qPCR Protocol Research Articles

Estimation of the number of Anisakis larvae in commercial fish using a descriptive model based on real‐time PCR

Seafood parasitation by Anisakis (Anisakidae) larvae has been reported in most of the oceans and seas worldwide. The presence of these nematodes in commonly consumed fish represents a potential hazard for consumers as they can provoke gastrointestinal symptoms and allergic reactions. In the present work, the capacity of a SYBR Green qPCR protocol to quantify Anisakis larvae in commercial fish was evaluated using experimentally spiked samples with different numbers (0-50) of A. simplex third-stage larvae (L3). To verify the agreement of the obtained results, 25 naturally infected fish specimens of Atlantic blue whiting underwent a parallel visual inspection. The logarithmic behavior of the Cq data obtained from the experimentally spiked samples allowed the development of a descriptive mathematical model that correlates the Cq value with the number of Anisakis larvae (R2 = 0.9908, CV = 2.37%). In the commercial blue whiting specimens there was a high correlation between the results of the molecular technique and the visual inspection (R2 = 0.9912); the Bland-Altman analysis showed that 94% of the differences were within the limits of agreement (-4.98 and 6.68), indicating the reliability of the descriptive mathematical model based on the SYBR Green qPCR technique. The descriptive function presented based on the SYBR Green qPCR assay is promising as a sensitive and accurate tool for measuring the Anisakis larval load in commercial fish, with a potential application not only in the food industry but also in prevention programs for public health. © 2020 Society of Chemical Industry.

Molecular investigation of zoonotic intracellular bacteria in Chilean bats

Intracellular pathogens were investigated for the first time in 55 Chilean bats belonging to six species. Using a conventional PCR protocol targeting a fragment of the ITS region, 21 bats (38 %) were positive for DNA of Bartonella sp. Molecular characterization of fragments of the gltA, rpoB and fstZ genes and subsequent phylogenetic analysis indicated the presence of diverse genotypes related to Bartonella from bats worldwide. DNA from C. burnetii was investigated using a real-time PCR (qPCR) protocol targeting the IS1111 gene and yielded positive results for 5 individuals (9%), being the first report of C. burnetii in wildlife in Chile. All bats were negative for Rickettsia sp., evaluated by qPCR for the gltA gene, confirming that bats do not act as important reservoirs for Rickettsia. This preliminary survey calls for more comprehensive studies on the epidemiology of these agents, including larger sample sizes, the evaluation of potential transmission routes and spillover potential.

Prognostic Molecular Signatures for Metastatic Potential in Clinically Low-Risk Stage I and II Clear Cell Renal Cell Carcinomas.

Introduction: For patients with localized node-negative (Stage I and II) clear cell renal cell carcinomas (ccRCC), current clinicopathological staging has limited predictive capability because of their low risk. Analyzing molecular signatures at the time of nephrectomy can aid in understanding future metastatic potential.Objective: Develop a molecular signature that can stratify patients who have clinically low risk ccRCC, but have high risk genetic changes driving an aggressive metastatic phenotype.Patients, Materials, and Methods: Presented is the differential expression of mRNA and miRNA in 44 Stage I and Stage II patients, 21 who developed metastasis within 5 years of nephrectomy, compared to 23 patients who remained disease free for more than 5 years. Extracted RNA from nephrectomy specimens preserved in FFPE blocks was sequenced using RNAseq. MiRNA expression was performed using the TaqMan OpenArray qPCR protocol.Results: One hundred thirty one genes and 2 miRNA were differentially expressed between the two groups. Canonical correlation (CC) analysis was applied and four CCs (CC32, CC20, CC9, and CC7) have an AUC > 0.65 in our dataset with similar predictive power in the TCGA-KIRC dataset. Gene set enrichment showed CC9 as kidney development/adhesion, CC20 as oxidative phosphorylation pathway, CC32 as RNA binding/spindle and CC7 as immune response. In a multivariate Cox model, the four CCs were able to identify high/low risk groups for metastases in the TCGA-KIRC (p < 0.05) with odds ratios of CC32 = 5.7, CC20 = 4.4, CC9 = 3.6, and CC7 = 2.7.Conclusion: These results identify molecular signatures for more aggressive tumors in clinically low risk ccRCC patients who have a higher potential of metastasis than would be expected.

