Digital Polymerase Chain Reaction Technology Research Articles

Comparison of digital PCR platforms using the molecular marker.

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

Traceability of the local cultivar ‘Caaveiro’ in flour mixtures used to produce Galician bread by simple sequence repeats and droplet digital polymerase chain reaction technology

SummaryThe analysis of simple sequence repeats (SSRs) and of bulk ground samples by droplet digital polymerase chain reaction (ddPCR) was investigated as an alternative to individual kernel testing for assessing the presence of local cultivars in common wheat (Triticum aestivum L.) varietal blends. The recent Protected Geographical Indication (PGI) of ‘Pan Galego’ (Galician bread) requires that the flour comprise a minimum 25% local wheat cultivars. As a test for compliance with this minimum level, wheat flours were prepared by mixing commercial flours with 0%, 5%, 20%, 25% and 30% ‘Caaveiro’ and 100% ‘Caaveiro’ and 100% commercial flours were used as controls. A second analysis was performed with a second set of wheat flours with 5% and 25% ‘Caaveiro’. These were mixed with two different commercial flours to assess the potential ability of five SSRs to identify the percentage of ‘Caaveiro’, constituting the first reference of the use of SSRs in the traceability of specific autochthonous cultivars in flour blends. ddPCR using the QX200 system platform was used to the targeted proportions across the simulated range with two out of five SSRs, indicating that they can be used in the traceability of ‘Caaveiro’ in mixed flours and breads.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (S/N) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high S/N ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes. Second, fluorescent-encoded beads (FEBs), coupled with the corresponding target sequence-specific capture probes through streptavidin-biotin conjugation, are used to partition amplicons via hybridization according to the Poisson distribution. Finally, FEBs mixed with digital PCR mixes are isolated into droplets generated via Sapphire chips (Naica Crystal Digital PCR system) to complete the digital PCR and result analysis. For proof of concept, we demonstrate that this method achieves high S/N ratios in a 5-plexed assay for influenza viruses and SARS-CoV-2 at concentrations below 10 copies and even close to a single molecule per reaction without cross-reaction, further verifying the possibility of clinical actual sample detection with 100% accuracy, which paves the way for the realization of digital PCR with ultrahigh coding capacity and ultra-sensitivity.

The development of real-time digital PCR technology using an improved data classification method

For digital polymerase chain reaction (PCR), data classification is always a crucial task. The dynamic real-time amplification process information of each partition is always ignored in typical digital PCR analysis, which can easily lead to inaccurate outcomes. In this work, an integrated device that offers real-time chip-based digital PCR analysis was established. In addition, an enhanced process-based classification model (PAM) was built and trained. And then the device and the analytical model were employed in classification tasks for different concentrations of Epstein-Barr Virus (EBV) plasmid quantification assays. The results indicated that the real-time analysis device achieved a linearity of 0.97, the classification method was able to distinguish the false-positive curves, and the recognition error of positive wells was decreased by 64.4% compared with typical static analysis techniques when low concentrations of samples were tested. With these advantages, it is supposed that the real-time digital PCR analysis apparatus and the improved classification method can be employed to enhance the performance of digital PCR technology.

[Clinical importance of screening differential gene set of monocytes based on single-cell sequencing and digital polymerase chain reaction technology for early diagnosis of sepsis].

To verify the specific differentiated subsets of monocytes in sepsis, and to screen and construct the differential gene set of monocytes used for early diagnosis of sepsis. Patients with sepsis admitted to Guangdong Provincial People's Hospital from June 2020 to March 2021 were enrolled, and peripheral blood mononuclear cells (PBMC) were extracted. Single-cell sequencing technology and pseudo-time analysis were used to verify the differential subsets of monocytes. Bioinformatics methods were used to analyze the expression of genes in differential subsets of monocytes and screen out differential genes for the preliminary construction of a candidate differential gene set. The digital polymerase chain reaction (PCR) technology was used to verify the candidate differential genes in PBMC of sepsis patients and sepsis human myeloid leukemia mononuclear cells (THP-1) models, and the Venn diagram was used to construct the final differential gene set of monocytes. Gene Expression Omnibus (GEO) database was used to validate the differential gene set of monocytes. (1) The results of cell annotation and pseudo-time analysis showed that the differentiation of NEAT1+CD163+ monocyte occurred in the early stage of sepsis was significantly different from other subsets, which validated that NEAT1+CD163+ monocyte was the characteristic subset in the pathological process of sepsis. (2) Twenty-two differential genes related to sepsis were screened out from the gene expression of NEAT1+CD163+ monocyte. After further verification by digital PCR, basic leucine zipper ATF-like transcription factor (BATF), JUNB proto-oncogene, carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4), chromosome 9 open reading frame 95 (C9orf95), G protein subunit alpha 15 (GNA15), complement C3a receptor 1 (C3AR1), transforming growth factor beta 1 (TGFB1) and mitochondrial carrier homolog 1 (MTCH1) were screened out to construct the final differential gene set of monocytes. (3) The external validation results showed that C9orf95 gene had no data in GSE154918 and GSE133822 from GEO, it was excluded during validation. In GSE154918, the expressions of BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1 in the sepsis group were significantly higher than those in the healthy control group (log2expression level: BATF was 12.78±0.08 vs. 11.39±0.35, JUNB was 16.88±0.07 vs. 16.04±0.03, CEACAM4 was 14.73±0.08 vs. 13.77±0.05, GNA15 was 13.16±0.06 vs. 12.30±0.04, C3AR1 was 14.62±0.13 vs. 12.87±0.05, TGFB1 was 16.95±0.05 vs. 16.57±0.36, MTCH1 was 14.80±0.02 vs. 14.61±0.15, all P < 0.05). In GSE133822, the expressions of BATF, CEACAM4, GNA15, and C3AR1 in the sepsis group were significantly higher than those in the health control group (log2expression level: BATF was 8.66±0.16 vs. 7.92±0.14, CEACAM4 was 9.20±0.16 vs. 8.36±0.20, GNA15 was 10.66±0.18 vs. 10.13±0.16, C3AR1 was 11.49±0.27 vs. 10.48±0.16, all P < 0.05), while the expressions of JUNB, TGFB1, and MTCH1 were not statistically different between two groups. The results of gene set variation analysis (GSVA) showed that the enrichment scores of monocytes differential gene set of sepsis group were significantly higher than those of the healthy control group in both GSE154918 (0.38±0.04 vs. -0.44±0.02) and GSE133822 (0.56±0.02 vs. 0.20±0.05, both P < 0.01). Receiver operator characteristic curve (ROC curve) analysis showed that the differential gene set of monocytes had a reliable diagnostic value for early sepsis with the area under ROC curve (AUC) of 0.993 [95% confidence interval (95%CI) was 0.980-1.000] in GSE154918 and 0.944 (95%CI was 0.873-1.000) in GSE133822. A differential gene set of monocytes (BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1) screened out by single-cell sequencing and digital PCR technology has a reliable diagnostic value for the early sepsis, and may provide a new idea for the early diagnosis of sepsis.

