Simultaneous Single-Channel Multiplexing and Quantification of Carbapenem-Resistant Genes Using Multidimensional Standard Curves

Rodríguez-Manzano, Jesús; Moniri, Ahmad; Malpartida-Cardenas, Kenny; Dronavalli, Jyothsna; Davies, Frances; Holmes, Alison; Georgiou, Pantelis

doi:10.1021/acs.analchem.8b04412

Cited by 21 publications

(26 citation statements)

References 22 publications

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“…There are multiple avenues for increasing the number of targets per experiment, such as: (i) Microfluidic solutions (e.g. SlipChip) which offer an affordable mechanism for generating multiple chambers [27][28][29][30] ; or (ii) data-driven approaches which have recently been shown to perform single-channel multiplexing in a single chamber for carbapenemase-producing organisms using only real-time amplification data [31][32][33] . Finally, it is important to further investigate the relationship between carrying the mcr−9 gene and its phenotypic implications, i.e.…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of Mobilized Colistin Resistance using a Nucleic Acid Based Lab-on-a-Chip Diagnostic System

Rodríguez-Manzano

Moser

Malpartida-Cardenas

et al. 2020

Sci Rep

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The increasing prevalence of antimicrobial resistance is a serious threat to global public health. One of the most concerning trends is the rapid spread of Carbapenemase-Producing Organisms (CPO), where colistin has become the last-resort antibiotic treatment. The emergence of colistin resistance, including the spread of mobilized colistin resistance (mcr) genes, raises the possibility of untreatable bacterial infections and motivates the development of improved diagnostics for the detection of colistin-resistant organisms. This work demonstrates a rapid response for detecting the most recently reported mcr gene, mcr−9, using a portable and affordable lab-on-a-chip (LoC) platform, offering a promising alternative to conventional laboratory-based instruments such as real-time PCR (qPCR). The platform combines semiconductor technology, for non-optical real-time DNA sensing, with a smartphone application for data acquisition, visualization and cloud connectivity. This technology is enabled by using loop-mediated isothermal amplification (LAMP) as the chemistry for targeted DNA detection, by virtue of its high sensitivity, specificity, yield, and manageable temperature requirements. Here, we have developed the first LAMP assay for mcr−9-showing high sensitivity (down to 100 genomic copies/reaction) and high specificity (no cross-reactivity with other mcr variants). This assay is demonstrated through supporting a hospital investigation where we analyzed nucleic acids extracted from 128 carbapenemase-producing bacteria isolated from clinical and screening samples and found that 41 carried mcr−9 (validated using whole genome sequencing). Average positive detection times were 6.58 ± 0.42 min when performing the experiments on a conventional qPCR instrument (n = 41). For validating the translation of the LAMP assay onto a LoC platform, a subset of the samples were tested (n = 20), showing average detection times of 6.83 ± 0.92 min for positive isolates (n = 14). All experiments detected mcr−9 in under 10 min, and both platforms showed no statistically significant difference (p-value > 0.05). When sample preparation and throughput capabilities are integrated within this LoC platform, the adoption of this technology for the rapid detection and surveillance of antimicrobial resistance genes will decrease the turnaround time for DNA detection and resistotyping, improving diagnostic capabilities, patient outcomes, and the management of infectious diseases. Antimicrobial resistance is a serious threat to global healthcare systems which poses a challenge for modern medicine and compromises effective infectious disease management. Of particular concern is the rapid spread of

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Section: Discussionmentioning

confidence: 99%

Rapid Detection of Mobilized Colistin Resistance using a Nucleic Acid Based Lab-on-a-Chip Diagnostic System

Rodríguez-Manzano

Moser

Malpartida-Cardenas

et al. 2020

Sci Rep

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“…Experimental results provided quantitative data with high sensitivity and specificity (100%) without cross-reactivity. TR-LAMP assays can be combined to develop a multiplex reaction azole-resistance detection, which will significantly speed up clinical diagnoses (20,52).…”

Section: Translating Tr-lamp Assays Onto Lab-on-a-chip Platformmentioning

confidence: 99%

“…This platform combines ion-sensitive field-effect transistors (ISFETs), fabricated in unmodified complementary metal-oxide semiconductor (CMOS) technology, with loop-mediated isothermal amplification (LAMP) and an AndroidOS application for data acquisition, visualization and cloud connectivity. Electrochemical sensing using ISFETs removes the need for bulky and complex optical equipment, while LAMP eliminates the requirement for thermocycling -reducing the hardware complexity, power consumption and size of the diagnostic platform (20)(21)(22)(23)(24).…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Azole-Resistant Aspergillus fumigatus in Clinical and Environmental Isolates by Use of a Lab-on-a-Chip Diagnostic System

Rodríguez-Manzano

Moser

et al. 2020

J Clin Microbiol

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Aspergillus fumigatus has widely evolved resistance to the most commonly used class of antifungal chemicals, the azoles. Current methods for identifying azole resistance are time-consuming and depend on specialized laboratories. There is an urgent need for rapid detection of these emerging pathogens at point-of-care to provide the appropriate treatment in the clinic and to improve management of environmental reservoirs to mitigate the spread of antifungal resistance. Our study demonstrates the rapid and portable detection of the two most relevant genetic markers linked to azole resistance, the mutations TR34 and TR46, found in the promoter region of the gene encoding the azole target, cyp51A. We developed a lab-on-a-chip platform consisting of: (1) tandem-repeat loop-mediated isothermal amplification, (2) state-of-the-art complementary metal-oxide-semiconductor microchip technology for nucleic-acid amplification detection and, (3) and a smartphone application for data acquisition, visualization and cloud connectivity. Specific and sensitive detection was validated with isolates from clinical and environmental samples from 6 countries across 5 continents, showing a lower limit-of-detection of 10 genomic copies per reaction in less than 30 minutes. When fully integrated with a sample preparation module, this diagnostic system will enable the detection of this ubiquitous fungus at the point-of-care, and could help to improve clinical decision making, infection control and epidemiological surveillance.

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“…Recently, a novel single fluorescent channel-based multiplexing method was proposed using intercalating dyes, and the result was processed based on multidimensional standard curves. 30 − 32 Moreover, the data set was achieved by commercial qPCR instruments, and the absolute quantification was determined, thus extending the use of these devices.…”

Section: Introductionmentioning

confidence: 99%

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

et al. 2021

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Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number ( cn ) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

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Simultaneous Single-Channel Multiplexing and Quantification of Carbapenem-Resistant Genes Using Multidimensional Standard Curves

Cited by 21 publications

References 22 publications

Rapid Detection of Mobilized Colistin Resistance using a Nucleic Acid Based Lab-on-a-Chip Diagnostic System

Rapid Detection of Mobilized Colistin Resistance using a Nucleic Acid Based Lab-on-a-Chip Diagnostic System

Rapid Detection of Azole-Resistant Aspergillus fumigatus in Clinical and Environmental Isolates by Use of a Lab-on-a-Chip Diagnostic System

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

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