Perfecta qPCR ToughMix Reagents | Taqman Probe qPCR Reagent | how much (2023)

Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic E. coli (aEPEC) in Swedish retail wheat flour

Robert Söderlund - 2023


Wheat flour has been identified as the source of several outbreaks of gastrointestinal illnesses caused by Shiga toxin-producing Escherichia coli (STEC). We analyzed the occurrence and genomic properties of STEC and related atypical enteropathogenic E. coli (aEPEC) in 200 Swedish-produced retail bags of wheat flour46 representing 87 products and 25 brands. Samples were spiked with modified TSB and examined with real-time PCR targeting stx1, stx2, eae, and serogroups O157, O121, and O26. Isolation was performed by IMS for suspected STEC/aEPEC O157, O121 and O26 and by colony group screening for other STEC. Real-time PCR after enrichment revealed that 12% of the samples were positive for the Shiga toxin genes (stx1 and/or stx2) and 11% were positive for inthymin (eae). Organic production, small-scale production, or whole grains did not significantly affect the presence or absence of the shiga toxin gene in a generalized linear mixed model analysis. Eight STEC isolates were recovered, all of which were negative for inthimin. Several Shiga toxin serotype/sequence type/subtype combinations were found, which were also found in flour samples from other European countries. Most of the recovered STEC types were associated with sporadic human STEC cases in Sweden, but no types known to cause flare-ups or severe disease (ie, HUS57) were found. The most common finding was O187:H28 ST200 with stx2g, with possible connections to cervid hosts. Wildlife associated with crop damage is a plausible explanation for at least part of the surprisingly high abundance of STEC in wheat flour.

Collection of oral bacteria at home using a lollipop-based microfluidic device

Wan-chen Tu - 2023


Our previous work featured CandyCollect1, a lollipop-inspired saliva collection device for diagnosing respiratory diseases. Here we carry out the first human study using this device to capture bacteria in saliva; We demonstrate that the CandyCollect device can be used to collect salivary bacteria from healthy adults, using Streptococcus mutans and Staphylococcus aureus as proof-of-concept commensal bacteria. We enrolled 14 healthy adults in a nationwide (US) remote study in which study packets containing CandyCollect devices and commercially available conventional oral swabs and saliva tubes were mailed to participants. Participants collected samples at home, completed usability and user preference surveys, and sent the samples to our lab for qPCR analysis. S. mutans and S. aureus are not universally present in all healthy adults.2-5 Our results showed that participants were exposed to a specific bacterium (S. mutans or S. aureus) using one or both of the commercially available methods CandyCollect devices showed 100% agreement with these results. Additionally, 26 respondents ranked the CandyCollect device as the preferred sampling method among the three sampling methods. We have also shown that the CandyCollect device has a shelf life of up to 1 year at room temperature, a convenient storage time for clinics or patients to keep the CandyCollect device and use it at any time. Taken together, we have shown CandyCollect to be an easy-to-use saliva collection tool that has the potential to be integrated into clinic visit and telemedicine diagnostic assays.

Point-of-Care Tests for Sensitive Detection of the African Swine Fever Virus Genome

Ahmed Elnagar - 2022


African swine fever (ASF) is a contagious viral haemorrhagic disease that affects domestic and wild pigs. The disease is mandatory notification to the World Organization for Animal Health (WOAH) and causes significant deaths and economic losses. There is currently no fully approved vaccine available. Therefore, early identification of the pathogen, African swine fever virus (ASFV), is of crucial importance for the implementation of control measures. PCR and real-time PCR are the standard methods recommended by WOAH for the direct detection of ASFV. However, in special field conditions or in simple or remote field laboratories, sophisticated equipment or even a stable power supply may not be available. In these circumstances, point-of-care systems may be established. With this in mind, a previously published rapid, reliable, and electricity-free (TripleE) extraction method was used to isolate viral nucleic acid from diagnostic samples. With this tool, nucleic acid extraction from up to eight diagnostic samples can be performed in less than 10 minutes. In addition, the possibility of completely dispensing with viral DNA extraction by so-called direct real-time PCR protocols using original ASFV samples diluted 1:40 in RNase-free water was explored. In addition, three real-time PCR cyclers developed for use in field conditions (IndiField, Liberty16, and UF-300 GenecheckerTM) were comparatively used for sensitive, high-speed detection of ASFV genomes with total PCR run times between 20 and 54 min. Depending on the viral DNA extraction/release method used and the point-of-care cycler used, a total detection time of 30 to 60 minutes was feasible for up to eight samples. As expected, limitations in analytical sensitivity were positively correlated with analysis time. These limitations are acceptable for ASFV diagnosis due to the expected high burden of the ASFV genome in diseased animals or carcasses.

A breakthrough for the identification and characterization of Shiga toxin-producing Escherichia coli in raw milk using long-read metagenomics

Sandra Jaudou - 2022


Shiga toxin-producing Escherichia coli (STEC) is a cause of severe human disease and is commonly associated with hemolytic uremic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause disease in humans. In addition to Shiga toxin (stx genes), many other factors have been reported, such as: B. Inthymin (eae gene), which is clearly an aggravating factor in the development of HUS. Current STEC detection methods are classically based on real-time PCR (qPCR) to detect the presence of the most important virulence markers (stx and eae). Although qPCR gives an idea of ​​the presence of these virulence markers, it is not suitable to confirm their presence in the same strain. Therefore, isolation steps are required to confirm STEC viability and characterize STEC genomes. Although STEC isolation is tedious and time consuming, metagenomics has the potential to speed up the process of characterizing STEC without isolation. Short read sequencing metagenomics has recently been applied for this purpose, but assembly quality and contiguity are affected by the high proportion of mobile genetic elements found in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics to identify eae-positive STEC strains using raw cow's milk as an etiologic matrix for foodborne STEC outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing, and generating an easy-to-use STEC characterization channel, an eae-positive STEC strain was directly identified after enrichment. Raw cow's milk samples artificially contaminated at a contamination level as low as 5. CFU ml−1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk combined with a complete process of STEC analysis from long read sequencing metagenomic data. This study shows the potential of innovative methodology to characterize STEC strains from complex matrices. However, further developments are needed for the application of this method in STEC monitoring.

An opinion on wastewater-based epidemiological surveillance (WBEM) with clinical diagnostic tests (CDT) to identify areas of high prevalence of COVID-19 infection in the community

Dr. Aminul-Islam - 2022


Wastewater-based epidemiological monitoring (WBEM) is an efficient surveillance tool during the COVID-19 pandemic, as it meets all the requirements for a complete surveillance system, including early warning, current trend tracking, prevalence of the disease, detection of genetic diversity, as well as the new emerging variants of SARS-CoV -2 with mutations from the wastewater samples. As a result, the clinical diagnostic test is being used despite several disadvantages, such as high diagnostic costs, reporting bias, and the difficulty of treating asymptomatic patients (silent spreaders of COVID-19 infection who do not have symptoms). of the illness). In this current review and opinion-based study, we first propose a combined approach to detect COVID-19 infection in communities using effluent and clinical sample testing, which may be feasible and effective as an emerging public health tool for national surveillance. long-term. system. Virus levels in wastewater samples can be used as indicators to monitor ongoing SARS-CoV-2 trends, predict asymptomatic carriers, and identify COVID-19 hotspots, while clinical samples help identify the mostly symptomatic positive cases to isolate them in the communities and validate the WBEM. protocol for mass vaccination including booster doses for COVID-19.