Digital PCR Fluorescence Image Segmentation Algorithm Based on Image Processing

Digital PCR (Droplet digital Polymerase Chain Reaction) technology is currently one of the mainstream technologies for the detection and quantification of nucleic acid samples. This technology is widely used in the field of molecular biology. This technology counts the number of negative and positive reaction chambers in fluorescent images, and realizes absolute quantification of nucleic acid molecules. Due to the influence of the sampling equipment, some fluorescent images have uneven illumination, and the sampled droplet images will have bad spots and large areas of bright spots. The traditional Otsu segmentation algorithm cannot achieve the ideal segmentation effect. Aiming at the problem of under-segmentation of fluorescent images with uneven illumination using the traditional Otsu method, this paper proposes an algorithm for local segmentation based on Otsu segmentation. This algorithm realizes the segmentation of fluorescent images with uneven illumination and detects bright spots in large areas. The problems of dead pixel removal and scratch processing have been well resolved.

Evaluation of HIF-1α and VEGF-A expression in radiation-induced cystitis: A case-control study.

ABSTRACTThe standard treatment for locally advanced cervical cancer (CC) is chemoradiotherapy. Once the bladder receives part of the radiation, a typical inflammatory condition that configures radiation-induced cystitis may develop. Chronic radiation-induced cystitis is commonly characterized by the bladder new submucosal vascularization, which is typically fragile and favors hematuria. The current study aims to investigate if Hypoxia-Induced Factor (HIF-1α) and its transcriptional target Vascular Endothelial Growth Factor A (VEGF-A) could be a primary pathway leading to increased submucosal vascularization. HIF-1α and VEGF-A mRNA levels in bladder core biopsies from CC patients treated with radiotherapy versus untreated (non-irradiated) patients were analyzed using a droplet digital polymerase chain reaction technology. Gene expression results showed that HIF-1α and VEGF-A had no significant differences between bladder samples from patients previously irradiated and untreated patient samples. However, a direct relationship between the degree of late morbidity and the expression of HIF-1α and VEGF-A has been demonstrated. Despite the lack of statistical significance precludes a definitive conclusion, the data presented herein suggests that further studies investigating the role of HIF-1α in bladder neovascularization in radiation-induced cystitis are highly recommended.

Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from the blood of Lyme disease patients by digital PCR.

Lyme disease patients would greatly benefit from a timely, sensitive, and specific molecular diagnostic test that can detect the causal agent Borrelia burgdorferi at the onset of symptoms. Currently available diagnostic methods recommended by the Centers for Disease Control and Prevention for Lyme disease involve indirect serological tests that rely on the detection of a host-antibody response, which often takes more than three weeks to develop. With this process, many positive cases are not detected within a timely manner, preventing a complete cure. In this study, we have developed a digital polymerase chain reaction (PCR) assay that detects Lyme disease on clinical presentation with a sensitivity two-fold higher than that of the currently available diagnostic methods, using a cohort of patient samples collected from the Lyme disease endemic state of Connecticut, USA, in 2016–2018. Digital PCR technology was chosen as it is more advanced and sensitive than other PCR techniques in detecting rare targets. The analytical detection sensitivity of this diagnostic assay is approximately three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients has hindered the clinical adoption of PCR-based diagnostic tests. However, this drawback was overcome by using a comparatively larger sample volume, applying pre-analytical processing to the blood samples, and implementing a pre-amplification step to enrich for B. burgdorferi-specific gene targets before the patient samples are analyzed via digital PCR technology. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma rather than whole blood. If detected in a timely manner, Lyme disease can be completely cured, thus limiting antibiotic overuse and associated morbidities.