Bonamia exitiosa in farmed native oysters Ostrea angasi in Australia: optimal epidemiological qPCR cut-point and clinical disease risk factors.

Bonamiosis has developed as a problem in Australian native oysters Ostrea angasi since the parasite Bonamia spp. was first detected in Port Phillip Bay, Victoria, in the early 1990s. At that time, large-scale mortalities in both farmed and wild oysters saw the demise of the pilot native oyster culture industry. More recent attempts to farm the species resulted in subclinical infections that progressed over time to clinical disease. The aim of this work was to establish what environmental factors result in the clinical manifestation of disease; determine the diagnostic sensitivity and diagnostic specificity of histopathological examination and a quantitative polymerase chain reaction (qPCR) test for the diagnosis of B. exitiosa infection in clinically diseased farmed native oysters; and calculate the optimal qPCR threshold cycle (CT) epidemiological cut-point for classification of positive and negative cases. After applying a range of stressors to tank-held oysters, results indicated a 58% increased risk (95% CI: 16%, 99%) of a Bonamia-infected oyster dying if the oyster was held at a higher temperature (p = 0.048). Starving and tumbling oysters, in isolation, was not significantly associated with clinical bonamiosis, but a Bonamia-infected oyster was at the greatest risk of death when increased water temperature was combined with both starvation and increased motion (p = 0.02; odds ratio = 3.47). The diagnostic sensitivity and specificity of the World Organisation for Animal Health qPCR protocol were calculated for increasing CT value cut-points from ≤25 to ≤40, with an optimal cut-point identified at ≤34.5 (specificity: 92.2; 95% posterior credible intervals [PCI]: 76.2, 99.8; Sensitivity: 93.5; 95% PCI: 84.7, 99.1).

Detection of Human polyomavirus 2 (HPyV2) in oyster samples in northern Brazil

BackgroundHuman polyomavirus 2 (HPyV2 or JCPyV) is persistent in the environment due to its excretion in urine and feces; it is detected in samples of wastewater, surface water and drinking water. A lack of basic sanitation and sewage collection results in the presence of this virus in food, especially in oysters, since they are bioaccumulators and are consumed in their natural form, thus posing a risk to human health.MethodsThis study investigated the frequency of HPyV2 in samples of oysters marketed in northeastern Pará State, Brazil, and optimized a real-time PCR (qPCR) protocol for the detection of an endogenous oyster control. A total of 217 oysters in 22 pools from five municipalities in the state of Pará were analyzed. Samples underwent dissection and total maceration of oyster tissue using a viral concentration technique, followed by DNA extraction with phenol-chloroform and amplification of the VP1 region for molecular detection via qPCR.ResultsHPyV2 was detected in 18.2% (4/22) of the pooled samples, with frequencies of 25, 20, 20 and 16% in the municipalities of Salinópolis, Augusto Corrêa, São Caetano de Odivelas and Curuçá, respectively. Notably, the sample pool from the municipality of Bragança did not have detectable HPyV2 and this was the only sampled location with a water treatment station. In this study, Crassostrea genus-specific primers (AFL52 ribosomal RNA gene) of oyster were developed for use as an endogenous control in the qPCR analysis, which will be useful for future studies.ConclusionsThe detection of HPyV2 in oyster samples commercialized in the state of Pará shows the circulation of this virus in the studied municipalities. Thus, it is necessary to implement measures for improving sewage collection and basic sanitation to avoid contamination of water and food with HPyV2.

Comparison of the modified agglutination test and real-time PCR for detection of Toxoplasma gondii exposure in feral cats from Phillip Island, Australia, and risk factors associated with infection.