Systematic stepwise screening of new microbial antagonists for the biological control of European cancer

G. Elena - 2022


Neonectria ditissima is the causative agent of the European canker. This pathogen causes cankers on apple branches and the main trunk, which can lead to the loss of the entire tree. Chemical control is the essential component in disease management and there are no suitable commercially available biological control products. This study aimed to select potential microbial antagonists against N. ditissima through a systematic stepwise screening program for the development of a new biocontrol product. A total of 520 potential candidates were isolated from apple branches showing cancer symptoms. Important properties for the application of microbial biocontrol agents were tested for each candidate: spore production, spore survival during storage, cold tolerance, drought tolerance, and UV-B resistance. Isolates that could germinate and grow at human body temperature were excluded. A total of 252 candidates met the specified criteria. The 520 candidates belonged to 44 different taxonomic groups, with Alternaria spp. (22.2%), Aureobasidium pullulans (16.1%), Cladosporium spp. (9.5%) and Fusarium spp. (9.0%). For each identified species, information was collected on possible pathogenicity and toxicity for humans, animals, plants and the environment, as well as biocontrol patents. A total of 158 candidates belonging to species that did not present potential risks or patent conflicts were evaluated based on their antagonistic behavior towards N. ditissima in plant bioassays. Each candidate was inoculated 24 h before in 'Elstar' apple tree branches inoculated with N. ditissima. The inoculated branches were incubated at 17 °C, 16 h of light per day and RH > 90 %. After four weeks, the expression of cancer symptoms was assessed visually. The ability of the candidates to reduce N. ditissima colonization on the branches was assessed by quantifying the concentration of N. ditissima DNA by qPCR. Four Clonostachys rosea candidates showed antagonistic properties; after 4 weeks of treatment, no cancer symptoms or small cortical cracks were observed in the inoculated branches and N. ditissima DNA was reduced by 90-99%. Subsequently, branches inoculated with a candidate Acanthomyces muscarius, A. pullulans and Cladosporium europaeum showed small cracks in the bark and a small swollen bark, and N. ditissima DNA was reduced by more than 90%. The systematic stepwise screening approach was a powerful strategy to find novel MBCAs against N. ditissima with antagonistic properties that also met important criteria related to commercial production and safety, as well as ecological requirements.

Monitoring of Campylobacter jejuni in a chicken infection model by measuring specific volatile organic compounds and by qPCR

Julia Hankel - 2022


Campylobacter is one of the major foodborne bacterial pathogens worldwide. Poultry are the host species of this pathogen with the greatest clinical impact. Herds become colonized with Campylobacter, leading to contamination of product entering the food chain. Rapid and reliable Campylobacter detection methods could support controls to minimize contamination risks within the food chain, making it easier to implement a culling logistics plan or other control options. The present study evaluates current and emerging detection technologies for C. jejuni in air samples in a single study design with predefined C. jejuni prevalences. Both non-invasive detection technologies in air samples by subsequent measurement of volatile organic compounds (VOCs) or by qPCR detected the presence of C. jejuni and were also able to distinguish between the number of C. jejuni-positive birds present in the design of the study. . However, fewer birds were diagnosed as positive for C. jejuni using electrostatic air samplers compared to the culture-based method. By measuring the VOCs it was possible to detect the presence of two positive birds in the room. This apparently high sensitivity still needs to be verified in field studies. Techniques such as these promising methods that may facilitate surveillance of C. jejuni in poultry flocks are desirable to reduce the risk of infection in humans.

Occurrence of Naegleria fowleri and fecal indicators in sediments from Lake Pontchartrain, Louisiana

Shalina A. Shahin - 2022


The presence of amoebas, Naegleria fowleri, was investigated in sediment samples from Lake Pontchartrain in Louisiana. This amoeba is pathogenic and can cause primary amebic meningoencephalitis. In this study, quantitative polymerase chain reaction methods were used to test the prevalence of Naegleria fowleri, HF183, and E. coli. N. fowleri was detected in 51.25% of our sediment samples. Illumina sequencing of sediment samples revealed ten distinct phyla, with cyanobacteria being most prevalent at sites that generally had the highest mean concentrations of N. fowleri. However, N. fowleri was negatively correlated with HF183 (r = -0.859, p < 0.001). When E. coli sediment concentrations were below 1.54 log GC/g, there was only a 37.5% chance that N. fowleri would be detected in the same sample. When E. coli concentrations in the sediment exceeded 2.77 log GC/g, the probability of detecting N. fowleri in the same sample increased to 90%, possibly indicating amoeba predation activity. The effect of temperature was found to vary in terms of the observed concentrations of N. fowleri and detection rates. Although sediment samples collected during periods of higher temperatures had significantly lower mean N. fowleri concentrations (2.7 log GC/g) compared to those collected at lower temperatures (3.7 log GC/g, t (39) = 4.167, p < 0.001), higher detection rates of N. fowleri were observed in bulk samples at higher temperatures (> 19.1 °C) than at lower temperatures (< 19.1 °C ).

(Video) Probe-based qPCR Approaches for Detection of SNPs and Validation of Molecular Diagnostics

Non-lethal sampling for the detection of Renibacterium Salmoninarum by qPCR for the diagnosis of bacterial kidney disease

Eva Jansson - 2022


Bacterial kidney disease (BKD) caused by Renibacterium Salmoninarum (Rs) can be transmitted both horizontally and vertically, and there is no available cure or prophylaxis. Control of BKD requires continuous surveillance, which is a challenge both in aquaculture and in conservation and restoration programs for salmonid strains. BKD is a notifiable disease in Sweden and is monitored under the mandatory health surveillance program using a polyclonal ELISA to detect the Rs p57 protein in the kidney. Fish must be killed for sampling, an obvious disadvantage, especially with valuable fingerlings. The present study demonstrates that gill/cloacal swabs collected in vivo for real-time PCR (qPCRgc) allow sensitive and specific detection of Rs. The sensitivity of qPCRgc was determined to be 97.8% (reliable interval (CI) 93.8-100%) compared to 98.3% (CI 92.7-100%) and 48.8%. % (ci 38.8%–58.8%) of kidney samples estimated for qPCR (qPCRk) or ELISA (ELISAk) using Bayesian latent class analysis (BLCA). Since the goal of the program is the eradication of BKD, the most sensitive test is preferable. Using qPCRgc instead of ELISAk results in a lower false negative rate and may be useful for monitoring in aquaculture and valuable fish breeding programs. However, a higher false positive rate justifies a lethal confirmatory test before capping a previously negative trade of Rs.