Highly Sensitive Quantification of Plasma Severe Acute Respiratory Syndrome Coronavirus 2 RNA Sheds Light on its Potential Clinical Value

BackgroundCoronavirus disease 2019 (COVID-19) is a global public health problem that has already caused more than 662,000 deaths worldwide. Although the clinical manifestations of COVID-19 are dominated by respiratory symptoms, some patients present other severe damage such as cardiovascular, renal and liver injury or/and multiple organ failure, suggesting a spread of the SARS-CoV-2 in blood. Recent ultrasensitive polymerase chain reaction (PCR) technology now allows absolute quantification of nucleic acids in plasma. We herein intended to use the droplet-based digital PCR technology to obtain sensitive detection and precise quantification of plasma SARS-CoV-2 viral load (SARS-CoV-2 RNAaemia) in hospitalized COVID-19 patients.MethodsFifty-eight consecutive COVID-19 patients with pneumonia 8 to 12 days after onset of symptoms and 12 healthy controls were analyzed. Disease severity was categorized as mild-to-moderate in 17 patients, severe in 16 patients and critical in 26 patients. Plasma SARS-CoV-2 RNAaemia was quantified by droplet digital Crystal Digital PCR™ next-generation technology (Stilla Technologies, Villejuif, France).ResultsOverall, SARS-CoV-2 RNAaemia was detected in 43 (74.1%) patients. Prevalence of positive SARS-CoV-2 RNAaemia correlated with disease severity, ranging from 53% in mild-to-moderate patients to 88% in critically ill patients (p=0.036). Levels of SARS-CoV-2 RNAaemia were associated with severity (p=0.035). Among nine patients who experienced clinical deterioration during follow-up, eight had positive SARS-CoV-2 RNAaemia at baseline while only one critical patient with undetectable SARS-CoV-2 RNAaemia at the time of analysis died at day 27.ConclusionSARS-CoV-2 RNAaemia measured by droplet-based digital PCR constitutes a promising prognosis biomarker in COVID-19 patients

Expression and function of an Hac1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae

Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation. However, low levels of foreign protein expression by S. cerevisiae have limited their widespread application. The ability of the endoplasmic reticulum (ER) to fold and process foreign proteins is an important factor restricting the expression of foreign proteins. In the current study, the effects of transcription factor Hac1p, which is involved in the unfolded protein response pathway, on S. cerevisiae-based expression of xylanase gene xynB from Aspergillus niger were examined. Overlap extension polymerase chain reaction (PCR), rDNA integration and droplet digital PCR technology were used to generate a S. cerevisiae strain (S8) containing eight copies of xynB, allowing high-yield secretory expression of xylanase. The effects of subsequent overexpression of HAC1 in strain S8 on the expression of genes associated with protein folding in the ER were then examined using the GeXP system. Results confirmed the constitutive secretory expression of the multiple copies of xynB following rDNA-based integration of the expression cassette, with a maximum xylanase yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL.

Abstract 2455: Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform

Abstract In this study we present a single-step, one-color quantification of mutant versus wild-type allele using a novel integrated, clinic-optimized digital PCR (dPCR) platform. Allelic variants in DNA or RNA can be distinguished and quantified using Polymerase Chain Reaction (PCR) in concert with intelligent primer and probe design. Most commonly, a researcher or clinician will use unlabeled forward and reverse primers that bind to conserved regions outside of the allelic variation in addition to two differently labeled hydrolysis probes that bind at the region of allelic variation. Here we describe a novel method that leverages the precision of digital PCR (dPCR) technology and requires only one-color fluorescence utilizing probe-free, intercalating dye chemistry. The expense in developing or purchasing a complete PCR assay is normally embedded in the cost of the fluorescently labeled probe. The method described here eliminates the need for the fluorescently labeled probe, thereby reducing chemistry cost. Furthermore, technical complexity is reduced by requiring only one-color fluorescence. In our assay, we use a common unlabeled reverse primer that binds downstream of the allelic variation. Two unlabeled, allele-specific forward primers bind preferentially to either the wild-type or mutant allele at the site of allelic variation. The mutant forward primer contains a non-annealing tail (ie: stretch of the DNA primer that does not anneal to the initial DNA template) that will cause the ultimate mutant PCR product to be longer than the wild-type product. Digital PCR is characterized by dividing up a bulk PCR reaction volume into thousands of fluidically isolated partitions or droplets. In the presence of an intercalating dye, a longer amplicon will result in a higher endpoint fluorescence in that specific dPCR partition. As such, using our novel assay, dPCR partitions containing mutant versus wild-type amplicons can be distinguished by differing levels of end-point partition fluorescence. In this study we used the BRAF gene and the clinically relevant BRAF V600E mutation to benchmark this assay using a novel integrated one-step dPCR platform. This dPCR platform consists of a single, fully-integrated instrument along with a novel micro-injection molded plastic consumable providing a simple, single-step workflow. The instrument is dry and contamination-free, making it an attractive instrument for easy transition to the clinic. Parameters including thermal-cycling time and primer concentration were serially optimized for the novel integrated dPCR platform. This work highlights that we can accurately and precisely quantify samples containing varying ratios of wild-type versus V600E BRAF down to 0.1% mutant/wild-type ratio using one color of fluorescence and a novel integrated single-step walkaway dPCR workflow. Citation Format: Megan E. Dueck, Christina Wood-Bouwens, Andrew Zayac, Robert Lin, Steve Gallagher, Hanlee Ji, Paul Hung. Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2455.