Toxoplasma gondii is considered a disease risk for many native Australian species. Feral cats are the key definitive host of T. gondii in Australia and therefore, investigating the epidemiology of T. gondii in cat populations is essential to understanding the risk posed to wildlife. Test sensitivity and specificity are poorly defined for diagnostic tests targeting T. gondii in cats and there is a need for validated techniques. This study focused on the feral cat population on Phillip Island, Victoria, Australia. We compared a novel real-time PCR (qPCR) protocol to the modified agglutination test (MAT) and used a Bayesian latent class modelling approach to assess the diagnostic parameters of each assay and estimate the true prevalence of T. gondii in feral cats. In addition, we performed multivariable logistic regression to determine risk factors associated with T. gondii infection in cats. Overall T. gondii prevalence by qPCR and MAT was 79.5% (95% confidence interval 72.6–85.0) and 91.8% (84.6–95.8), respectively. Bayesian modelling estimated the sensitivity and specificity of the MAT as 96.2% (95% credible interval 91.8–98.8) and 82.1% (64.9–93.6), and qPCR as 90.1% (83.6–95.5) and 96.0% (82.1–99.8), respectively. True prevalence of T. gondii infection in feral cats on Phillip Island was estimated as 90.3% (83.2–95.1). Multivariable logistic regression analysis indicated that T. gondii infection was positively associated with weight and this effect was modified by season. Cats trapped in winter had a high probability of infection, regardless of weight. The present study suggests qPCR applied to tissue is a highly sensitive, specific and logistically feasible tool for T. gondii testing in feral cat populations. Additionally, T. gondii infection is highly prevalent in feral cats on Phillip Island, which may have significant impacts on endemic and introduced marsupial populations.

Comparison of Bacterial DNA Profiles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries.

Infection and inflammation are well recognized causes of spontaneous preterm delivery (PTD) (<37 gestational weeks) and adverse infant outcomes. To date, there has been very little investigation into bacterial communities in amniotic fluid using next generation sequencing technology. In particular, it is important to characterize amniotic fluid bacterial profiles in complicated pregnancies as well as in asymptomatic women to identify predictive bacterial DNA signatures. Here, 1198 mid-trimester amniotic fluid samples from a cohort of Swedish women undergoing mid-trimester genetic amniocentesis were screened for bacterial DNA using qPCR protocols specifically designed to reduce the impacts of reagent contamination and human DNA mispriming. The majority of samples were devoid of detectable bacterial DNA; however, approximately a fifth of the cohort (19.9%) were 16S rRNA gene positive in duplicate screening. Among these, nine women had a spontaneous PTD. These nine women were matched with 18 healthy women with a delivery at term. We used PacBio SMRT technology, coupled with appropriate negative extraction and PCR controls, to sequence the full-length 16S rRNA gene in this subset of 27 women. The amniotic fluid samples contained low-abundance and low-diversity bacterial DNA profiles. Species typically associated with spontaneous PTD were absent. We were not able to identify any differences in the amniotic fluid bacterial DNA profiles of women with a subsequent spontaneous PTD compared to women who delivered at term. The findings suggest that, in a minor proportion of pregnancies, DNA from non-pathogenic bacteria may be present in the amniotic fluid far earlier than previously reported. Early detection of bacterial DNA in the amniotic fluid was, in this study, not associated with spontaneous PTD.

Detection of Viable Xanthomonas fragariae Cells in Strawberry Using Propidium Monoazide and Long-Amplicon Quantitative PCR.

Xanthomonas fragariae causes angular leaf spot in strawberry. The pathogen's association with its host tissue is thought to be a condition for its survival. Consequently, transmission of the pathogen to field production sites occurs almost exclusively through the movement of contaminated planting stock. The aim of this study was to develop a propidium monoazide (PMA)-quantitative PCR (qPCR) protocol for specific detection of viable X. fragariae cells. The qPCR procedure was developed for two different primer pairs: one producing a long amplicon (863 bp) and the other a short amplicon (61 bp). Both pairs were tested on mixtures of viable and heat-killed bacteria cells, bacteria-spiked strawberry petiole samples, and petioles collected from symptomatic, inoculated plants. The results showed that long-amplicon PMA-qPCR enabled specific and sensitive detection of X. fragariae with a detection limit of 103 CFU/ml, and it significantly improved PMA efficiency in differentiating viable from dead bacterial cells relative to short-amplicon PMA-qPCR. Based on the delta threshold cycle (Ct) values (i.e., the difference in Ct values between PMA-treated and nontreated samples), the long-amplicon PMA-qPCR was able to suppress the detection of dead X. fragariae cells 1.9- to 3.1-fold across all petiole samples tested. The quantification results from PMA-qPCR for mixtures of viable and dead cells were highly correlated with the predicted bacterial concentrations in a linear relationship (R2 = 0.981). This assay can be useful for identifying inoculum sources in the strawberry production cycle, which may lead to improved disease management strategies.

