Comparison of total and endometrial circulating cell-free DNA in women with and without endometriosis.

RESEARCH QUESTION: Do women with laparoscopically confirmed endometriosis have higher plasma concentrations of circulating cell-free DNA (cirDNA) than those without endometriosis? DESIGN: Prospective study of women aged 18-45 years undergoing benign gynaecological laparoscopy at two tertiary hospitals. Venous blood was collected immediately before surgery, and women were allocated to the endometriosis or control groups based on surgical findings. Total plasma cirDNA and cirDNA integrity were measured by quantitative polymerase chain reaction (qPCR) targeting short (115 bases) and long (247 bases) ALU segments. Endometrial-derived cirDNA was measured by qPCR of bisulfite-treated cirDNA using primers selective for a FAM101A sequence uniquely unmethylated in endometrial tissue. Five cirDNA parameters were compared between the control and endometriosis cohorts: total cirDNA concentration, long-stranded cirDNA concentration, integrity ratio, endometrial cirDNA concentration and endometrial cirDNA proportion. RESULTS: Twenty-eight endometriosis and 15 control samples were included. Women with and without endometriosis had cirDNA concentrations of 2.24 ± 0.89 ng/ml and 2.56 ± 0.92 ng/ml, respectively. Analysis by phenotype of endometriosis revealed a significantly higher endometrial cirDNA concentration in women with superficial disease (n = 10) compared with deep endometriosis (n = 18) (mean difference 0.14 ng/ml; 95% CI 0.15 to 0.26; P = 0.025), but not with controls. CONCLUSIONS: No significant differences were found in any of the cirDNA parameters between women with and without endometriosis. The low statistical power and heterogenous pelvic pathology in the control group render it difficult to determine whether the negative results reflect a true lack of increase in cirDNA in endometriosis.

Affinity Selections of DNA-Encoded Chemical Libraries on Carbonic Anhydrase IX-Expressing Tumor Cells Reveal a Dependence on Ligand Valence.

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; KD =8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.

Cell-free DNA is abundant in ascites and represents a liquid biopsy of ovarian cancer.

OBJECTIVE: Malignant ascites is a common clinical feature of ovarian cancer and represents a readily accessible sample of tumour cells and tumour DNA. This study aimed to characterise the cell-free DNA (cfDNA) in ascites in terms of its size profile, stability and cell-free tumour DNA (cftDNA) content. METHODS: Cell spheroids, loose cells and cell-free fluid was collected from ascites from 18 patients with ovarian cancer. cfDNA was isolated and assessed for size by electrophoresis, concentration by fluorometry,cftDNA content by methylation specific qPCR of HOXA9 and IFFO1 promoter regions and by targeted sequencing. Stability was assessed after ascites fluid was stored at 4 °C for 24 and 72 h before fractionating. RESULTS: The concentration of cfDNA in ascites ranged from 6.6 to 300 ng/mL. cfDNA size distribution resembled blood plasma-derived cfDNA, with major peaks corresponding to mono- and di-nucleosome DNA fragments. High molecular weight cfDNA was observed in 7 of 18 patients and appeared to be associated with extracellular vesicles. IFFO1 and HOXA9 methylation was proportionately higher in cfDNA than spheroid- and loose-cell fractions and was not observed in healthy primary cells. Variant allele frequency was highest in cfDNA compared to single cells and spheroids from ascites. Though cancer cell numbers in ascites declined to near zero in recurrent ascites from one patient undertaking chemotherapy, cftDNA could still be sampled. cfDNA size, concentration and tumour content was stable over 72 h. CONCLUSION: cfDNA in ovarian cancer ascites demonstrates inter-patient variability, yet is consistently a rich source of cftDNA, which is a stable substrate. This supports the wider clinical use of ascites in the molecular analysis of ovarian cancer.

Genotyping Sri Lankan women with polycystic ovary syndrome (PCOS): towards a novel screening tool.