Digital polymerase chain reaction technology - recent advances and future perspectives.

Digital polymerase chain reaction (dPCR) technology has remained a "hot topic" in the last two decades due to its potential applications in cell biology, genetic engineering, and medical diagnostics. Various advanced techniques have been reported on sample dispersion, thermal cycling and output monitoring of digital PCR. However, a fully automated, low-cost and handheld digital PCR platform has not been reported in the literature. This paper attempts to critically evaluate the recent developments in techniques for sample dispersion, thermal cycling and output evaluation for dPCR. The techniques are discussed in terms of hardware simplicity, portability, cost-effectiveness and suitability for automation. The present paper also discusses the research gaps observed in each step of dPCR and concludes with possible improvements toward portable, low-cost and automatic digital PCR systems.

Fungal Genomic DNA Extraction Methods for Rapid Genotyping and Genome Sequencing.

Isolation of fungal genomic DNA of high quality is required for a number of downstream biotechnology-derived applications such as genome sequencing, microarrays, and digital PCR technologies, to only name a few. In most cases, not only a high molecular weight DNA of superior grade is required but also large quantities. On the other hand, a number of laboratory experiments, such as polymerase chain reaction (PCR) for medical diagnostic or for genotyping, have to be conducted in a limited amount of time and can provide complete results with the use of lower quality DNA. We describe here two different fungal DNA extraction approaches, which are applicable to a wide range of fungal species.First, we adapted a DNA extraction method for PCR-based genotyping which allows analysis of single to hundreds of colonies simultaneously. Cells are disrupted in the presence of sodium dodecyl sulfate and Proteinase K which are then removed by precipitation and centrifugation. The cleared lysate is used for PCR reaction.Secondly, we describe a method to obtain genome sequencing quality grade DNA from fungal liquid cultures. Mycelia are harvested by either filtration or centrifugation. Cells are mechanically disrupted by liquid nitrogen grinding, followed by genomic DNA extraction using the QIAGEN's DNeasy® Plant Kit. The quality and quantity of genomic DNA is monitored by fluorometry.

Low-cost quantitative detection of nucleic acid using microbeads and microcolumn array chip

Abstract High sensitive digital polymerase chain reaction (PCR) is one of the novel technology in molecular biology and has become more and more important in the arena of nucleic acid quantification. However, existing digital PCR technologies usually require specialized platform like high sensitive fluorescence microscope or flow cytometer, which has increased the detection costs and limited their further applications. Here we developed a simple and low cost nucleic acid quantification method based on beads, emulsion, amplification, and magnetics (BEAMing), microsphere and microcolumn array chip. We used the streptavidin (SA) modified polystyrene microspheres to capture the biotin covered target beads obtained via BEAMing technology, which could represent the population of target DNA. The microspheres-beads complexes were then collected and trapped in a microcolumn array chip and observed under a normal microscope. Because the number of microspheres-beads complexes had a relationship of coupled Poisson distribution with the number of target DNA, we could easily infer the number of target DNA through counting the number of chip-trapped microspheres. Our method has reduced the costs and lowers down the difficulty of digital PCR technology, which may promote the applications of digital PCR in research and clinical field.