An evaluation of direct PCR assays for the detection and quantification of Porphyromonas gingivalis.

Porphyromonas gingivalis has been linked to the development and progression of oesophageal squamous cell carcinoma (ESCC), and is considered to be a high-risk factor for ESCC. Currently, the commonly used methods for P. gingivalis detection are culture or DNA extraction-based, which are either time and labour intensive especially for high-throughput applications. We aimed to establish and evaluate a rapid and sensitive direct quantitative polymerase chain reaction (qPCR) protocol for the detection of P. gingivalis without DNA extraction which is suitable for large-scale epidemiological studies. Paired gingival swab samples from 192 subjects undergoing general medical examinations were analysed using two direct and one extraction-based qPCR assays for P. gingivalis. Tris-EDTA buffer-based direct qPCR (TE-direct qPCR), lysis-based direct qPCR (lysis-direct qPCR) and DNA extraction-based qPCR (kit-qPCR) were used, respectively, in 192, 132 and 60 of these samples for quantification of P. gingivalis. The sensitivity and specificity of TE-direct qPCR was 95.24% and 100% compared with lysis-direct qPCR, which was 100% and 97.30% when compared with kit-qPCR; TE-direct qPCR had an almost perfect agreement with lysis-direct qPCR (κ = 0.954) and kit-qPCR (κ = 0.965). Moreover, the assay time used for TE-direct qPCR was 1.5 h. In conclusion, the TE-direct qPCR assay is a simple and efficient method for the quantification of oral P. gingivalis and showed high sensitivity and specificity compared with routine qPCR.

Urinary TMAO Levels Are Associated with the Taxonomic Composition of the Gut Microbiota and with the Choline TMA-Lyase Gene (cutC) Harbored by Enterobacteriaceae.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

A direct qPCR screening approach to improve the efficiency of Mycoplasma bovis isolation in the frame of a broad surveillance study

Culturing Mycoplasma bovis is laborious and unpredictable with most laboratories relying on molecular methods for its detection and identification. However, bacterial culture is still necessary to relate phenotypic characteristics to genotypic traits within and between individual strains. Thus, the main objective of this study was to develop a procedure that saved time and consumables during the culturing of M. bovis within the scope of a broad antimicrobial resistance surveillance project. Deep nasopharyngeal swabs (DNPS) collected from feedlot cattle upon arrival at 10 Southern Alberta feedlots were enriched in broth and an aliquot of the culture was directly used in a M. bovis-specific quantitative PCR (qPCR) assay. Only qPCR-positive cultures were plated onto agar media for the isolation of M. bovis. The detection of M. bovis from broth culture by direct-culture-qPCR proved to be more sensitive (1.61 × 102 CFU/mL) than using a commercial kit (1.61 × 103 CFU/mL) to extract DNA from pure cultures of M. bovis. When isolation of M. bovis from broth-enriched DNPS (n = 208 samples) was used as the gold standard for diagnostics, the qPCR screening approach showed 100% sensitivity, 87.27% specificity, and a kappa index = 0.87 (strong agreement). In contrast, qPCR of DNPS samples (n = 58) exhibited 100% sensitivity, 42.86% specificity, and a kappa index = 0.49 (weak agreement). The qPCR protocol described here together with a high throughput direct-culture-qPCR approach for sample testing made it possible to reduce the labor and cost of M. bovis isolation by eliminating the need to process 97.3% of M. bovis-negative samples. This was possible through the use of qPCR Ct values as a predictive tool of the likelihood of M. bovis isolation. This new procedure could be evaluated for its use in antimicrobial resistance surveillance programs that focus on Mycoplasma species.