Introduction: Polycystic ovary syndrome (PCOS) is the multigenic, endocrine disorder of young women. Inheritance of PCOS is likely to be oligogenic and genetic basis remains largely unknown. Screening the candidate genes of PCOS and their SNPs individually is time consuming. Hence, developing a tool that would help in screening multiple candidate genes simultaneously is essential to determine the exact genetic basis of PCOS. Objectives: This study aimed to develop a simple and cost-effective genetic screening tool to simultaneously genotype 16 single nucleotide polymorphisms (SNPs) of PCOS. Methods: The genetic screening tool was developed using allele specific real time quantitative PCR (AS-qPCR) in 96 well PCR plate. Eight SNPs identified in our previous study as well as 8 SNPs identified from other reported studies that had a strong association in the etiology of PCOS were used to develop the tool. Samples from our previous study were reanalyzed using the developed genetic screening tool. Genetic screening tool results were validated with Sanger sequencing. Results: Totally 10 AS-qPCR runs (160 reactions = 16SNPs*10runs) were performed using the developed tool and all except 3 genotype results agreed with Sanger sequencing. The tool showed 100% specificity and 96% sensitivity. Conclusion: The developed genetic screening tool has excellent potential in determining the genotype of multiple SNPs of PCOS simultaneously. This tool is highly suitable for developing countries as a cost effective and accurate early genetic screening test for PCOS. Thus, provides a reliable, fast and user-friendly genotyping method facilitating a wider implication in clinical practice.

Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment.

Different methodological approaches are available to assess DNA methylation biomarkers. In this study, we evaluated two sodium bisulfite conversion-dependent methods, namely pyrosequencing and methylation-specific qPCR (MS-qPCR), with the aim of measuring the closeness of agreement of methylation values between these two methods and its effect when setting a cut-off. Methylation of tumor suppressor gene p16/INK4A was evaluated in 80 lung cancer patients from which cytological lymph node samples were obtained. Cluster analyses were used to establish methylated and unmethylated groups for each method. Agreement and concordance between pyrosequencing and MS-qPCR was evaluated with Pearson's correlation, Bland-Altman, Cohen's kappa index and ROC curve analyses. Based on these analyses, cut-offs were derived for MS-qPCR. An acceptable correlation (Pearson's R2 = 0.738) was found between pyrosequencing (PYRmean) and MS-qPCR (NMP; normalized methylation percentage), providing similar clinical results when categorizing data as binary using cluster analysis. Compared to pyrosequencing, MS-qPCR tended to underestimate methylation for values between 0 and 15%, while for methylation >30% overestimation was observed. The estimated cut-off for MS-qPCR data based on cluster analysis, kappa-index agreement and ROC curve analysis were much lower than that derived from pyrosequencing. In conclusion, our results indicate that independently of the approach used for estimating the cut-off, the methylation percentage obtained through MS-qPCR is lower than that calculated for pyrosequencing. These differences in data and therefore in the cut-off should be examined when using methylation biomarkers in the clinical practice.

Company matters: The presence of other genotypes alters traits and intraspecific selection in an Arctic diatom under climate change.

Arctic phytoplankton and their response to future conditions shape one of the most rapidly changing ecosystems on the planet. We tested how much the phenotypic responses of strains from the same Arctic diatom population diverge and whether the physiology and intraspecific composition of multistrain populations differs from expectations based on single strain traits. To this end, we conducted incubation experiments with the diatom Thalassiosira hyalina under present-day and future temperature and pCO2 treatments. Six fresh isolates from the same Svalbard population were incubated as mono- and multistrain cultures. For the first time, we were able to closely follow intraspecific selection within an artificial population using microsatellites and allele-specific quantitative PCR. Our results showed not only that there is substantial variation in how strains of the same species cope with the tested environments but also that changes in genotype composition, production rates, and cellular quotas in the multistrain cultures are not predictable from monoculture performance. Nevertheless, the physiological responses as well as strain composition of the artificial populations were highly reproducible within each environment. Interestingly, we only detected significant strain sorting in those populations exposed to the future treatment. This study illustrates that the genetic composition of populations can change on very short timescales through selection from the intraspecific standing stock, indicating the potential for rapid population level adaptation to climate change. We further show that individuals adjust their phenotype not only in response to their physicochemical but also to their biological surroundings. Such intraspecific interactions need to be understood in order to realistically predict ecosystem responses to global change.