Profiling of Multiomics Biomarkers Using Particle Detection Counters and FLIM Spectral Microscopy

Tam Vu - 2021


Bulk and in situ multioma biomarker profiling provides important information enabling basic research and clinical applications. Unfortunately, most existing profiling methods are limited due to low multiplexing, sensitivity, cost, or assay complexity. This dissertation aims to develop two core technologies that address 1) bulk profiling problems with sensitivity and low performance, and 2) in-place profiling problems with low multiplexing capabilities, cost, and limited performance. To address the first issue, this paper presents a novel liquid biopsy approach using a platform technology called Integrated Full Digital Droplet Detection (IC3D). This integrated approach combines microfluidic droplet delivery technology, fluorescent multiplex PCR chemistry, and our own unique and rapid particle counting technology to enable the ultra-sensitive and ultra-fast detection of colorectal cancer-specific genomic biomarkers from minimally accurate blood samples. processed. xvTo address the second issue, this paper presents a new spatial multi-omics technology called the multi-omics single scanning assay with integrated combinatorial analysis (MOSAICA), which allows a) in situ labeling of molecular markers (eg, B. mRNA, proteins) in cells or tissues integrated with combinatorial and lifetime-encoded fluorescence spectral probes, and b) analysis and imaging of time-resolved spectra and fluorescence to enable rapid, high-throughput, and low-cost spatial profiling of multiomic biomarkers. By using intensity and time domains for labeling and imaging, this technology attempts to simultaneously discriminate a broad repertoire of spectral and lifetime components within the same pixel or image of a sample to enable much greater multiplexing capabilities with standard optical systems. Together, these two technologies represent simple, versatile, and scalable tools that enable faster, more sensitive, and/or multiplexed protein/transcriptome analysis.

Design of bacterial strain-specific qPCR assays using NGS data and publicly available resources and applying them to monitoring biocontrol strains

Iker Hernandez - 2020


Biological control is emerging as a viable alternative to chemical pesticides in agriculture. Measurement of microbial biological control agent (mBCA) populations in the environment is essential for an accurate assessment of environmental and health risks and for optimizing the use of an mBCA-based crop protection product. We hereby show a workflow to obtain a large number of qPCR markers suitable for robust strain-specific quantification. The workflow starts with whole genome sequencing data and consists of four phases: (i) identification of strain-specific sequences, (ii) development of specific primer/probe sets for qPCR, and (iii) empirical verification of performance of the essay. The first two levels involve only computational work, but are intended for researchers with little or no experience in bioinformatics: only knowledge of the BLAST suite tools and working with spreadsheets is required; Familiarity with the Galaxy environment and next generation sequencing concepts is recommended. All bioinformatics work can be implemented using publicly available resources and a normal desktop computer (regardless of operating system) connected to the Internet. The workflow tested five bacterial strains from four different genera under development as mBCAs and produced thousands of candidate markers and a triple qPCR assay for each mBCA candidate. qPCR assays have been successfully tested in different types of soil, water from different sources, and with samples from different plant tissues. The mBCA detection limits and population dynamics on the different matrices are similar to those of qPCR assays performed by other means. In summary, a new accessible, inexpensive and robust workflow to obtain a large number of strain-specific qPCR markers is presented.

Detection of freshwater mussels (Unionidae) using environmental DNA in river systems

Luis Gasparini - 2020


Environmental DNA (eDNA) methods are being developed for use in conservation biology to improve traditional methods of studying species. Validation of eDNA methods in different environmental contexts is required if it is to be widely used. One possible application of eDNA methods is the detection of freshwater mussels (Bivalvia: Unionidae), which are among the most threatened species in North America. Traditional study methods for unionids are highly invasive and can be difficult to perform due to problems with morphological identification and their cryptic use of habitat. eDNA methods may offer a non-invasive, highly specific, and highly sensitive alternative. Here we examine the efficacy of eDNA methods for the detection of an endangered Union species, the wavy lampshell (Lampsilis fasciola), in moderate-discharge lotic systems. We developed a new qPCR assay for the detection of L. fasciola eDNA that included a custom internal positive control to verify PCR inhibition. We used different experimental densities of caged L. fasciola samples as a point source for eDNA in two rivers of the Grand River Basin in southern Ontario. Sampling was carried out at fixed intervals downstream of the cage using specially designed sampling devices. Detection occurred in the cage (ie, at the point of eDNA detachment) but not downstream at distances ≥ 10 m in river discharges of approximately 1632–2332 l/s. The results indicate that eDNA dilutes rapidly in rivers with moderate discharge and that high-resolution spatial sampling may be required to obtain meaningful eDNA-based distribution data for unionids and other sessile organisms present at low densities in systems. lotics.

T cell receptor cleavage circles in neonates with heart defects

Kiran A. Gul – 2020


In the fetus, both the thymus and the conotruncus of the heart arise from the cardiac neural crest. Newborn screening for severe T-cell lymphopenia can detect newborns with congenital heart defects. In this study, we evaluated the occurrence of T-cell lymphopenia in neonates with or without 22q11.2 (del) deletion syndrome who had heart defects. This retrospective cohort study included 125 patients with heart defects. T-cell receptor excision circles (TREC), a measure of T-cell lymphopenia, were quantified by RT-PCR using stored newborn screening blood samples. Three groups of patients were compared: non-conotruncal defects (n = 57), conotruncal defects (n = 42) and 22q11.2 del with conotruncal defects (n = 26). Significantly lower TREC values ​​were found in patients with 22q11.2 del and conotruncal heart defects compared with patients with non-syndromic (p < 0.001) and non-conotruncal (p < 0.001) conotruncal defects. In contrast, no significant differences were found between patients with non-syndromic and non-conotruncal conotruncal heart diseases (p = 0.152). Low REBT levels were obtained in neonates who underwent cardiac surgery/intervention within 2 weeks of birth and in neonates with fatal outcome (p = 0.02), regardless of patient group. A correlation was found between low TREC numbers and oxygen saturation, SpO2 below 95% (p = 0.017). SpO2 was significantly lower in the non-syndromic conotruncal group compared to the non-conotruncal group (p < 0.001) and 22q11.2 del (p = 0.015). No correlation was found between low neonatal TREC and infections requiring hospitalization later in life (p = 0.135). Patients with 22q11.2 del and conotruncal defects have significantly lower TREC values ​​compared with patients with heart defects without this syndrome.

MAPK-induced miR-29 inhibits melanoma progression by targeting MAFG

Olga Vera, Good Book - 2020


The miR-29 tumor suppressor microRNA family is encoded by two groups, miR-29b1∼a and miR-29b2∼c, which are regulated by tumor suppressor and oncogenic stimuli, including p53. Here we investigate whether oncogenic stress induced by MAPK hyperactivation regulates miR-29 abundance and how this signaling axis affects melanoma development. Using mouse embryonic fibroblasts and human melanocytes, we found that oncogenic MAPK signaling stimulates p53-independent and p53-dependent transcription of pri-miR-29b1∼a and pri-miR-29b2∼c, respectively. Expression analysis revealed that while pri-miR-29a∼b1 remains elevated, pri-miR-29b2∼c levels decrease during melanoma progression. Using a rapid mouse modeling platform, we show that in vivo knockout of miR-29 accelerates the development of overt melanoma and decreases overall survival. We have identified MAFG as a relevant miR-29 target that has oncogenic potential in melanocytes and is required for melanoma cell growth. Our results suggest that MAPK-driven induction of miR-29 presents a tumor suppressor barrier when targeting MAFG, which is overcome by attenuating miR-29b2∼c expression.