Detection and absolute quantification of betasatellites associated with okra yellow vein mosaic disease by qPCR

Okra yellow vein mosaic disease (OYVMD) causes serious loss in okra production in Sri Lanka. Therefore, screening of resistant okra verities is an essential need to control the disease. As the available qualitative and semi-quantitative methods failed to detect latent infection the present study aimed to develop a quantitative PCR (qPCR) assay to detect and quantify one of the OYVMD causing agent, symptom modulating satellite molecules. A pair of primers targeting a portion of βC1 gene of BYVMBs was designed and used to quantify of BYVMBs by absolute quantification method using SYBR Green I chemistry. Standard curves were prepared using series of dilutions of known copy number plasmids carrying target sequence. The mean amplification efficiency was 95% and the coefficient of determination was 0.994. The method was tested to find out the relation between symptoms and betasatellite titre in range of severity of OYVMD symptoms; the betasatellite titre increased with increasing severity. Interestingly, the method was able to detect BYVMBs present in apparently healthy plants growing in an infected field at a concentration which was not able to detect in end point PCR. Betasatellite titre was also measured in different ages of leaves and different positions. On average, the betasatellite titre in younger leaves was higher than in mature leaves and there were no significant variations in betasatellite titre in different position in each leaf. The assay was also tested as a tool to screen for resistant okra varieties; among the eight varieties tested no BYVMBs were detected in variety Maha F1. Varieties TV8 and MI5 had significantly higher copy number than rest of the varieties. The qPCR protocol described in this study is a useful method to detect and quantify BYVMBs in okra, especially for plant samples with betasatellite titre lower than the detection limit of conventional methods.

Detection and Quantification of Spongospora subterranea Sporosori in Soil by Quantitative Real-Time PCR.

Powdery scab on potato tubers is caused by the obligate soilborne biotroph Spongospora subterranea and is known to cause substantial losses in potato production. The pathogen also infects roots of susceptible hosts, forming galls which can negatively affect root function. S. subterranea is also the vector of Potato mop-top virus, which causes a tuber necrosis disease that can, depending on temperature and cultivar, render potato tubers unmarketable. In this study, we adapted a published protocol to develop a sensitive and robust quantitative real-time PCR (qPCR) assay using specific primers and probes for detecting and quantifying S. subterranea sporosori in soil types that differ in physical properties, including organic matter content and soil pH. For the first time, an external control was utilized and applied directly to the soil prior to DNA extraction, which facilitated normalization of S. subterranea sporosori soil levels from sample to sample. The duplex qPCR protocol was demonstrated to be highly sensitive, capable of detecting and quantifying as few as 1 sporosorus/g of soil, with consistently high qPCR efficiency and the coefficient of determination (R2) values ranging from 94 to 99% and 0.98 to 0.99, respectively. The protocol was successfully implemented in enumerating S. subterranea sporosori in naturally infested field soil collected from several states and in artificial potting mixes with high organic matter content ranging from 64 to 71%. The qPCR method developed can be useful for potato growers to avoid agricultural soils highly infested with S. subterranea and in the development of risk assessment models in the future that incorporate cultivar susceptibility to powdery scab and soil infestation levels.

Molecular Monitoring of Chronic Myeloid Leukemia.

Molecular diagnosis and measurement of minimal residual disease (MRD) in patients with chronic myeloid leukemia (CML) is essential for clinical management. In the era of tyrosine kinase inhibitor therapy molecular tests including BCR-ABL1 transcript monitoring and kinase domain mutation analysis are the main tools used to inform choice of treatment, appropriate dosage and even whether therapy can be safely withdrawn. Quantitation of BCR-ABL1 oncogene transcript by real-time quantitative PCR (qPCR) is currently the gold-standard method for monitoring as it provides superior sensitivity over karyotyping and fluorescent in situ hybridization (FISH). Here we describe step-by-step methods of RNA conversion to cDNA along with the qPCR protocol which is used in one of the main reference laboratories for this test.

1325P - Validation of a clinicopathological and gene expression profile (CP-GEP) model for sentinel lymph node metastasis in primary cutaneous melanoma