Preferential Plastid Retention by the Acquired Phototroph Mesodinium chamaeleon.

The ciliate genus Mesodinium contains species that rely to varying degrees on photosynthetic machinery stolen from cryptophyte algal prey. Prey specificity appears to scales inversely with this reliance: The predominantly phototrophic M. major/rubrum species complex exhibits high prey specificity, while the heterotrophic lineages M. pulex and pupula are generalists. Here, we test the hypothesis that the recently described mixotroph M. chamaeleon, which is phylogenetically intermediate between M. major/rubrum and M. pulex/pupula, exhibits intermediate prey preferences. Using a series of feeding and starvation experiments, we demonstrate that M. chamaeleon grazes and retains plastids at rates which often exceed those observed in M. rubrum, and retains plastids from at least five genera of cryptophyte algae. Despite this relative generality, M. chamaeleon exhibits distinct prey preferences, with higher plastid retention, mixotrophic growth rates and efficiencies, and starvation tolerance when offered Storeatula major, a cryptophyte that M. rubrum does not appear to ingest. These results suggest that niche partitioning between the two acquired phototrophs may be mediated by prey identity. M. chamaeleon appears to represent an intermediate step in the transition to strict reliance on acquired phototrophy, indicating that prey specificity may evolve alongside degree of phototrophy.

Early Detection of Food Safety and Spoilage Incidents Based on Live Microbiome Profiling and PMA-qPCR Monitoring of Indicators.

The early detection of spoilage microorganisms and food pathogens is of paramount importance in food production systems. We propose a novel strategy for the early detection of food production defects, harnessing the product microbiome. We hypothesize that by establishing microbiome datasets of proper and defective batches, indicator bacteria signaling production errors can be identified and targeted for rapid quantification as part of routine practice. Using the production process of pastrami as a model, we characterized its live microbiome profiles throughout the production stages and in the final product, using propidium monoazide treatment followed by 16S rDNA sequencing. Pastrami demonstrated product-specific and consistent microbiome profiles predominated by Serratia and Vibrionimonas, with distinct microbial signatures across the production stages. Based on the established microbiome dataset, we were able to detect shifts in the microbiome profile of a defective batch produced under lactate deficiency. The most substantial changes were observed as increased relative abundances of Vibrio and Lactobacillus, which were subsequently defined as potential lactate-deficiency indicators. PMA-qPCR efficiently detected increased levels of these species, thus proving useful in rapidly pinpointing the production defect. This approach offers the possibility of the in-house detection of defective production events with same-day results, promoting safer food production systems.

Microbial Community Establishment, Succession, and Temporal Dynamics in an Industrial Semi-Synthetic Metalworking Fluid Operation: A 50-Week Real-Time Tracking.

Microorganisms colonizing modern water-based metalworking fluids (MWFs) have been implicated in various occupational respiratory health hazards to machinists. An understanding of the exposure risks from specific microbial groups/genera/species (pathogenic or allergenic) and their endotoxins and the need for strategies for effective, timely fluid management warrant real-time extended tracking of the establishment of microbial diversity and the prevailing fluid-related factors. In the current study, the microbial community composition, succession, and dynamics of a freshly recharged industrial semi-synthetic MWF operation was tracked in real-time over a period of 50 weeks, using a combination of microbiological and molecular approaches. Substantial initial bacterial count (both viable and non-viable) even in the freshly recharged MWF pointed to the inefficiency of the dumping, cleaning, and recharge (DCR) process. Subsequent temporal analysis using optimized targeted genus/group-specific qPCR confirmed the presence of Pseudomonads, Enterics, Legionellae, Mycobacteria (M. immunogenum), Actinomycetes, and Fungi. In contrast, selective culturing using commercial culture media yielded non-specific isolates and collectively revealed Gram-negative (13 genera representing 19 isolates) and Gram-positive (2 genera representing 6 isolates) bacteria and fungi but not mycobacteria. Citrobacter sp. and Bacillus cereus represented the most frequent Gram-negative and Gram-positive isolates, respectively, across different media and Nectria haematococca isolation as the first evidence of this fungal pathogen colonizing semi-synthetic MWF. Unbiased PCR-DGGE analysis revealed a more diverse whole community composition revealing 22 bacterial phylotypes and their succession. Surges in the endotoxin level coincided with the spikes in Gram-negative bacterial population and biocide additions. Taken together, the results showed that semi-synthetic MWF is conducive for the growth of a highly diverse microbial community including potential bacterial and fungal pathogens, the current DCR practices are inefficient in combating microbial reestablishment, and the practice of periodic biocide additions facilitates the build-up of endotoxins and non-viable bacterial population.