Rickettsia felis has been identified in two fatal cases of acute meningoencephalitis

Arthur HP Mawuntu – 2020


Background Rickettsia felis has recently emerged as a cause of human disease worldwide. It usually causes low-grade undifferentiated fever and has been implicated in several cases of non-fatal neurologic disease in Mexico and Sweden. Little is known about its distribution and pathogenicity in Southeast Asia. MAIN METHODS/RESULTS We retrospectively analyzed cerebrospinal fluid (CSF) or serum from 64 adult patients admitted to a hospital in North Sulawesi, Indonesia, with acute neurological disease. Rickettsia felis DNA was identified in the cerebrospinal fluid of two fatal cases of meningoencephalitis by multilocus sequence typing, semi-nested PCR followed by Sanger sequencing. DNA from both cases had 100% sequence homologies with the R. felis reference strain URRWXCal2 for the 17 kDa and ompB genes and 99.91% with gltA. Indonesia suggests that the distribution and pathogenicity of this emerging vector-borne bacterium could be higher than commonly assumed. Rickettsiae are generally susceptible to tetracyclines, and better knowledge of the endemicity of R. felis in Indonesia should lead to better treatment of some acute neurological cases.

The α-specific retinoic acid receptor (RAR) agonist Am80 (tamibarotin) and other RAR agonists potently inhibit hepatitis B virus transcription from cccDNA

Shirin Nkongolo - The Best of Shirin Nkongolo


Chronic human hepatitis B virus (HBV) infection is a major global health problem. The hepatitis D virus (HDV) is a satellite of HBV that uses the HBV coat proteins to enter and leave the cell. Using infection systems encoding the HBV/HDV receptor human sodium taurocholate cotransporter polypeptide (NTCP), we screened 1181 FDA-approved drugs using markers for interference with HBV and HDV infection. As a major success, we identified acitretin, a retinoid, as an inhibitor of HBV replication and HDV release. Based on this, other retinoic acid receptor (RAR) agonists with different specificities were found to disrupt HBV replication, confirming that the retinoic acid receptor pathway regulates replication. Of the eight agonists studied, the RARα-specific agonist Am80 (tamibarotene) was the most active. Am80 reduced the secretion of HBeAg and HBsAg with IC50s < 10 nM in differentiated HepaRG-NTCP cells. Similar effects have been observed in primary human hepatocytes. In HepG2-NTCP cells, Am80-mediated profound inhibition required prolonged treatment of up to 35 days. Am80 treatment of cells with an established pool of HBV cccDNA resulted in a reduction in secreted HBsAg and HBeAg, which correlated with reduced levels of intracellular viral RNA but not cccDNA copy number. The effect lasted more than 12 days after stopping the drug. HBV genotypes B, D and E were similarly inhibited. In contrast, Am80 did not affect HBV replication in transfected cells or in HepG2.2.15 cells carrying an integrated HBV genome. Taken together, our results indicate a persistent inhibition of HBV transcription by Am80, which could be used for drug retargeting.

(Video) Fighting African Swine Fever I ASF Strategy - Sharing experiences from European outbreaks

Differentiation between wild boars and domestic pigs in food by identifying two gene loci using real-time PCR

Maria Kaltenbrunner - 2019


Studies show that many meat products are not authentic, mainly because the types of meat differ from those listed on food labels. Currently, DNA-based methods play the most important role in authenticating meat varieties. Discrimination between wild boar and domestic pig meat in food is challenging, as it is a distinction at the subspecies level. We have developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and one duplex real-time PCR assay targeting SNP g.299084751 C>T in the NR6A1 gene on chromosome 1. Singleplex real-time PCR gave some ambiguous results for Mangalica and Krškopolje pig breeds and German wild boar individuals, duplex real-time PCR assay especially for Turopolje pig breed. We show that the probability of misclassification can be significantly reduced when considering the results of singleplex real-time PCR assays and duplex real-time PCR assays. 86 (91.5%) out of a total of 94 individuals, including 64 domestic pigs (14 different breeds and 6 crosses) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were correctly classified.

Vector competition, vector capacity of Nyssorhynchus darlingi and basic reproduction number of Plasmodium vivax in agricultural settlements in the Amazon region of Brazil

Maria Anice M. Sallus - 2019


Background Brazilian malaria control programs successfully reduced incidence and mortality rates between 2005 and 2016. Since 2017, an increase in malaria has been reported throughout the Amazon. Few field studies focus on the main malaria vector in highly to moderately endemic areas, Nyssorhynchus darlingi, as the main entomological component of malaria risk, and on the spread metrics of Plasmodium vivax in rural communities in the Amazon, from households from 26 communities. in five communities of the Brazilian states of Acre, Amazonas and Rondônia, with API (> 30). In addition, data on the number of locally acquired symptomatic infections were used in mathematical model analyzes performed to determine Ny. Darlingi vector competence and vector ability for P. vivax; and to calculate the reference reproductive number for P. vivax. Results Entomological indices and malaria metrics varied between sites: Prevalence of P. vivax infection in New York. darlingi, from 0.243% in Mâncio Lima, Acre to 3.96% in Machadinho D'Oeste, Rondônia; daily rate of human bites per person from 23 ± 1.18 in Cruzeiro do Sul, Acre, to 66 ± 2.41 in Lábrea, Amazonas; Vector competence from 0.00456 in São Gabriel da Cachoeira, Amazonas to 0.04764 in Mâncio Lima, Acre; vector capacity from 0.0836 in Mâncio Lima to 1.5 in Machadinho D’Oeste. The estimated R0 for P. vivax (PvR0) was 3.3 in Mâncio Lima, 7.0 in Lábrea, 16.8 in Cruzeiro do Sul, 55.5 in São Gabriel da Cachoeira and 58.7 in Machadinho D'Oeste . Correlation between the prevalence of P. vivax in Ny. Darlingi and vector competence were nonlinear, whereas the association between P. vivax mosquito prevalence, vector capacity, and R0 was linear and positive. Conclusions Despite the low vector competence of Ny. Darlingi to P. vivax, the proliferation of parasites in the human population is enhanced by the high human bite rate and relatively high vector capacity. High PvR0 levels indicate hyperendemia in Machadinho D'Oeste and São Gabriel da Cachoeira, at levels similar to those of P. falciparum in sub-Saharan Africa. Mass detection of parasite reservoirs, effective antimalarial drugs, and vector control interventions will be necessary to reduce transmission in rural communities in Amazonia, Brazil.

