Analysis of the competitiveness between a non-aflatoxigenic and an aflatoxigenic Aspergillus flavus strain on maize kernels by droplet digital PCR

AbstractNon-aflatoxigenic Aspergillus flavus strains are used as a biocontrol system on maize fields to decrease the aflatoxin biosynthesis of aflatoxigenic A. flavus strains. A. flavus strain AF36 was the first commercially available biocontrol strain and is authorized for use on maize fields by the US Environmental Protection Agency, e.g., in Texas and Arizona. A droplet digital PCR (ddPCR) assay was developed to analyze the mechanisms of competition and interaction of aflatoxigenic and non-aflatoxigenic A. flavus strains. This assay enables the parallel identification and quantification of the biocontrol strain A. flavus AF36 and the aflatoxigenic A. flavus strain MRI19. To test the assay, spores of both strains were mixed in varying ratios and were incubated on maize-based agar or maize kernels for up to 20 days. Genomic equivalent ratios (genome copy numbers) of both strains were determined by ddPCR at certain times after incubation and were compared to the spore ratios used for inoculation. The aflatoxin biosynthesis was also measured. In general, A. flavus MRI19 had higher competitiveness in the tested habitats compared to the non-aflatoxigenic strain, as indicated by higher final genomic equivalent ratios of this strain compared to the spore ratios used for inoculation. Nevertheless, A. flavus AF36 effectively controlled aflatoxin biosynthesis of A. flavus MRI19, as a clear aflatoxin inhibition was already seen by the inoculation of 10% spores of the biocontrol strain mixed with 90% spores of the aflatoxigenic strain compared to samples inoculated with only spores of the aflatoxigenic A. flavus MRI19.

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Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽

10.1161/hyp.68.suppl_1.p613 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Ryousuke Satou ◽

Akemi Katsurada ◽

Kayoko Miyata ◽

Andrei Derbenev ◽

Andrea Zsombok

Keyword(s):

Copy Number ◽

System Analysis ◽

Renin Angiotensin System ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Angiotensin System ◽

Renin Angiotensin ◽

Copy Numbers ◽

Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

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Inducers of Aflatoxin Biosynthesis from Colonized Maize Kernels Are Generated by an Amylase Activity from Aspergillus flavus

Phytopathology ◽

10.1094/phyto.1997.87.2.164 ◽

1997 ◽

Vol 87 (2) ◽

pp. 164-169 ◽

Cited By ~ 59

Author(s):

C. P. Woloshuk ◽

J. R. Cavaletto ◽

T. E. Cleveland

Keyword(s):

Aspergillus Flavus ◽

Amylase Activity ◽

Carbon Sources ◽

Gene Promoter ◽

Starch Degradation ◽

Aflatoxin Biosynthesis ◽

Minimum Concentration ◽

Chromatography Analysis ◽

Gus Reporter ◽

Maize Kernels

Aflatoxin biosynthesis was induced by compounds in filtrates (EF) obtained from cultures consisting of ground maize kernels colonized by Aspergillus flavus. The inducing activity increased to a maximum at 4 days of incubation and then decreased. Amylase activity was detected in the EF, suggesting that the inducers are products of starch degradation (glucose, maltose, and maltotriose). Analysis of the enzyme by isoelectric focusing electrophoresis indicated a single α-amylase with a pI of 4.3. No maltase or amyloglucosidase was detected in the EF. High-pressure liquid chromatography analysis of the EF indicated the presence of glucose, maltose, and maltotriose in near-equal molar concentrations (about 15 mM). With a β-glucuronidase (GUS) reporter assay consisting of A. flavus transformed with an aflatoxin gene promoter-GUS reporter gene fusion to monitor induction of aflatoxin biosynthesis, the minimum concentration of glucose, maltose, or maltotriose that induced measurable GUS activity was determined to be 1 mM. These results support the hypothesis that the best inducers of aflatoxin biosynthesis are carbon sources readily metabolized via glycolysis. They also suggest that α-amylase produced by A. flavus has a role in the induction of aflatoxin biosynthesis in infected maize kernels.

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Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments

Journal of Water and Health ◽

10.2166/wh.2017.259 ◽

2017 ◽

Vol 15 (4) ◽

pp. 505-508 ◽

Cited By ~ 7

Author(s):

Gulshan Singh ◽

Ayanda Sithebe ◽

Abimbola M. Enitan ◽

Sheena Kumari ◽

Faizal Bux ◽

...

Keyword(s):

Environmental Samples ◽

Informal Settlement ◽

Quantitative Polymerase Chain Reaction ◽

River Sediments ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Analytical Sensitivity ◽

Aquatic Sediments ◽

Pcr Inhibitors ◽

Copy Numbers

Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves. Information is virtually absent where ddPCR has been applied to detect pathogenic organisms in aquatic sediments. This study compared the efficacy of ddPCR with qPCR, for quantification of Salmonella in sediments from the Palmiet River near an informal settlement in Durban, South Africa. ddPCR significantly improved both analytical sensitivity and detection of low concentrations of Salmonella as compared to qPCR. The expected copy numbers measured from both qPCR and ddPCR showed good R2 values (0.999 and 0.994, respectively). The site mostly affected by the informal settlements exhibited Salmonella in the range of 255 ± 37 and 818 ± 30 Salmonella/g (p ≤ 0.0001) in qPCR and ddPCR, respectively. The improved detection of Salmonella in sediments with ddPCR makes it a promising technical method for the quantification of Salmonella in multifarious environmental samples.

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Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall

PLoS ONE ◽

10.1371/journal.pone.0170767 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0170767 ◽

Cited By ~ 6

Author(s):

Delicia Shu Qin Ooi ◽

Verena Ming Hui Tan ◽

Siong Gim Ong ◽

Yiong Huak Chan ◽

Chew Kiat Heng ◽

...

Keyword(s):

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Buccal Cells ◽

Copy Numbers ◽

Gene Copy Numbers

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Applying Standard Clinical Chemistry Assay Validation to Droplet Digital PCR Quantitative Liquid Biopsy Testing

Clinical Chemistry ◽

10.1373/clinchem.2018.291278 ◽

2018 ◽

Vol 64 (12) ◽

pp. 1732-1742 ◽

Cited By ~ 17

Author(s):

Dragana Milosevic ◽

John R Mills ◽

Michael B Campion ◽

Noemi Vidal-Folch ◽

Jesse S Voss ◽

...

Keyword(s):

Dynamic Range ◽

Clinical Laboratory ◽

Limit Of Detection ◽

Clinical Chemistry ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Performance Criteria ◽

Limit Of Quantification ◽

Assay Validation ◽

Copy Numbers

Abstract BACKGROUND Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.

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