A vertebrate-specific qPCR assay as an endogenous internal control for robust species identification.

The identification of vertebrate species is important in numerous fields including archaeology, ecology, as well as food and forensic sciences. Real-time quantitative PCR (qPCR) assays specific for one vertebrate species are promising approaches for species identification, although there are several drawbacks such as difficulty determining whether the detected DNA is authentic or a contaminant. Here, we describe a qPCR assay specific for vertebrate mitochondrial DNA (mtDNA) which can overcome these drawbacks. Since we found that mitochondrial 16S rRNA contains regions that are perfectly (not highly) conserved across virtually all vertebrates, but are variable in invertebrates, we were able to design a vertebrate-specific qPCR assay by placing primers/probe within these regions. The specificity and accuracy of this assay were validated with representative vertebrate and invertebrate samples. This assay detected DNA from all vertebrate samples, but not from any invertebrate samples. In addition, this assay was able to quantify vertebrate mtDNAs as accurately as previously reported species-specific qPCR assays. The results demonstrated it is feasible to quantify vertebrate mtDNA specifically and accurately in a sample. This means that it is possible to determine the ratio of specific vertebrate species mtDNA to total vertebrate mtDNA in a sample. In conjunction with this assay as an endogenous internal control, species-specific qPCR assays will allow for the robust identification of vertebrate species.

DYSF promotes monocyte activation in atherosclerotic cardiovascular disease as a DNA methylation-driven gene.

Dysferlin (DYSF) has drawn much attention due to its involvement in dysferlinopathy and was reported to affect monocyte functions in recent studies. However, the role of DYSF in the pathogenesis of atherosclerotic cardiovascular diseases (ASCVD) and the regulation mechanism of DYSF expression have not been fully studied. In this study, Gene Expression Omnibus (GEO) database and epigenome-wide association study (EWAS) literatures were searched to find the DNA methylation-driven genes (including DYSF) of ASCVD. The hub genes related to DYSF were also identified through weighted correlation network analysis (WGCNA). Regulation of DYSF expression through its promoter methylation status was verified using peripheral blood leucocytes (PBLs) from ASCVD patients and normal controls, and experiments on THP1 cells and Apoe-/- mice. Similarly, the expressions of DYSF related hub genes, mainly contained SELL, STAT3 and TMX1, were also validated. DYSF functions were then evaluated by phagocytosis, transwell and adhesion assays in DYSF knock-down and overexpressed THP1 cells. The results showed that DYSF promoter hypermethylation up-regulated its expression in clinical samples, THP1 cells and Apoe-/- mice, confirming DYSF as a DNA methylation-driven gene. The combination of DYSF expression and methylation status in PBLs had a considerable prediction value for ASCVD. Besides, DYSF could enhance the phagocytosis, migration and adhesion ability of THP1 cells. Among DYSF related hub genes, SELL was proven to be the downstream target of DYSF by wet experiments. In conclusion, DYSF promoter hypermethylation upregulated its expression and promoted monocytes activation, which further participated in the pathogenesis of ASCVD.

Rapid Diagnosis of Central Nervous System Scedosporiosis by Specific Quantitative Polymerase Chain Reaction Applied to Formalin-Fixed, Paraffin-Embedded Tissue.