Real-time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine applications

Dongmei Li - 2019


Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage in the horticultural industry. The immature stages of these two fruit fly species have been caught several times in New Zealand. It was not possible to determine the species based on morphological characteristics; therefore, it is important to develop an assay for their identification to the species level. Here real-time PCR assays for the rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity through the successful amplification of the two target species, with no cross-reactions observed between the tephritid species tested. In silico testing of the primer and probe binding sites of the two assays also demonstrated the specificity of the assays, as there were no discrepancies in the binding regions of the target species, but there were 1 to 4 mismatches in the target species. binding regions of the non-target species. fruit fly species. The detection thresholds for the two assays are as low as 10 copies/µl of target DNA, indicating that the assays have very high sensitivity. The application of real-time PCR assays has been of great assistance to the routine pest identification and monitoring program at the New Zealand border. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations around the world.

Digital loop-mediated isothermal amplification on a commercial membrane

Xingyu Lin - 2019


In this work, we report digital loop-mediated isothermal amplification (LAMP) or reverse transcription LAMP (RT-LAMP) on a commercial membrane without the need for complex chip fabrication or the use of specialized equipment. Due to the pore size distribution, the digital LAMP theoretical error in these membranes was analyzed using a combination of random distribution model and multi-volume theory. A rapid etching process was developed for effective droplet formation on commercially etched polycarbonate (PCTE) membrane. Each pore acts as an individual nanoreactor for the amplification of single DNA. Absolute quantification of bacterial genomic DNA was performed with a dynamic range of 11 to 1.1 × 105 copies/μL. Single step digital RT-LAMP was also successfully performed on the membrane for quantification of MS2 virus in the effluent. With the introduction of new probes, positive pores can be easily distinguished from negative ones with a 100-fold difference in fluorescence intensity. Finally, the cost of a disposable diaphragm is less than $0.10/pc, which to our knowledge is the most cost effective way to run digital LAMP. The membrane system offers point-of-care or general laboratory users possibilities to perform digital quantification, single cell analysis or other bioassays in a cost-effective, flexible and simplified manner.

A brief overview of the environmental DNA (eDNA) of non-bird reptiles, with a case study of eDNA of colored tortoises (Chrysemys picta) under field conditions

Clare IM Adams - 2019


Environmental DNA (eDNA) is a non-invasive molecular tool increasingly used to detect the presence of species and monitor populations. In this article, we review the current state of work on non-avian reptile eDNA in aquatic systems and present a field experiment to detect the presence of eDNA from colored turtles (Chrysemys picta). To date, eDNA studies of turtles and snakes have shown mixed results in detecting the presence of these animals under field conditions. However, some cases of low recognition rates and lack of recognition do occur in these non-avian reptiles, particularly the squamous ones. We investigated eDNA quantification of non-avian reptiles by sampling four lens ponds at different densities (0 kg/ha, 6 kg/ha, 9 kg/ha and 13 kg/ha) of painted turtles for three months Detecting differences in the use of eDNA using a qPCR assay that amplifies the COI gene from the mtDNA genome. Only one sample of the highest density pool amplified eDNA for a positive detection. However, eDNA concentration estimates from pond eDNA were ranked in correlation with turtle density. We present the "shedding hypothesis", the possibility that animals with tough, keratinized skin do not lose as much DNA as mucus-coated organisms, as a potential challenge for eDNA studies. Despite the challenges of eDNA inhibition and availability in water samples, we remain confident that eDNA can be used to detect freshwater turtles in the field. We provide important recommendations for biologists who want to use eDNA methods to detect non-avian reptiles.

Quantification of Covalently Gated Circular Hepatitis B Virus DNA in Infected Cell Culture Models Using Quantitative PCR

Bingqian Qu - 2019


The persistence of human hepatitis B virus (HBV) requires the maintenance of covalently closed circular DNA (ccc), the episomal genomic reservoir, in the nuclei of infected hepatocytes. Removal of cccDNA is an important goal of future curative therapies currently under development. Hybridization and amplification-based assays for cccDNA quantification are currently used in cell culture-based in vitro studies. Southern blotting, the current gold standard, is time consuming and impractical for large numbers of samples. PCR-based methods show limited specificity when there are excessive amounts of replicative HBV intermediates. We recently developed a real-time quantitative PCR protocol in which total cellular DNA plus all forms of viral DNA are extracted through a silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA, while cccDNA remains intact and can be reliably quantified by PCR. This method was used to measure the kinetics of cccDNA accumulation in various in vitro infection models and the effect of antivirals on cccDNA. It allowed the detection of cccDNA in non-human cells (primary macaque and porcine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporter polypeptide (NTCP). Here we present a detailed protocol of this method, including a workflow diagram, schematic diagram, and illustrations to calculate "cccDNA copies per (infected) cell".

Link of resistome and plasmidome to the microbiome

Thibault Stalder- 2019


The rapid spread of antibiotic resistance among bacterial pathogens is a serious threat to human health. Although several environments have been identified as reservoirs for antibiotic resistance genes (ARGs), we lack knowledge about the origins of these ARGs and their spread from the environment to the clinic. This is due in part to our inability to identify the natural bacterial hosts for ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrins. Here we show that the in vivo Hi-C proximity ligation method can reconstruct a known plasmid-host association from an effluent community and identify the host range in situ of ARGs, plasmids, and integrins by physically associating with their host chromosomes. Hi-C discovered known and new associations between ARGs, mobile genetic elements, and host genomes, thus validating this method. We showed that IncQ plasmids and class 1 integrons had the widest host range in this effluent and identified bacteria from Moraxellaceae, Bacteroides, and Prevotella, and particularly Aeromonadaceae, as the most likely reservoirs of ARGs in this community. Better identification of natural carriers of ARG will help in the development of strategies to limit the spread of resistance to pathogens.

Development of event-specific qPCR detection methods for GM alfalfa events J101, J163 and KK179

Patrick Gurtler - 2019


Genetically modified alfalfa has been approved for cultivation in several countries since 2005. On the other hand, cultivation and export to the European Union is not permitted and therefore no certified reference material or official methods are available. event-specific verification. Therefore, based on the patent sequence information, event-specific real-time PCR detection methods were developed targeting the alfalfa genome junctional sequence and the transgenic insert of the respective events J101, J163 and KK179. Newly developed plasmids were used as reference material for assay optimization and internal validation. Plasmid standards were quantified by digital droplet PCR and LOD95%, PCR efficiency, robustness, and assay specificity were determined by real-time PCR. A 95% LOD of 10 copies per PCR reaction was observed and PCR efficiencies of 95-97% were achieved. Different real-time PCR instruments and PCR conditions were applied to test the robustness of the assays, using DNA at a concentration of 30 copies per μL for each GM alfalfa event. All replicates were positive regardless of instrument or PCR conditions. For the experimental test of specificity, DNA from certified reference material from several genetically modified crops and reference material from the three events was used. No non-specific amplification signal was observed in any of the assays. The validation results corresponded to the "Minimum Performance Requirements for GMO Analytical Test Methods" of the European Network of GMO Laboratories. In addition, an interlaboratory comparison study was performed to demonstrate the transferability and applicability of the methods and to verify assay performance parameters.