Basic Science Luminal

Journal of Gastroenterology and HepatologyVolume 32, Issue S2 p. 15-17 Supplement ArticleFree Access Basic Science Luminal First published: 17 August 2017 https://doi.org/10.1111/jgh.13888Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Mucosal thickening following oral administration of emu oil represents a process of normal intestinal growth in rats SJ Barker1,2, GS Howarth1,2,3, BL Scherer4, LC Chartier1,2, KY Cheah2, KA Lymn3 and S Mashtoub1,2,5 1Discipline of Physiology, Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia; 2Gastroenterology Department, Women's and Children's Hospital, Adelaide, South Australia, Australia; 3School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy, South Australia, Australia; 4Commonwealth Scientific and Industrial Research Organisation (CSIRO), Adelaide, South Australia, Australia; 5School of Medicine, University of Western Australia, Fiona Stanley Hospital, Perth, Western Australia, Australia Background and Aim: Emu oil (EO) has demonstrated therapeutic properties in models of intestinal damage. Previously, oral EO administration lengthened small intestinal villi and crypts in normal rats, although it remains unclear whether this represents a process of normal or aberrant growth. We sought to determine the impact of EO on enterocyte kinetics in normal healthy rats during EO administration and following cessation of EO treatment. Methods: Dark Agouti rats (n = 8 per group/time point) were randomly assigned to four groups and orally gavaged daily with either water (1 mL), olive oil (OO; 1 mL) or EO (low dose, 0.5 mL; or high dose, 1 mL) for 10 days. Groups of rats were euthanized on Days 10 and 17. Jejunal and ileal sections were stained with hematoxylin and eosin for quantification of crypt depth, crypt cell counts, and crypt cell diameter. Data were analyzed using a one-way anova with Tukey's post-hoc test and were expressed as mean ± SEM. P < 0.05 was considered significant. OO was used as a control oil on the basis of its similar oleic acid composition to EO. Results: On Day 10, only high-dose EO increased jejunal and ileal crypt depth compared with water controls (P < 0.05; jejunum: water, 155 ± 2 μm; OO, 169 ± 6 μm; low-dose EO, 157 ± 5 μm; high-dose EO, 175 ± 6 μm; ileum: water, 115 ± 6 μm; OO, 135 ± 6 μm; low-dose EO, 130 ± 6 μm; high-dose EO, 140 ± 4 μm). Following oil withdrawal, crypt depths returned to normal values. On Day 10, jejunal cell numbers per crypt increased in OO (59 ± 1) and high-dose EO (59 ± 1) groups compared with water controls (54 ± 1; P < 0.05), whereas, in the ileum, only high-dose EO (50 ± 1) resulted in increased cell numbers compared with water controls (42 ± 2; P < 0.05). After withdrawal of oils, jejunal and ileal cell numbers per crypt returned to normal water control values. On Days 10 and 17, jejunal and ileal average cell diameter was unaffected in all oil-treated rats compared with water controls (P > 0.05). Conclusions: Restoration of normal intestinal growth parameters following cessation of EO administration provides support for its safety as an oral supplement for bowel disorders. Oncogenic BRAF mutation induces widespread DNA hypermethylation in a murine model for human serrated colorectal neoplasia C Bond1, C Liu1,2, F Kawamata1,3, D McKeone1, S Jamieson1, S Pearson1, S Woods4, T Lannagan4, L Fennell1, W Fernando1, M Bettington5, D Worthley4, B Leggett1,2,6 and V Whitehall1,2,7 1Conjoint Gastroenterology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 2University of Queensland, Brisbane, Queensland, Australia; 3Hokkaido University, Hokkaido, Japan; 4Gastrointestinal Cancer Biology Group, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia; 5Envoi Specialist Pathologists, Brisbane, Queensland, Australia; 6Department of Gastroenterology and Hepatology, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia; 7Pathology Queensland, Brisbane, Queensland, Australia Background and Aim: The serrated colorectal neoplasia pathway describes the progression of morphologically serrated polyps to cancer and accounts for about one-third of all colorectal cancer cases. Human serrated polyps are characterized by an activating mutation of the BRAF oncogene and widespread DNA methylation changes termed the CpG island methylator phenotype. A causative versus synergistic relationship between BRAF mutation and this methylator phenotype has not been determined. We aimed to address this by developing a murine model for serrated neoplasia driven by BRAF mutation. Methods: BrafV637E conditionally active mice were crossed with intestine-specific, inducible Villin-CreERT2 mice to direct the BRAF mutation to the intestine at 2 weeks of age. The proximal ilieum and/or proximal colon were sampled at defined time points, including 10 days, 10 weeks, 5 months, 8 months, 10 months, 12 months, and 14 months. Macroscopic lesions larger than 10 mm were bisected for molecular and histological assessment. The entire remaining intestine was fixed and examined histologically. DNA methylation was investigated for 94 genes known to be methylated in colorectal cancer using Epitect MethylII Complete PCR Arrays (Qiagen). The MEK inhibitor, PD0325901, was administered for 10 days (25 mg/kg/day) in Braf mutant mice, with subsequent histological and methylation analyses. Results: Braf mutant mice displayed histological changes analogous to the human serrated neoplasia pathway. Extensive intestinal hyperplasia developed by 10 days after induction of the BRAF mutation. By 10 weeks, 50% of mice had developed areas of crypt dilation reminiscent of human sessile serrated adenomas. By 8 months, the majority of mice had murine serrated adenomas with dysplasia, and invasive cancer developed in 40% of mice by 14 months. Compared with age-matched control mice, Braf mutant mice showed significant, gene-specific increases in DNA methylation from 5 months (P < 0.0001). Following