Scedosporium (S.) apiospermum is a typical mold causing cerebral abscesses, often after near-drowning. Infections are associated with high morbidity and mortality due to diagnostic challenges including the need for prolonged incubation of cultures. In addition, histopathological differentiation from other filamentous fungi, including Aspergillus fumigatus, may not be possible, excluding early specific diagnosis and targeted therapy. Polymerase chain reaction (PCR) on tissue samples can rapidly identify fungi, leading to an earlier adequate treatment. Due to an extensive spectrum of causative fungi, broad-range PCRs with amplicon sequencing have been endorsed as the best DNA amplification strategy. We herein describe a case with brain abscesses due to S. apiospermum in a 66-year-old immunocompromised female patient. While broad-range PCR failed to identify a fungal pathogen from a cerebral biopsy demonstrating hyaline mold hyphae, specific quantitative PCR (qPCR) identified Scedosporium and ruled out Aspergillus, the most prevalent agent of central nervous system mold infection. A panel of specific qPCR assays, guided by the morphology of fungal elements in tissue or as a multiplex assay, may be a successful molecular approach to identify fungal agents of brain abscesses. This also applies in the presence of negative broad-range fungal PCR, therefore providing diagnostic and therapeutic potential for early specific management and improvement of patient clinical outcome.

Evaluation of simple sequence repeats (SSR) and single nucleotide polymorphism (SNP)-based methods in olive varieties from the Northwest of Spain and potential for miniaturization.

Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.

Detection of EGFR mutations in liquid biopsy samples using allele-specific quantitative PCR: A comparative real-world evaluation of two popular diagnostic systems.

PURPOSE: The detection of epidermal growth factor receptor (EGFR) mutations in plasma cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. We compared the diagnostic accuracy of two commonly used in vitro diagnostic-certified allele-specific quantitative PCR assays for detecting plasma cfDNA EGFR mutations. METHODS: We analyzed EGFR mutations in plasma cfDNA from 90 NSCLC patients (stages I-IV) before treatment (n â€‹= â€‹60) and after clinical progression on EGFR tyrosine kinase inhibitors (n â€‹= â€‹30) using the cobas EGFR mutation test v2 (Roche Molecular Systems, Inc.) and therascreen EGFR Plasma RGQ PCR kit (Qiagen GmbH). RESULTS: There was higher concordance between plasma cfDNA and matched tumor tissue EGFR mutations with cobas (66.67%) compared with therascreen (55.93%). The concordance rate increased to 90.00% with cobas (Cohen's kappa coefficient, κ â€‹= â€‹0.80; p â€‹< â€‹0.0001) and 73.33% with therascreen (κ â€‹= â€‹0.49; p â€‹= â€‹0.0009) in advanced NSCLC patients. In treatment-naïve patients, cobas was superior to therascreen (sensitivity: 82.35% vs. 52.94%; specificity: 100% vs. 100%). In patients with clinical progression on EGFR tyrosine kinase inhibitors, EGFR exon 20 p.T790M was detected in 30% and 23% of cfDNA samples by cobas and therascreen, respectively. CONCLUSIONS: Cobas was superior to therascreen for detection of plasma EGFR mutations in advanced NSCLC. Plasma cfDNA EGFR mutation analysis is complex; therefore, the diagnostic accuracy of commercially available assays should be validated.

Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae.

Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8-99.3%; R 2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicability of our protocol for field samples, we analyzed three different Hong Kong coral samples. Six Symbiodiniaceae genera were detected in Acropora valida, Oulastrea crispata, and Platygyra acuta, with Breviolum, Effrenium, Fugacium, and Gerakladium sp. being reported for the first time. Our results suggest that aggressively disrupting cells to ensure thorough cell lysis, estimating cell loss and DNA loss, and validating qPCR assays are critical for success. The number of species examined here is limited, but the primers are potentially applicable to most species in respective genera, and the protocol and the approach to develop it provide a base and template toward a standardized procedure for quantitatively characterizing Symbiodiniaceae communities in corals.

A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus.

Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses.

A fast and reliable method for detecting SNP rs67384697 (Hsa-miR-148a binding site) by a single run of allele-specific real-time PCR.