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A European collaborative study to evaluate the performance of different PCR methods for the diagnosis of Mycoplasma bovis

Henk J. Wisselink - 2019


Background: Several species-specific PCR assays based on a variety of target genes are currently used to diagnose Mycoplasma bovis infections in herds of cattle with respiratory disease and/or mastitis. With this variety of methods and the development of new methods and formats, regular performance comparisons are necessary to ensure diagnostic quality. The present study compares the PCR methods currently used in six national veterinary institutes across Europe. Three different sample panels were assembled and tested to assess the analytical specificity, analytical sensitivity, and comparability of the different PCR methods. Where appropriate, the results were also compared with those obtained by isolation by culture. The sensitivity and comparability panels consisted of bronchoalveolar fluid samples from fattening calves, artificially contaminated or naturally infected, and therefore the comparison of the different methods covered the entire workflow from DNA extraction to PCR analysis. Results: Participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or endpoint PCRs targeting four different genes, and iii) six different real-time PCR platforms. Only one commercial kit was evaluated; all other PCR assays were internal tests adapted from published methods. The analytical specificity of the different PCR methods was comparable with the exception of one laboratory that tested positive for Mycoplasma agalactiae. Weak positive results were often obtained with Ct values ​​between 37 and 40 for non-target Mycoplasma strains. The limit of detection (LOD) ranged from 10 to 103 cfu/mL to 103 and 106 cfu/mL for the real-time and endpoint assays, respectively. Cultures have also been shown to detect concentrations as low as 102 CFU/ml. Although Ct values ​​in naturally infected samples showed significant interlaboratory and interassay variation, the final interpretation of sample results (positive versus negative) was essentially the same between different laboratories. Conclusion: With a few exceptions, all methods were used routinely in the participating laboratories and showed comparable performance, guaranteeing the quality of diagnoses despite the variety of methods.

Efecto de la termoterapia en la acquisition de Candidatus Liberibacter Asiaticus por la pulga asiatica de los citrusos (Hemiptera: Liviidae)

Alicia J. Kelley - 2019


The Asian citrus amphipod, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the most damaging insect pest of citrus disease due to its role as a vector of Candidatus Liberibacter asiaticus (Las), the bacterial causative agent of Huanglongbing. Trees infected with Las decline rapidly and fruit production decreases until the tree eventually dies. For disease management, few treatment options are available for infected trees. A technique called "thermotherapy" is being developed to reduce bacterial titers in infected trees; however, the effect of these treatments on the Las transmission cycle is unknown. Field and laboratory tests were performed to determine if thermotherapy treatment reduced the acquisition of Las by D. citri. Trees in the field were treated with a mobile heat treatment facility. Potted trees in the laboratory were treated in a steam chamber. We monitored acquisition rates in D. citri after heat treatment of Las-positive Citrus sinensis (L.) (Rutaceae). Psyllium acquisition and Las titers in thermotherapy-treated trees were compared with untreated Las-positive and Las-negative trees. Our results confirmed the effectiveness of whole tree thermotherapy on Las in potted citrus trees. In contrast, thermotherapy did not significantly reduce plant Las titers or Las acquisition by D. citri under field conditions. These results suggest that further development of field application methods is needed to determine the utility of thermotherapy as a tool for the management of Huanglongbing.

Using discrete and online ATP measurements to assess growth potential after ozonation and (non-)biological treatment of drinking water

Canada Andrés de Vera - 2019


Water companies must control microbial regeneration in the distribution system to protect public health. In this study, an adenosine triphosphate (ATP)-based biomass production potential assay using native bacterial communities was used to assess regrowth potential after ozonation with biofiltration (BF) or prolonged chlorination (SCl2). Two large-scale water treatment plants with different upstream processes were compared (i.e., WTP-BF: ozonation, coagulation/flocculation, biofiltration, UV radiation, chlorination; and WTP-SCl2: ozonation, chlorination, coagulation/flocculation, filtration, chlorination). Characterization of native bacteria by 16S rRNA gene sequencing, qPCR, and cellular ATP (cATP) revealed changes in microbial diversity throughout treatment, detachment of biomass from biofilters (cATP effluent = 30 ± 1 ng/L) and disinfection by chlorine (cATP < 1ng/l). he). For both treatment plants, the 14-day cumulative biomass production (CBPt¼Ptt¼0ATPðtÞDt) was higher for the ozonated water samples (CBP14¼1.2103e3.0103d ngATP/L). CBP continued to increase with increasing ozone dose due to the production of more biodegradable carbon. The promotion of carbon growth was confirmed by the consumption of ozonation by-products (carboxylic acids, aldehydes) and the increase in CBP (9.5102-2.9103 d ngATP/L) after the addition of 50-300 mg C /L of acetate. Ozone followed by prolonged chlorination (WTP-SCl2) effectively controlled biomass growth during the treatment process (CBP14<10 d ngATP/L). In contrast, ozone followed by biofiltration (WTP-BF) reduced regrowth potential by 30 % (CBP14 influent biofilter = 1.3103 d ngATP/L; CBP14 effluent biofilter = 9.3102 d ngATP/L). After adding chlorine to the biofilter outflow, CBP14 was reduced to < 10 d ng ATP/L. Finally, in-line ATP measurements confirmed the discrete measurements and improved the identification of the cATP peak and growth phases of native bacteria.

Using environmental DNA to extend the early detection window for Dreissenid mussels

Adam J. Sepúlveda - 2019


Tools are needed to support early detection of invasive three-sided mussels to prevent their spread into western North America. In this study, we assess whether environmental DNA (eDNA) can extend the seasonal window for early detection of dreissenids beyond plankton tows, which are restricted to warmer seasons where mussel larvae are present. We focused eDNA sampling efforts at several locations in Tiber Reservoir, Montana, where Dreissenid mussel abundance is thought to be low. Samples were collected in June and October 2017, when the water temperature was lower than the optimal temperature for dreissenids to breed, and in July 2017, when the water temperature was warmer and more conducive to breeding. We detected DNA from dreisenid mussels in June, July, and October, although observations of nordreissenid mussels were made in 2017 using non-molecular tools. A subset of the eDNA positive and negative samples were tested by an independent laboratory and the results were confirmed. We then estimated the effort required to detect Dreissenid DNA with a probability of 95% at each site within the Tiber Reservoir and found that up to 27, 14, and 34 samples had to be taken in June, July, and October, respectively. To further validate the usefulness of eDNA, we also present complementary eDNA results from other waters in the Flathead Reservoir, Montana, where dreissenid mussels have never been recorded, and from waters with established populations of zebra mussels in the upper Mississippi River. recorded in the spring. The water temperatures sampled were cooler than the thermal optimum for dreissenid breeding. All samples from the Flathead Reservation were negative for Dreissenid mussel DNA, while all samples from the Upper Mississippi were positive. This study adds to a growing body of research showing that eDNA is a highly sensitive tool for monitoring dreissenid mussels in newly invaded waters, including cooler seasons when non-molecular tools are likely to be less effective or more difficult to use.

