Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Park, Sol; Rana, Anita; Sung, Way; Munir, Mariya

doi:10.3390/applmicrobiol1030028

Cited by 10 publications

(7 citation statements)

References 117 publications

(181 reference statements)

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“…Droplet digital PCR generates up to 20,000 droplets and a PCR reaction occurs in each droplet. In terms of sensitivity, the lower limit of detection of ddPCR was 10 times more efficient than that of qPCR 49 . Therefore, we believe that HT-qPCR is useful for quantifying a wide range of genes simultaneously, but the results obtained should be confirmed with another method, such as ddPCR.…”

Section: Discussionmentioning

confidence: 98%

Organic amendment treatments for antimicrobial resistance and mobile element genes risk reduction in soil-crop systems

Jauregi

González

Garbisu

et al. 2023

Sci Rep

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Agricultural fertilization with organic amendments of animal origin often leads to antibiotic resistance dissemination. In this study, we evaluated the effect of different treatments (anaerobic digestion, biochar application, ozonation, zerovalent iron nanoparticle application, and spent mushroom substrate addition) on the resistome in dairy cow manure-derived amendments (slurry, manure, and compost). Anaerobic digestion and biochar application resulted in the highest reduction in antibiotic resistance gene (ARG) and mobile genetic element (MGE) gene abundance. These two treatments were applied to cow manure compost, which was then used to fertilize the soil for lettuce growth. After crop harvest, ARG and MGE gene absolute and relative abundances in the soil and lettuce samples were determined by droplet digital PCR and high-throughput qPCR, respectively. Prokaryotic diversity in cow manure-amended soils was determined using 16S rRNA metabarcoding. Compared to untreated compost, anaerobic digestion led to a 38% and 83% reduction in sul2 and intl1 absolute abundances in the soil, respectively, while biochar led to a 60% reduction in intl1 absolute abundance. No differences in lettuce gene abundances were observed among treatments. We conclude that amendment treatments can minimize the risk of antibiotic resistance in agroecosystems.

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Section: Discussionmentioning

confidence: 98%

Organic amendment treatments for antimicrobial resistance and mobile element genes risk reduction in soil-crop systems

Jauregi

González

Garbisu

et al. 2023

Sci Rep

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“…The qPCR is prone to quantitation bias and errors that includes (i) dependence on accurate quantification of calibrators to generate standard curve, with an assumption of no loss of calibrator molecules (ii) potential for errors during initial preparation and quantification of the standards, resuspensions, serial dilutions etc., and (iii) during PCR amplification itself. 47 In addition, the qPCR reaction efficiency is vulnerable to the presence of inhibitors (from blood sample, reagents, salts, anti-coagulants, etc. [29][30][31] On the contrary, the absolute quantification of gene copy numbers by ddPCR is independent of the use of standard curve and the partition of the reaction in individual droplets largely overcomes the limitations mentioned above for qPCR.…”

Section: Discussionmentioning

confidence: 99%

Bioanalytical Methods for Characterization of CAR‐T Cellular Kinetics: Comparison of PCR Assays and Matrices

Masilamani

Jawa

Dai

et al. 2023

Clin Pharma and Therapeutics

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Recently, multiple chimeric antigen receptor T‐cell (CAR‐T)‐based therapies have been approved for treating hematological malignancies, targeting CD19 and B‐cell maturation antigen. Unlike protein or antibody therapies, CAR‐T therapies are “living cell” therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T‐cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR‐T therapy trial. In addition, our studies show that the qPCR‐based amplification of transgene levels was well‐correlated, independent of DNA sources (either CD3+ T‐cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR‐T dosing prior to expansion and during long‐term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

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“…manure samples, both methods will be influenced and likely disproportionately by the sample of manures, where inhibitors (which vary among manure samples) may prevent adequate amplification (Sidstedt et al, 2020;Waseem et al, 2020;Park et al, 2021). We provide a comparison of these methods to target ARGs in our swine manure samples below.…”

Section: Discussionmentioning

confidence: 99%

Comparison of antibiotic resistance genes in swine manure storage pits of Iowa, USA

Neher¹,

Soupir²,

Andersen³

et al. 2023

Front. Antibiot.

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Antimicrobial resistance (AMR) can develop in deep-pit swine manure storage when bacteria are selectively pressured by unmetabolized antibiotics. Subsequent manure application on row crops is then a source of AMR into soil and downstream runoff water. Therefore, understanding the patterns of diverse antibiotic resistance genes (ARGs) in manure among different farms is important for both interpreting the results of the detection of these genes from previous studies and for the use of these genes as bioindicators of manure borne antibiotic resistance in the environment. Previous studies of manure-associated ARGs are based on limited samples of manures. To better understand the distribution of ARGs between manures, we characterized manures from 48 geographically independent swine farms across Iowa. The objectives of this study were to characterize the distribution of ARGs among these manures and to evaluate what factors in manure management may influence the presence of ARGs in manures. Our analysis included quantification of two commonly found ARGs in swine manure, ermB and tetM. Additionally, we characterized a broader suite of 31 ARGs which allowed for simultaneous assays of the presence or absence of multiple genes. We found the company integrator had a significant effect on both ermB (P=0.0007) and tetM gene concentrations (P=0.0425). Our broad analysis on ARG profiles found that the tet(36) gene was broadly present in swine manures, followed by the detection of tetT, tetM, erm(35), ermF, ermB, str, aadD, and intl3 in samples from 14 farms. Finally, we provide a comparison of methods to detect ARGs in manures, specifically comparing conventional and high-throughput qPCR and discuss their role in ARG environmental monitoring efforts. Results of this study provide insight into commonalities of ARG presence in manure holding pits and provide supporting evidence that company integrator decisions may impact ARG concentrations.

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Competitiveness of Quantitative Polymerase Chain Reaction (qPCR) and Droplet Digital Polymerase Chain Reaction (ddPCR) Technologies, with a Particular Focus on Detection of Antibiotic Resistance Genes (ARGs)

Cited by 10 publications

References 117 publications

Organic amendment treatments for antimicrobial resistance and mobile element genes risk reduction in soil-crop systems

Organic amendment treatments for antimicrobial resistance and mobile element genes risk reduction in soil-crop systems

Bioanalytical Methods for Characterization of CAR‐T Cellular Kinetics: Comparison of PCR Assays and Matrices

Comparison of antibiotic resistance genes in swine manure storage pits of Iowa, USA

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