BackgroundAs primary cutaneous melanoma patients with a positive sentinel lymph node (SLN, stage III) are now considered candidates for adjuvant systemic therapy, a SLN biopsy (SLNB) is indicated in more patients. However, SLNB is an invasive procedure and is negative in approximately 80% of patients. Therefore, there is a need for a non-invasive test to accurately identify patients with primary cutaneous melanoma without nodal metastases. Here we describe the first independent validation of a recently developed CP-GEP model to predict nodal metastasis. This risk model combines Breslow thickness, age, and gene expression variables from the primary melanoma. MethodsThis study focused on all patients >18 years who underwent a SLNB at the Erasmus Medical Center (between January 2007 and December 2017), within 90 days after diagnosis of primary cutaneous melanoma. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) primary cutaneous melanomas, reversed transcribed into cDNA and subsequently analyzed for the expression of 8 target genes involved in melanoma metastasis (ITGB3, PLAT, SERPINE2, GDF15, TGFBR1, LOXL4, IL8, MLANA) using an optimized qPCR protocol. ResultsFFPE tissue samples from 211 patients were analyzed using the CP-GEP model. At diagnosis, the median age was 55 years (interquartile range [IQR] 45-65), and the median Breslow thickness was 2.1mm (IQR 1.4-3.4). Most patients presented with a T2 or T3 melanoma, accounting for 94 and 70 patients, respectively. Overall, 27.5% of patients had a positive SLN. The CP-GEP model had a negative predictive value (NPV) of 89.4%. In patients with stage T1-T2 melanoma, the model was able to achieve an SLNB reduction rate of 40.1% with an NPV of 90.7%. ConclusionsThe CP-GEP model is a non-invasive and validated tool that is able to predict nodal metastasis in an independent Dutch population. Consequently, this risk model is able to accurately identify patients with primary cutaneous melanoma that can safely forego SLNB. Therefore, the CP-GEP model is a promising tool for patient care, preventing unnecessary surgery in the majority of patients. Legal entity responsible for the studyErasmus University Medical Center, Department of Dermatology. FundingSkylineDx. DisclosureJ.T. Dwarkasing: Shareholder / Stockholder / Stock options, Full / Part-time employment: SkylineDx BV. D. Tempel: Shareholder / Stockholder / Stock options, Full / Part-time employment: SkylineDx BV. L. Bosman: Shareholder / Stockholder / Stock options, Full / Part-time employment: SkylineDx. A.A.M. Van der Veldt: Advisory / Consultancy: BMS; Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pierre Fabre; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Ipsen. All other authors have declared no conflicts of interest.

Decreased expression of microRNAs targeting type-2 diabetes susceptibility genes in peripheral blood of patients and predisposed individuals.

Certain microRNA molecules (miRNAs) that target genes involved in beta-cell growth and insulin resistance are found deregulated in patients with type-2 diabetes mellitus (T2D) and correlate with its complications. However, the expression profile of miRNAs that regulate genes bearing T2D-related single-nucleotide polymorphisms has been hardly studied. We recently reported that the mRNA patterns of specific T2D-susceptibility genes are impaired in patients, and associate with disease parameters and risk factors. The aim of this study was to explore the levels of miRNAs that target those genes, in peripheral blood of patients versus controls. A panel of 14 miRNAs validated to target the CDKN2A, CDK5, IGF2BP2, KCNQ1, and TSPAN8 genes, was developed upon combined search throughout the DIANNA TarBase v7.0, miRTarBase, miRSearch v3.0-Exiqon, miRGator v3.0, and miRTarget Link Human algorithms. Specifically developed poly(A)polyadenylation(PAP)-reverse transcription(RT)-qPCR protocols were applied in peripheral blood RNA samples from patients and controls. Possible correlations with the disease, clinicopathological parameters and/or risk factors were evaluated. T2D patients expressed decreased levels of let-7b-5p, miR-1-3p, miR-24-3p, miR-34a-5p, miR-98-5p, and miR-133a-3p, compared with controls. Moreover, these levels correlated with certain disease features including insulin and % HbA1c levels in patients, as well as BMI, triglycerides' levels and family history in controls. A T2D-specific expression profile of miRNAs that target disease-susceptibility genes is for the first time described. Future studies are needed to elucidate the associated transcription-regulatory mechanisms, perchance involved in T2D pathogenesis, and to evaluate the potential of these molecules as possible biomarkers for this disorder.