Immunoserology of European sea bass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus) as a non-lethal diagnostic tool for viral nerve necrosis

Koby Tarrab - 2018


Viral Nerve Necrosis (VNN) is a deadly fish disease that has spread throughout the world over the past two decades, causing severe losses in aquaculture. Diagnosis of infection is usually made by harvesting brain tissue, often at the cost of culling valuable fish. In order to develop a non-lethal diagnostic method, immune responses to experimental nerve necrosis virus (NNV) infection were studied in sea bass Dicentrarchus labrax and white grouper Epinephelus aeneus, the two species most susceptible to infection. disease. RT-qPCR demonstrated the presence of NNV in the fish brain within 24 h post infection, with viral titers remaining high up to 30-35 days post infection. In the blood of D. labrax, the virus was detectable within the first 5 days, after which its presence decreased rapidly. The expression of the Mx gene was correlated with the presence of the virus in the blood and brain. An indirect ELISA was developed that quantified anti-NNV IgM in fish blood. In D. labrax, the anti-NNV IgM titer increased significantly within 5 days post-infection and the presence of specific IgM was detectable for 180 days. A sandwich ELISA for E. aeneus was developed. In the latter species, the anti-NNV IgM titer increased significantly in the first 12 days and was detectable for a further 208 days. The sandwich ELISA can be used as a diagnostic tool to detect NNV exposure in all fish species for which specific antibodies against their IgM are not yet commercially available. Our immunoserological method can be used reliably to diagnose VNN infection and does not require euthanasia of the fish.

Preweaning Heifer Management on US Dairy Farms: Part IV Factors Associated with the Presence of Escherichia coli O157 in Preweaning Dairy Heifers

C. Stenkamp-Strahm - 2018


Dairy calves shed pathogenic Escherichia coli O157 (O157) in feces and are a potential route of exposure to human infection. As part of the National Animal Health Surveillance System (NAHMS) Dairy 2014 study, we evaluated agricultural, animal, and environmental factors associated with the presence of O157 in dairy calves. For this O157 study, calves from 100 dairies in 13 states were enrolled. Each operation collected calf data from birth to weaning over an 18-month period. A single fecal sample was collected from 487 calves in the western states and 871 calves in the eastern states (total n=1,358), and O157 was detected in 2.5% (n=34) of the fecal samples. Descriptive statistics and univariate detection were used to determine which agricultural practices, environmental factors, and calf health interventions are associated with O157 detection. Multilevel logistic models controlling the dairy farm were built using backward elimination of the filtered variables. The final O157 main effect model included variables for colostrum source, temperature humidity index (THI), and serum IgG concentration. Higher serum IgG was associated with a lower probability of O157 shedding, while calves fed their own dam's colostrum had a higher probability of O157 shedding than calves fed colostrum from combined sources. Interaction models showed that THI levels modified the effect of colostrum source on O157 excretion; Calves with a THI indicative of heat stress showed a significantly higher presence of O157 when fed colostrum from a first lactation dam. The THI level also modified the effects of serum IgG. Calves with thermoneutral or heat stress THI levels had a higher presence of O157 with low (<10 g/l) or fair (10-15 g/l) serum IgG levels compared to calves with excellent levels Serum IgG (≥15 g/l) levels These results highlight factors affecting the presence of O157 in pre-weaning calves and can be used to guide practices that mitigate shedding through improved husbandry.

Activation of the M3 subtype muscarinic receptor stimulates intracellular calcium oscillations and aldosterone production in HAC15 cells of the human adrenal cortex

Latha M. Malaiyandi - 2018


Previous work in bovine and rodent models shows that cholinergic agonists modulate steroid hormone secretion from the adrenal cortex. In this study, we used live cell Ca2+ imaging to examine cholinergic activity in the human adrenocortical carcinoma cell line HAC15. The cholinergic agonists carbachol and acetylcholine elicited heterogeneous Ca2+ oscillations, which were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, whereas preferential blockade of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine slightly increased aldosterone secretion in HAC15 cells, and this effect was also reduced by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, whereas mRNA for M1 and M5 was much lower. In summary, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.

Adequacy of oral saliva sampling at the group level in ruminant populations for the detection of contagious nodular skin disease virus

K. Dietze - 2018


The geographic spread of contagious nodular skin disease (LSD) from the Middle East to the European Union again highlighted the need for appropriate disease screening tools applicable to host animal populations where access to individual animals it's hard. This is of particular importance considering that the clinical manifestation of LSD is often mild, making early detection of the disease difficult in the above conditions. Based on the positive experience with saliva samples at the herd level for pathogen detection, as they are known to work in herds and wild boars, the concept was transferred to ruminants. Two groups of six cattle were experimentally infected with contagious nodular skin disease virus (LSDV) under controlled conditions. Blood, oropharyngeal, and nasal swabs were collected at regular intervals. Group samples were obtained by placing cotton gauze around a salt block commonly provided as a dietary supplement. Gauze pieces with visible signs of tampering were tested in parallel with individual animal samples. Genome payload analysis using qPCR technology revealed a detection window for LSDV from day 2 post-infection to day 28 post-infection, the end of the animal experiment. At the individual level, detection times varied by animal and sample type and included intermittent detections. The cumulative nature of the alternative sampling method makes it suitable for detecting LSDV DNA at the group level, even at times of infection when selective sampling of individuals from a group, as is normally done in LSD surveillance, would likely have failed in any case. the detection.

Salmonella-mediated inflammation eliminates fructose-asparagine competitors in the intestine.

Jikang Wu - 2018


Salmonella enterica triggers intestinal inflammation to access nutrients. One of these nutrients is fructose asparagine (F-Asn). The availability of F-Asn to Salmonella during infection is dependent on the pathogenicity islands SPI1 and SPI2, which in turn are required to elicit inflammation. Here we find that F-Asn is present in mouse chow at approximately 400 pmol/mg dry weight. F-Asn is also present in the intestinal tract of germ-free mice at 2700 pmol/mg dry weight and in the intestinal tract of conventional mice at 15 pmol/mg. These results suggest that the mouse gut microbiota consumes F-Asn. We used highly labeled F-Asn precursors to monitor their formation in the intestine in the presence or absence of inflammation and none were observed. Finally, we found that some members of the Clostridia class encode F-Asn utilization pathways and are deleted from highly inflamed Salmonella-infected mice. Overall, our studies identify the source of F-Asn as the diet and that Salmonella-mediated inflammation is required to eliminate competitors and allow the pathogen almost exclusive access to this nutrient.