treatment with the MEK inhibitor, there was attenuation of the hyperplastic phenotype and a reduction of DNA methylation in a gene-specific manner Conclusions: Using an in vivo model, we observed the temporal accumulation of DNA methylation changes in hyperplastic epithelium in direct response to mutation of the BRAF oncogene. This murine model morphologically and molecularly recapitulates the human serrated neoplasia pathway and establishes a causative role for BRAF mutation in establishing a methylator phenotype. Human amnion epithelial cells reduce intestinal inflammation in a dextran sulfate sodium-induced murine model of acute colitis N Kuk1,2, J Correia1,2, M Alhomrani1,2,3, A Hodge1,2, R Lim3, W Sievert1,2 and G Moore1,2 1Centre for Inflammatory Disease, Monash University, Melbourne, Victoria, Australia; 2Gastroenterology and Hepatology Unit, Monash Health, Melbourne, Victoria, Australia; 3Hudson Institute of Medical Research, Melbourne, Victoria, Australia Introduction: Ulcerative colitis is a form of inflammatory bowel disease (IBD) characterized by inflammation and ulceration of the colon. Common symptoms include diarrhea, hematochezia, and weight loss. Immunosuppressive therapy is associated with numerous side effects, and a small subset of patients remain refractory to treatment. Sourced from placentas, human amnion epithelial cells (hAECs) possess anti-inflammatory properties. Combined with an excellent safety profile, these stem cells are a promising tool for inflammatory diseases. We examined the efficacy of hAECs in a dextran sulfate sodium (DSS)-induced murine model of acute colitis. Methods: C57BL/6J mice received 2% DSS (w/v) in their normal drinking water for 7 days. At Day 2, Group 1 (n = 17) received an intravenous injection of 2 × 106 hAECs. At Day 4, Group 2 (n = 17) received an intravenous injection of 2 × 106 hAECs. Control mice (n = 17) on DSS water were untreated. All mice were monitored daily and culled at Day 8. The extent of colitis severity was determined histologically through hematoxylin and eosin staining of the colon. Pro-inflammatory cytokines (IL-1β and tumor necrosis factor [TNF]-α) were measured through enzyme-linked immunosorbent assays. Neutrophil infiltration in the colon was evaluated with a myeloperoxidase (MPO) activity assay. Results: Control mice receiving DSS water lost significantly more body weight (–17.4%) compared with hAEC-treated mice (Group 1: –5.6%, P < 0.0001; Group 2: –8.8%, P < 0.0001). Similarly, controls had shorter colons (5.3 cm) compared with Group 1 (6.7 cm, P < 0.001) and Group 2 (6.3 cm, P < 0.01). hAEC-treated mice had significantly lower histological severity scores (Group 1: 5.3, P < 0.0001; Group 2: 8.33, P < 0.001) compared with controls (12). IL-1β in control mice (289.6 pg/mg protein) was significantly higher compared with Group 1 (166.5 pg/mg protein, P < 0.01) and Group 2 (202.5 pg/mg protein, P < 0.05). Likewise, TNF-α was higher in untreated control mice (26.8 pg/mg protein) compared with Group 1 (17.25 pg/mg protein, P < 0.01) and Group 2 (21.76 pg/mg protein, P < 0.05). There were no differences in hAEC-treated mice except mice treated at Day 2 had a lower histological severity score than mice at Day 4 (P < 0.01). Conclusion: hAECs exert a protective effect in DSS colitis and reduce the severity of disease, by reducing the level of neutrophil infiltration into the colon and decreasing concentrations of pro-inflammatory cytokines. Although the therapeutic effects of hAECs in IBD are promising, further research into their mechanism of actions is warranted. Gremlin 1-expressing intestinal reticular stem cells give rise to cancer-associated fibroblasts in a mouse model of colorectal cancer TRM Lannagan1, SL Woods1, T Wang1, R Somashekar1, M Yang1, JQ Ng1, K Gieniec1, YK Lee1, L Toh1, A Quek1, L Vrbanac1, N Suzuki1, M Ichinose1, Y Tailor2, Y Hayakawa4, S Asfaha3, SJ Leedham5, TC Wang2 and DL Worthley1 1School of Medicine, University of Adelaide and South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia; 2Columbia University, New York, New York, USA; 3University of Western Ontario, London, Ontario, Canada; 4University of Tokyo, Tokyo, Japan; 5Gastrointestinal Stem Cell Biology Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK All developing and adult organs are supported by connective tissues. We recently demonstrated that Gremlin 1-expressing cells in the bone (osteochondroreticular stem cells) and the bowel (intestinal reticular stem cells) are connective tissue stem cells, during development and healing. The contribution of these stem cells to the desmoplasia surrounding gastrointestinal and skeletal cancers, however, is unknown. In this study, we first established that typical markers of bone marrow skeletal stem cells also identify colony forming unit-fibroblasts (CFU-Fs) in the tumor microenvironment (identified by CD45-Ter-119-CD31-CD1040a+CD105+). Next, we tested the local origins of cancer-associated fibroblasts in a carcinogenesis (AOM/DSS) mouse model of colorectal cancer. Using the Grem1-creERT;R26-LSL-ZsGreen; Acta2-RFP transgenic mouse to lineage trace and report the connective tissues in the bowel, we found that Grem1-expressing and Acta2-expressing cells each contribute to reactive cancer-associated fibroblasts. In particular, we observed that Grem1 lineage traced cells gave rise to Acta2-expressing cells in the tumor microenvironment. We have additionally undertaken BrdU labeling (4–8 weeks) to confirm if these truly are new cells. We are also currently further characterizing these CAFs by co-staining with other known CAF markers. While neoplastic cells appear to make a significant contribution to cancer-associated fibroblasts in some other cancers, using an epithelial specific Cre (K19-cre) there was no contribution of epithelium to Acta2-expressing cells in our AOM-DSS colorectal cancer models. Thus, targeting the intestinal