A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters

Kolm, Claudia; Martzy, Roland; Brunner, Kurt; Mach, Robert L.; Krska, Rudolf; Heinze, Georg; Sommer, Regina; Reischer, Georg H.; Farnleitner, Andreas H.

doi:10.1021/acs.est.7b01074

Cited by 12 publications

(10 citation statements)

References 49 publications

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“…The analytical sensitivity of the XAC1051-2qPCR and the XAC1051-F/R PCR were estimated by determining the 95% limit of detection (LOD95%), i.e., the concentration at which a detection probability of 95% is expected [ 55 – 57 ] as explained previously [ 42 ].…”

Section: Methodsmentioning

confidence: 99%

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

et al. 2020

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Background Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols. Results Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml− 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml− 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays. Conclusions We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

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Section: Methodsmentioning

confidence: 99%

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

et al. 2020

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“…However, this analysis requires a qPCR thermal cycler capable of monitoring the amplification procedure in real time. To avoid the necessity of complex equipment, our group developed a LAMP [ 20 ] and an HDA assay [ 21 ] that target the same DNA sequence and were shown to have comparable specificity and sensitivity as the USEPA assay. While both new methods can be performed entirely on a heating block, the LAMP assay is faster with a reaction time of only 45 min, whereas the HDA assay has a significantly lower detection limit.…”

Section: Case Study: Towards Implementing a Field-applicable Workflowmentioning

confidence: 99%

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis

et al. 2019

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Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field. Graphical abstract

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“…The field of microbial molecular diagnostics comprises various methods for the specific detection of nucleic acids (NAs) from different microorganisms 1,2 – e.g., human pathogens in clinical and environmental samples 3–5 , faecal indicator bacteria in water 6,7 , or harmful microbial agents in food and feed 8,9 . However, to detect the desired sequence of a certain NA (DNA or RNA), preceding steps are necessary to isolate the genetic material from the respective cells.…”

Section: Introductionmentioning

confidence: 99%

Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

Martzy

Bica-Schröder

Pálvölgyi

et al. 2019

Sci Rep

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The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.

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A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters

Cited by 12 publications

References 49 publications

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis

Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

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