Development of a new qPCR method for the detection and specific quantification of genetically modified maize MON863

Patrick Gurtler - 2018


Analysis of non-transgenic seed samples for the presence of MON863-GM maize revealed unexpected amplification signals using an official qPCR method. These gain signals only occurred when different master mix products were used as in the original validation process. DNA sequence data from a non-specific amplicon could be mapped to a maize mitochondrial DNA reference sequence. Oligo sequence analysis revealed that the forward primer and probe can hybridize to maize mitochondrial DNA, resulting in non-specific amplification signals in qPCR. Therefore, we developed and validated a new qPCR method for the MON863 event with a LOD of 5 copies per reaction. The method has a high level of specificity, since no non-specific amplification signal was detected after analysis of the reference material for several genetically modified plants and conventional maize samples. Robustness tests were performed in two different laboratories and no impact on method performance was observed when using different master mixes and qPCR devices, or when varying oligonucleotide concentrations.

Efficacy of pneumococcal conjugate vaccine-13 (PCV13) against invasive pneumococcal disease in children in the Dominican Republic

Sarah Tomczyk - 2018


There are limited data on the efficacy of 13-valent pneumococcal conjugate vaccine (PCV13) in low-resource settings and PCV-naïve populations. The Dominican Republic introduced PCV13 in September 2013 with a 2+1 calendar (2, 4 and 12 months) without a recovery campaign. We evaluated the efficacy of PCV13 against vaccine-type invasive pneumococcal disease (IPD) in children in the Dominican Republic.

A protocol to quantify bacterial 16S rDNA in total plasma as a marker of microbial translocationlive

Wei Jiang - 2018


A protocol to quantify bacterial 16S rDNA in total plasma as a marker of microbial translocation in vivo

Development of a quantitative loop-mediated isothermal amplification assay for field detection of Erysiphe necator

Lindsey D. Thiessen - 2018


Plant pathogen detection systems have been useful tools to monitor the presence of inoculum and initiate management plans. More recently, a loop-mediated isothermal amplification (LAMP) assay has been successfully developed for field use in the grape powdery mildew pathosystem; However, false negative or false positive results have been common in tests conducted by breeders due to the difficulty in detecting magnesium pyrophosphate precipitate at low DNA concentrations. Wine growers in Oregon's Willamette Valley evaluated a quantitative LAMP assay (qLAMP) using a fluorescence resonance energy transfer-based probe. Customized impaction spore samplers were installed at one research vineyard and six commercial vineyard sites and tested biweekly by the laboratory and growers. qLAMP trials conducted by breeders used a beta version of Smart-DART-LAMP portable reaction devices (Diagenetix, Inc., Honolulu, HI, USA) compatible with Android 4.4 enabled, Nexus 7 tablets were connected with Bluetooth for the exit . Quantification by a quantitative PCR assay was assumed to be correct to compare quantification by laboratory and producer qLAMP assays. Breeders could run and interpret the qLAMP results; However, quantification of the Erysiphenecator inoculum was not reliable with the Beta Smart DART devices. The developed qLAMP assay was sensitive to one spore in early testing of the assay, but was reduced to more than 20 spores by the end of the study. The qLAMP assay is unlikely to be a suitable management tool for powdery mildew of grapes due to the loss of sensitivity and decreased cost and portability of other more reliable molecular tools.

Climate change favors certain fungal communities in boreal marshes

Asma Asemaninejad - 2018


Fungi play a central role in the carbon sequestration potential of boreal peatlands through the decomposition process. Therefore, climate-related changes in the diversity and composition of fungal communities in peatlands could have significant impacts on the release of carbon from these ecosystems, particularly in the subsurface peatland, which is a major global reservoir of carbon. We used Illumina MiSeq rDNA sequencing to study fungal communities at 18 months in intact peat mesocosms exposed to conditions associated with Canada's future climate, including: warming, increased atmospheric CO2, and lower groundwater tables. Warming was the main driver of changes in fungal communities at three depths of the peat profile, with dominant groups of Ascomycota and Basidiomycota becoming more homogeneous under warming conditions. However, specific changes in fungal functional groups were temperature dependent, with potential cellulose decomposers and mycorrhizal root-associated fungi of Basidiomycota dominating under +4 °C heating, while potential lignocellulose decomposers and root-associated fungi Ascomycota mycorrhizal species predominated below +8 °C. C heating. These climate change-induced changes in fungal community structure in favor of recalcitrant compound decomposers, observed along a depth gradient, may reduce long-term carbon storage of boreal peatlands under future change scenarios. climate.

Efficacy of hyperbaric oxygen therapy in the eradication of bacterial biofilm

Nicolás E. Sanford - 2018


Objective: Chronic wounds often require multiple concurrent treatments, such as debridement, decompression, and systemic and/or topical antibiotics. The aim of this study was to investigate the effectiveness of hyperbaric oxygen therapy (HBOT) in reducing or eliminating bacterial biofilms in vitro and in vivo. Method: Efficacy was determined using in vitro cultured biofilms directly exposed to HBOT for 30, 60, and 90 minutes, followed by assessment of cell viability by propidium monoazide polymerase chain reaction (PMA-PCR). The efficacy of HBOT in vivo was assessed by searching our chronic patient wound database and comparing healing time between patients who did and did not receive HBOT as part of their treatment. Results: In vitro data showed small but significant decreases in cell viability at the 30 and 90 minute time points in the HBOT group. In vivo data showed a reduction in bacterial load in patients undergoing HBOT and a shorter treatment duration ~1 week. In addition, patients' chronic wounds experienced a significant onset of anaerobic bacteria and fungi between intermittent HBOT treatments. Conclusion: The data indicates that HBOT possesses some level of ability to kill biofilms. Additionally, as an adjunct to standard care, more favorable patient outcomes are achieved through faster healing time, reducing the likelihood of complications. Furthermore, the data provided insights into biofilm adaptation to the challenges of this treatment strategy, which should be taken into account when treating chronic wounds. Further studies will be needed to evaluate the benefits and mechanisms of HBOT not only for patients with chronic wounds but also for other chronic infections caused by bacterial biofilms.

Red deer (Cervus elaphus) specific real-time PCR assay for the detection of food adulteration

Maria Kaltenbrunner - 2018


We present a deer-specific real-time PCR assay that, in combination with a previously published reference real-time PCR assay, enables quantification of deer content in food products. Therefore, it can be used to detect adulteration of food. The deer-specific real-time PCR assay primer/probe system amplifies an 87 bp fragment of the C iota protein kinase gene. To eliminate cross-reactivity with closely related species, the forward primer was designed to contain an intentional base mismatch adjacent to a deer-specific base. The deer-specific real-time PCR assay did not cross-react with 23 animal and 50 plant species tested. The LOD and LOQ determined by analysis of a serially diluted DNA extract containing 1% (w/w) red deer DNA in porcine DNA were 0.05% and 0.4%, respectively. Precision has been validated by testing DNA pools, meat extract pools, meat pools, and venison sausage models with known red deer content. The best precision was obtained when the calibration mixture was similar in composition and concentration to the animal species of interest in the analyzed sample. High recoveries were obtained not only for raw samples, but also after heat treatment including broths (15 min at 75-78 °C), boiling (90 min at 100 °C), and microwave treatment (15 s, 40 s or 2 min 0.650 W). ). The deer-specific real-time PCR assay was shown to be resistant to small variations in reaction volume or hybridization temperature and the use of a different real-time PCR instrument.

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