Surface expression of human leukocyte antigen (HLA)-class I molecules is critical for modulating T/natural killer lymphocytes' effector functions. Among HLA molecules, HLA-C, the most recently evolved form of class I antigens, is subjected to both transcriptional and multiple post-transcriptional regulation mechanisms affecting its cell surface expression. Among the latter a region placed in the 3' untranslated region of HLA-C transcript contains the single nucleotide polymorphism (SNP) rs67384697 "G-ins/del" that has been found to be strictly associated with surface levels of HLA-C allomorphs because of the effect on the binding site of a microRNA (Hsa-miR-148a). Higher expression of HLA-C has been proved to influence HIV-1 infection via a better control of viremia and a slower disease progression. More importantly, the analysis of SNP rs67384697 "G-ins/del" combined with the evaluation of the HLA-Bw4/-Bw6 C1/C2 supratype, as well as the killer immunoglobulin-like receptor genetic asset, has proved to be pivotal in defining the status of Elite Controllers in the Caucasian population. Here we describe a new reliable and fast method of allele-specific real-time PCR to monitor the integrity/disruption of the binding site of the microRNA Hsa-miR-148a in a high-throughput format that can be easily applied to studies involving large cohorts of individuals.

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method.

Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method-the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

Detection of Chlamydiaceae in ocular swabs from Australian pre-export feedlot sheep.

Infectious Ovine Keratoconjunctivitis (IOK) is a contagious ocular disease of sheep. A range of organisms have been observed as the aetiological agents of IOK. In this study, the presence of chlamydial pathogens (C. pecorum, C. abortus, C. psittaci) in conjunctival swabs was tested for. The swabs were collected from sheep with varying grades of IOK in an Australian pre-export feedlot. The sheep had been rejected from a shipment because of the eye disease. The relative contribution of chlamydial pathogens to IOK and the rejection of animals was evaluated. In total, 149 conjunctival swabs were taken from rejected sheep (IOK Grades 1 to 6; n = 126) as well as those with healthy eyes (Grade 0; n = 23). Screening for chlamydial pathogens was done using species-specific qPCR assays. Chlamydial DNA was detected in 35.6% (53/149) of conjunctival samples. C. pecorum was the most predominant species with an overall prevalence of 28.9% (43/149). C. psittaci prevalence was 6.7% (10/149). Both organisms were detected in healthy as well as IOK-affected eyes. All swabs tested negative for C. abortus. The results from this study demonstrate that Chlamydia spp can be readily detected in sheep presenting with IOK. The zoonotic C. abortus was not detected in any of the samples in this study, providing further evidence to the suggestion that this pathogen remains absent from Australia. Although the exact contribution of Chlamydia spp in the IOK pathogenesis is unclear, such studies are anticipated to be of benefit to Australian domestic and live export production systems.

Chlamydial infection and on-farm risk factors in dairy cattle herds in South East Queensland.

Chlamydial infections in dairy cattle are common and have been sporadically associated with reduced performance and severe disease manifestations. While chlamydial infections are well described in sheep, very little is known about the epidemiology of these infections in dairy cattle in Australia. In this study, we screened for chlamydial infections and assessed on-farm risks in dairy cattle herds from Southeast Queensland (SE Qld) region of Australia. In total, 228 paired vaginal and rectal swabs were collected from 114 visually healthy dairy cows from four farms in SE Qld. Risk factors were rated by observational study and included: hygiene and cleanliness of cows, walkway and parlour, incidence of perinatal mortality, external replacements, mode of breeding, calving pen management, heat reduction strategies, and feed ration usage. Testing for chlamydial pathogens (Chlamydia pecorum, Chlamydia psittaci and Chlamydia abortus) was done using species-specific quantitative polymerase chain reaction (qPCR) assays. Detected rates of chlamydial infection were evaluated against the on-farm risk factors. C. pecorum infection was widespread in all four farms, with 56.1% (64/114) of individual animals shedding this organism from vaginal and rectal, or both sites. C. abortus and C. psittaci were not detected in any animals. No association was found to exist with risk factors and C. pecorum infection rates in our study, however the number of Chlamydia positive animals was statistically different between the herds. This study suggests that subclinical chlamydial infections may impact on dairy herd health at the production level rather than affecting individual animal.