reticular stem cells, in the setting of gastrointestinal cancer, is a potential therapeutic target. Finally, we intend to compare the capacity of these Grem1-lineage traced CAFs to support the in vitro growth of colorectal normal and neoplastic organoids compared with other colonic mesenchymal cell types, as well as examine the secreted factors from these cells that are relevant to tumor initiation and spread. Metastatic gastric signet ring cell carcinoma organoids generated from normal mouse stomach N Suzuki1,2,3, M Hata1, Y Hikiba3, S Ihara1,3, S Kosuke1,3, H Nakagawa1, Y Hirata1, M Ichinose-Suzuki2, SL Woods2, DL Worthley2, Y Hayakawa1 and K Koike1 1Department of Gastroenterology, University of Tokyo, Tokyo, Japan; 2University of Adelaide and South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia; 3Division of Gastroenterology, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan Background: Signet ring cell carcinoma (SRCC) of the stomach has a particularly poor prognosis. This is due to frequent metastasis for this tumor type. Reduced expression of CDH1 (E-cadherin) is associated with the development of SRCC. However, there are still many unclear points in the development of SRCC. In recent years, it has become possible to culture normal digestive tract cells in vitro using organoid technology. Previously, we reported that normal transgenic bile duct organoids can be transformed by genetic modification with Lentivirus.1 Therefore, we examined whether it is possible to cause gastric organoids to become cancerous by Kras, TGFBR2 and Cdh1 gene modification. Methods: Cdh1 flox/flox mice were crossed with Kras-LSL-G12D and TGFBR2 flox/flox to generate Kras-LSL-G12D;TGFBR2 flox/flox:Cdh1 flox/flox mice. We isolated organoids from these mice and infected with Lenti-Cre virus to induce gene recombination. We confirmed the Kras gene recombination by polymerase chain reaction and also TGBR2 and Cdh1 by western blot analysis. Organoids after recombination were transplanted subcutaneously into the back of nude mice. Histological examination was performed on these organoids and transplanted tissues. Results: We were able to confirm Kras, TGFBR2, and Cdh1 gene recombination and make KrasG12D; TGFBR2 KO: Cdh1 KO gastric organoids (KTC-organoids). In the KTC-organoids, some cells spilling into the lumen and enlargement of the nucleus was seen. KTC-organoids also lost their adherens junction. Transplanted KTC-organoids were engrafted subcutaneously in the back of nude mice with the establishment of 100% (8/8). In addition, blood ascites and peritoneal metastasis were seen 2 months after transplantation. Pathological analysis of the subcutaneous and metastatic tumors diagnosed SRCC. Conclusion: Normal mouse gastric organoids develop into SRCC that can cause peritoneal metastasis by inducing Kras-G12D, TGFBR2-KO, and Cdh1-KO. This is the first model of SRCC that can cause the peritoneal metastasis of mice. References 11 Nakagawa H, Suzuki N, Hirata Y, et al. Biliary epithelial injury-induced regenerative response by IL-33 promotes cholangiocarcinogenesis from peribiliary glands. Proc. Natl. Acad. Sci. U.S.A. 2017; 114: E3806– 15. The development of a state-of the art, high-sensitivity stool DNA test to improve screening for serrated polyps and early lesions TRM Lannagan1, YK Lee1, K Gieniec1, T Wang1, R Somashekar1, M Yang1, JQ Ng1, L Vrbanac1, M Ichinose1, N Suzuki1, M.L. Bettington2,3, BA Leggett2, VL Whitehall2, DL Worthley1 and SL Woods1,† 1School of Medicine, University of Adelaide and Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia; 2QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 3Envoi Specialist Pathologists, Brisbane, Queensland, Australia †Contributed equally. The serrated or alternate pathway to colorectal cancer (CRC) accounts for 25% of all CRC. Serrated polyps frequently have sessile (flat) morphology, are much less likely to bleed and therefore be detected by fecal immunochemical testing, and are less conspicuous at colonoscopy. As such, patients with these early cancers often present or are diagnosed later, creating a major impediment to successful removal and treatment. Not surprisingly, this group contains treatment-resistant and worst-prognosis subtypes, as well as being over-represented in interval polyps and cancers. Serrated CRC and the development and detection of these lesions are the focus of this project. We model serrated CRC through combining recent advances in stem cell biology, genome editing, and small animal colonoscopy. We incorporate serrated specific genetic changes into primary colon organoids, orthotopically inject the resulting “serratoid” lines into recipient mouse colon and follow tumor formation and progression in vivo. The beauty of our approach using genome editing is that, unlike traditional transgenic approaches, we can rapidly incorporate multiple and previously untested genetic alterations associated with serrated CRC. Preclinical models have also yet to be effectively used to develop new strategies for CRC screening. To this end, we are testing a new, non-invasive, highly sensitive method for detecting early genetic changes, such as BrafV600E, found within these hard-to-detect serrated lesions. We use the most sensitive digital polymerase chain reaction technology on the market (RainDrop; RainDance Technologies) to analyze stool DNA samples from our preclinical models for serrated specific genetic changes. We envisage this could one day lead to a test that will complement Australia's current National Bowel Cancer Screening Program, to improve the detection of these hidden polyps and cancers. Citing Literature Volume32, IssueS2Special Issue: Gastroenterological Society of Australia Australian Gastroenterology Week Precision Medicine in Gastroenterology, Gold Coast Convention & Exhibition Centre, Gold Coast, Queensland, 20–22 Aug 2017August 2017Pages 15-17 ReferencesRelatedInformation

