Thirty-minute screening of antibiotic resistance genes in bacterial isolates with minimal sample preparation in static self-dispensing 64 and 384 assay cards

Abstract

In a clinical setting, molecular assays such as polymerase chain reaction offer a rapid means to infer or confirm identity and therapeutic decisions. Accordingly, a number of molecular assays targeting identity and antibiotic resistance (AR) genes have been developed; however, these methods can be technically complex and relatively expensive. Herein, we describe a diagnostic concept utilizing isothermal amplification technology with non-purified heat-lysed cells and self-dispensing cards for testing multiple primers in parallel. This proof-of-concept study, performed with Staphylococcus aureus isolates and associated AR genes, was compared with culture-based susceptibility and quantitative PCR (qPCR). Results demonstrate reduced sample processing steps resulting in a turnaround time (starting from bacterial culture to ending in the antibiotic resistance gene profile) in less than 30 min. For antibiotics tested in which an associated AR gene was targeted on the Gene-Z card, 69 % (18/26) of culture-based resistance events were positive for related AR genes. A comparison of loop-mediated isothermal amplification (LAMP) and qPCR assays targeting the same antibiotic resistance genes showed a 98.2 % agreement in terms of presence and absence calls. Identity-based discrepancies between conventional (phenotypic) and molecular (genotypic) results were further resolved, and we were able to demonstrate higher accuracy in identification with the molecular analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6774-z) contains supplementary material, which is available to authorized users.

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

“…Alternative genotypic methods (detecting genes responsible for known mechanisms of resistance) are more rapid than culture-based approaches. [10] However, these resistance genes constitute only a fraction of all possible mechanisms of resistance, [11] and new forms of resistance evolve quickly. [12] Therefore, predicting resistance by analyzing a few known resistance genes is not a general solution.…”

Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater

“…For some organisms and targets, direct LAMP amplification without DNA lyses may cause reduction in quantitative capacity or sensitivity. For example, previous studies with Staphylococcus aureus observed a 1 min difference between crudely lysed and non lysed cells (Kostic et al, 2015). Perhaps one of the greatest limitations of LAMP is increased possibility of contamination to subsequent reactions, due to the large concentration of generated amplicons.…”

“…In addition, LAMP analysis is faster than qPCR because amplification can be accomplished without DNA extraction or using a crude lysate (Kostic et al, 2015; Modak et al, 2016). Previous studies demonstrated LAMP assays for quantification of Dehalococcoides spp.…”

Quantification of microRNAs directly from body fluids using a base-stacking isothermal amplification method in a point-of-care device

“…Gene-Z performance has been demonstrated for many applications including bacterial pathogens important to water safety, human health, and assessing bioremediation performance in contaminated acquifers (Kostic et al 2015; Stedtfeld et al 2014). The work described here is its first demonstration for microRNA detection.…”

“…However, isothermal polymerases (e.g. Bst) are more robust and less impacted by inhibitory substrates compared to PCR polymerases (Kostic et al 2015; Stedtfeld et al 2014). Thus, an isothermal direct amplification approach has the potential to reduce analysis time and costs, without isolation and purification, and is therefore well suited for use outside of laboratories with specialized infrastructures (Njiru 2012).…”