Soil temperature and hydric potential influences the monthly variations of soil Tuber aestivum DNA in a highly productive orchard

Tuber aestivum, also known as the summer or Burgundy truffle, is an ectomycorrhizal Ascomycete associated with numerous trees and shrubs. Its life cycle occurs in the soil, and thus soil parameters such as temperature and water availability could influence it. T. aestivum cultivation has started in several countries, but ecological and agronomic requirements for the establishment and management of orchards are largely unknown. The aims of this work were: 1) to design a specific qPCR protocol using genomic data to trace and quantify T. aestivum DNA in the soil; and 2) to assess the monthly soil DNA dynamic according to soil parameters (i.e. soil hydric potential and temperature) in this orchard. The study was conducted in a highly productive T. aestivum orchard (hazels, oaks, pines, lime and hornbeam). The production started five years after the plantation and then increased exponentially to reach a maximum of 320 kg/ha in 2017. The soil hydric potential and temperature partially explained the monthly T. aestivum soil DNA variability. The data presented here offer new insights into T. aestivum ecology and cultivation.

Real-time PCR methods for detecting Salmonella spp. in food after different DNA extraction procedures

The aim of this paper was to evaluate two real-time PCR (qPCR) protocols for the detection of Salmonella spp. in minced meat and chicken neck skin, after DNA extraction using the InstaTM Gene matrix (BioRad, USA) and DNA extraction based on thermal cell lysis. The applied molecular methods were sensitive and specific for the rapid detection of Salmonella spp. in minced meat and chicken neck skin. The qualitative results were identical regardless of the applied DNA extraction or qPCR protocols. Lower Cq values were achieved after DNA extraction using the InstaTM Gene matrix.

Correlation of Parasite Burden, kDNA Integration, Autoreactive Antibodies, and Cytokine Pattern in the Pathophysiology of Chagas Disease.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), is the main parasitic disease in the Western Hemisphere. Unfortunately, its physiopathology is not completely understood, and cardiomegaly development is hard to predict. Trying to explain tissue lesion and the fact that only a percentage of the infected individuals develops clinical manifestations, a variety of mechanisms have been suggested as the provokers of CD, such as parasite persistence and autoimmune responses. However, holistic analysis of how parasite and host-related elements may connect to each other and influence clinical outcome is still scarce in the literature. Here, we investigated murine models of CD caused by three different pathogen strains: Colombian, CL Brener and Y strains, and employed parasitological and immunological tests to determine parasite load, antibody reactivity, and cytokine production during the acute and chronic phases of the disease. Also, we developed a quantitative PCR (qPCR) protocol to quantify T. cruzi kDNA minicircle integration into the mammalian host genome. Finally, we used a correlation analysis to interconnect parasite- and host-related factors over time. Higher parasite load in the heart and in the intestine was significantly associated with IgG raised against host cardiac proteins. Also, increased heart and bone marrow parasitism was associated with a more intense leukocyte infiltration. kDNA integration rates correlated to the levels of IgG antibodies reactive to host cardiac proteins and interferon production, both influencing tissue inflammation. In conclusion, our results shed light into how inflammatory process associates with parasite load, kDNA transfer to the host, autoreactive autoantibody production and cytokine profile. Altogether, our data support the proposal of an updated integrative theory regarding CD pathophysiology.

Quantitative molecular diagnosis of levamisole resistance in populations of Haemonchus contortus

Parasitism by Haemonchus contortus is one of the main limiting factors in small ruminant production around the globe. Although several studies suggest the use of integrated management practices, these parasites have been controlled essentially with synthetic anthelmintic drugs. The resistance mechanism against the imidazothiazole derivative levamisole in Haemonchus contortus has not been fully described. Recently, resistance was associated with a 63bp deletion in the Hco-acr-8b gene that encodes a subunit for a nicotinic acetylcholine receptor. This study aimed to standardize a real time PCR (qPCR) protocol for levamisole resistance diagnosis in H. contortus populations based on this polymorphism and use it to characterize 23 field H. contortus populations obtained from different localities of Ceará State, Northeast Brazil. In addition, two populations of H. contortus were used as a standard of susceptibility and resistance, Inbred Strain Edinburgh (ISE) and Kokstad, respectively. Larval development tests (LDT) were performed on five field isolates and both EC50 and EC95 were estimated. LDT EC95 values provided a wider interval between susceptible and resistant populations than EC50 values (EC95 = 1.96–57.93 μM; EC50 = 0.05–0.39 μM), and were found to be more appropriate for differentiating them. Real time PCR results showed resistance allele frequencies ranged from 20.9 to 76.7%. Our results suggest that levamisole resistance may be present in field populations but it is not as widespread as benzimidazole resistance. This methodology may be useful to monitor levamisole resistance in field populations of H. contortus.