Digital PCR: Endless Frontier of 'Divide and Conquer'.

Digital polymerase chain reaction (PCR) is becoming ever more recognized amid the overwhelming revolution in DNA quantification, genomics, genetics, and diagnostics led by technologies such as next generation sequencing and studies at the single-cell level. The demand to quantify the amount of DNA and RNA has been driven to the molecular level and digital PCR, with its unprecedented quantification capability, is sure to shine in the coming era. Two decades ago, it emerged as a concept; yet one decade ago, integration with microfluidics invigorated this field. Today, many methods have come to public knowledge and applications surrounding digital PCR is mounting. However, to reach wider accessibility and better practicality, efforts are needed to tackle the remaining problems. This perspective looks back at several inspiring and influential digital PCR approaches in the past and tries to provide a futuristic picture of the trends of digital PCR technologies to come.

AKT1 (E17K) mutation profiling in breast cancer: prevalence, concurrent oncogenic alterations, and blood-based detection.

BackgroundThe single hotspot mutation AKT1 [G49A:E17K] has been described in several cancers, with the highest incidence observed in breast cancer. However, its precise role in disease etiology remains unknown.MethodsWe analyzed more than 600 breast cancer tumor samples and circulating tumor DNA for AKT1E17K and alterations in other cancer-associated genes using Beads, Emulsions, Amplification, and Magnetics digital polymerase chain reaction technology and targeted exome sequencing.ResultsOverall AKT1E17K mutation prevalence was 6.3 % and not correlated with age or menopausal stage. AKT1E17K mutation frequency tended to be lower in patients with grade 3 disease (1.9 %) compared with those with grade 1 (11.1 %) or grade 2 (6 %) disease. In two cohorts of patients with advanced metastatic disease, 98.0 % (n = 50) and 97.1 % (n = 35) concordance was obtained between tissue and blood samples for the AKT1E17K mutation, and mutation capture rates of 66.7 % (2/3) and 85.7 % (6/7) in blood versus tissue samples were observed. Although AKT1-mutant tumor specimens were often found to harbor concurrent alterations in other driver genes, a subset of specimens harboring AKT1E17K as the only known driver alteration was also identified. Initial follow-up survival data suggest that AKT1E17K could be associated with increased mortality. These findings warrant additional long-term follow-up.ConclusionsThe data suggest that AKT1E17K is the most likely disease driver in certain breast cancer patients. Blood-based mutation detection is achievable in advanced-stage disease. These findings underpin the need for a further enhanced-precision medicine paradigm in the treatment of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2626-1) contains supplementary material, which is available to authorized users.

Human papillomavirus detection in a "Digital" age.

It is increasingly important to identify the presence of human papillomavirus (HPV) in patients with oropharyngeal squamous cell carcinoma, because HPV status is now useful for clinical trial stratification, prognostic determination, diagnosis in patients with neck masses, and identification of the primary tumor site. Therefore, it is important that the technique used for identification be feasible, accurate, reproducible, and cost effective. The authors summarize these aspects of HPV detection and the use of newer digital polymerase chain reaction technology for this purpose. See also pages.

Picoliter-Droplet Digital Polymerase Chain Reaction-Based Analysis of Cell-Free Plasma DNA to AssessEGFRMutations in Lung Adenocarcinoma That Confer Resistance to Tyrosine-Kinase Inhibitors

The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC). Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues. cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%) were positive for the T790M mutation, with fractions of T790M (+) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance. Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs. Noninvasive monitoring of the predominance of tumors harboring the secondary T790M mutation in the activating mutation in EGFR gene is necessary for precise and effective treatment of lung adenocarcinoma. Because cells harboring the T790M mutation are resistant to epidermal growth factor receptor-tyrosine-kinase inhibitors (TKIs), the predominance of tumor cells harboring the T790M mutations influences the choice of whether to use conventional or next-generation TKIs. Digital polymerase chain reaction-based examination of cfDNA is a promising method; however, its feasibility, including its consistency with examination of rebiopsied tumor tissue, has not been fully proven. Here, picoliter-droplet digital polymerase chain reaction technology is presented as a candidate method for testing cfDNA and assessing the predominance of T790M-mutant tumors.

Digital PCR and Applications

Polymerase chain reaction (PCR), is a simple, effective and widely used enzymatic technic especially in the field of molecular biology which provides the opportunity to amplify a specific DNA fragment from DNA complex pool. Several PCR techniques such as realtime PCR, quantitative and qualitative PCR, Reverse Transcriptase PCR, Nested PCR and multiplex PCR have been developed since the first invention of PCR. Due to the various difficulties of the PCR techniques developed so far, widening the application fields and finding out more advanced level of PCR techniques have become the first priority of the researchers. Digital PCR (dPCR) technology has been developed as a new method to permit the evaluation of the small changes in the copy number variations of the rare mutations, differences between the gene expression changes or state of methylation. Digital PCR is a PCR based new technique for the sensitive measurement of number of DNA copies, it provides opportunity to do large number of PCR with a few number of sample dilution and it has so many small compartments where seperate PCRs are executed in each. At the same time, dPCR leaves some of the quantitative methods behind with the performance of determining the quantity of small amount of genetic material within seconds. This method shows high OZET Polimeraz zincir reaksiyonu (PZR), DNA kompleks