Table S2), the transporter gene of the efflux pump, AmrAB-OprA, and the housekeeping gene control, mmsA (targeted by the 266152 assay [56]). The reaction mixtures included 1ϫ PCR buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 1.2 M Q-solution, 0.08 U/l HotStarTaq, and primer concentrations of 0.2 M for amrB or 0.3 M for mmsA.…”

PCR-based Methodologies Used to Detect and Differentiate theBurkholderia pseudomalleicomplex:B. pseudomallei,B. mallei, andB. thailandensis

“…The test's accuracy approached 100% (Trung et al, 2011;Kaestli et al, 2012;Price et al, 2012) with only one strain decreasing the assay's sensitivity. This false negative was a B. pseudomallei strain which had a reduced genome that lacked some virulence loci, including orf2 .…”

“…In a follow-up study, four qPCR procedures Price et al, 2012), including BurkDiff, were evaluated for their ability to detect and differentiate Burkholderia using purified DNA . BurkDiff was the most reliable test for B. pseudomallei detection, having a qPCR accuracy of 100%, but was also the most difficult test to interpret due to probe cross hybridization.…”

“…Several clinical evaluations note that the difference in specimen collection methods (with some specimens collected during treatment) may have affected sensitivity. Kaestli et al was able to standardize some of the more heavily evaluated procedures (Thibault et al, 2004;Merritt et al, 2006;Novak et al, 2006;Neubauer et al, 2007;Supaprom et al, 2007;Tuanyok et al, 2007;Price et al, 2012) and showed that Novak et al's assay was the most reliable on clinical samples. Novak et al's test would also be useful in detecting B. pseudomallei in purified DNA samples, since all follow-up studies have indicated an accuracy approaching 100%.…”

Simultaneous Identification of Haemophilus Influenzae and Haemophilus Haemolyticus using Real-Time PCR

“…H. influenzae ATCC 49247 was used as the control for fucP and siaT, and H. haemolyticus future science group www.futuremedicine.com ATCC 33390 the control for hypD. LOQ values were determined for each assay based on previously described criteria [41], with minor modifications. Briefly, replicates at a given dilution with a cycles to threshold (C T ) standard deviation (σ) of ≥0.8 were considered to exceed the LOQ, with one or more amplification failures also deemed a LOQ failure.…”