Use Of qPCR Research Articles

Evaluation of candidate reference genes for quantitative real-time PCR analysis in a male rat model of dietary iron deficiency

BackgroundQuantitative real-time polymerase chain reaction (qPCR) is a reliable and efficient method for quantitation of gene expression. Due to the increased use of qPCR in examining nutrient-gene interactions, it is important to examine, develop, and utilize standardized approaches for data analyses and interpretation. A common method used to normalize expression data involves the use of reference genes (RG) to determine relative mRNA abundance. When calculating the relative abundance, the selection of RG can influence experimental results and has the potential to skew data interpretation. Although common RG may be used for normalization, often little consideration is given to the suitability of RG selection for an experimental condition or between various tissue or cell types. In the current study, we examined the stability of gene expression using BestKeeper, comparative delta quantitation cycle, NormFinder, and RefFinder in a variety of tissues obtained from iron-deficient and pair-fed iron-replete rats to determine the optimal selection among ten candidate RG.ResultsOur results suggest that several commonly used RG (e.g., Actb and Gapdh) exhibit less stability compared to other candidate RG (e.g., Rpl19 and Rps29) in both iron-deficient and iron-replete pair-fed conditions. For all evaluated RG, Tfrc expression significantly increased in iron-deficient animal livers compared to the iron-replete pair-fed controls; however, the relative induction varied nearly 4-fold between the most suitable (Rpl19) and least suitable (Gapdh) RG.ConclusionThese results indicate the selection and use of RG should be empirically determined and RG selection may vary across experimental conditions and biological tissues.

Abstract P366: The Impact Of Estrogen Signaling On Gut Epithelial Cells

Cardiovascular disease (CVD) is the leading cause of death in women worldwide. Menopause is associated with increased susceptibility to CVD, but the mechanism is currently unknown. Studies have suggested that loss of estrogen signaling in the intestine during menopause may lead to worse myocardial infarction (MI) outcomes in female mice. Our lab has shown that suppressing inflammation in the gut using pre- and pro-biotics protects the heart from ischemic heart disease caused by MI through unknown means. We propose that alterations in gut microbiota are due to destruction of intestinal pattern recognition receptors (PRRs) such as toll-like receptor 4 (TLR4) and TLR5. Studies show that estrogen signaling modulates PRRs in other tissues, but this has yet to be investigated in gut epithelial cells. To elucidate the mechanisms of estrogen receptor function in the gut as a direct mediator of vascular health, we are utilizing an immortalized human cell line of colorectal adenocarcinoma brush border expressing (Caco2 BBE) cells. We hypothesized that estrogen treatment in gut epithelial cells will lead to increased expression of PRRs and decreased inflammation in Caco2 BBE cell lines. To investigate expression and localization of estrogen receptors (ERs), Caco2 BBE cells at different confluency were treated with estradiol (E2) or DMSO control for 24hrs, then either RNA or protein were harvested for use in qPCR and western blot, or cells were formalin fixed for use in immunohistochemistry. In each case, ERα was not detected, but ERβ was present at varying concentrations. Data also showed increased expression of TLR5 and TLR7 in E2 treated cells, while TNF-α and IL-1 were decreased. In conclusion, data show potential confluency dependent ERβ expression and suggest that ERα may not be present in our Caco2 BBE cell line. Our hypothesis is also supported by data for PPRs and inflammatory markers. Though these results are revealing, future studies are needed to further characterize Caco2 BBE cells in the context of ER signaling.

Rapid Antibiotic Susceptibility Testing of Tier-1 Agents Bacillus anthracis, Yersinia pestis, and Francisella tularensis Directly From Whole Blood Samples.

Rapid antibiotic susceptibility tests, performed directly on whole blood samples, will offer great clinical advantages. This issue is of considerable importance when it comes to bioterror pathogens where prompt antibiotic treatment should be offered to infected patients as well as prophylaxis to suspected exposed individuals. Herein, we describe a novel and rapid method, named MAPt, that is based on the direct application of a blood sample onto solid agar that has been embedded with different concentrations of the tested antibiotic. Following a short incubation, bacterial growth is monitored by qPCR. The method was applied on blood cultures and whole blood samples inoculated with the Tier-1 pathogens Bacillus anthracis, Yersinia pestis, and Francisella tularensis. The use of agar medium, which better supports the growth of bacteria at low concentrations, together with the use of qPCR, which provides sensitivity and specificity, allowed minimal inhibitory concentration (MIC) determination to a wide range of bacterial concentrations, ranging from ∼5 × 102 cfu/ml up to 108 cfu/ml. The omission of the enrichment procedure in blood culture and the isolation step, both required in standard antibiotic susceptibility tests (ASTs), allowed a dramatic reduction in time to answer, from a few days to a few hours. The total time required for MIC determination was ∼6 h for fast-growing bacteria, such as B. anthracis, and 12–16 h for slow-growing bacteria, represented by Y. pestis and F. tularensis. Accordingly, MAPt may offer health authorities means for public preparedness in the case of a bioterror attack as well as prompt clinical treatment options in common blood stream infections.

Detection of quantitative and qualitative for p.aeruginosa in contaminated and non-contaminated soil with oil residues targeting gyrb gene by QPCR

Comparison of P. aeruginosa counts is found in contaminated and non- contaminated soil with oil residue using a real-time quantitative PCR by Direct DNA extraction from soil samples as well as indirect DNA extraction from bacterial isolates. Sampling were collected randomly from ten different regions of the Ministry of Science &Technology during the period from October to December 2019. Twenty soil samples were investigated for hydrocarbon tolerance in selective broth containing hydrocarbon source, and then isolates diagnosed based on phenotype, microscopic, and biochemical tests, were used. Direct DNA extraction from soil samples using DNeasy Power Soil Pro Kit for the rapid detection of P. aeruginosa from soil, as well as wizard DNA extraction method from bacterial isolation. Quantitative and Qualitative PCR was performed with the Magnetic Induction Cycler (Mic), DNA extraction from soil and bacterial isolates amplified and detected using fluorescent reporter dye probes specific for Pseudomonas aeruginosa DNA and Internal Control IC. A total of twenty bacterial isolates were obtained from the different soil samples according to phenotyping tests, and also, we obtain 20 DNA samples from direct DNA extraction from soil, the results of purity were (1.70- 1.95) for wizard method and (1.46- 2.35) for direct extraction from soil. Pseudomonas aeruginosa counts in contaminated soil samples (8 x 107 Copies/μL) showed higher compared with the non- contaminated soil samples (2 x 102 Copies/μL) at maximum use of qPCR and soil DNA. Rapid and specific test (less than 4 hours) using Rt-PCR by targeting gyrB gene for Pseudomonas aeruginosa detection in contaminated and non-contaminated soil samples, combined with correctly adjusted samples processing methods, as well as soil DNA extraction were made which were applied within the sensitivity required for methods.

Salinity-induced Changes in Gene Expression in the Streptophyte Alga Chara: The Critical Role of a Rare Na+ -ATPase.

The primarily freshwater genus Chara is comprised of many species that exhibit a wide range of salinity tolerance. The range of salt tolerance provides a good platform for investigating the role of transport mechanisms in response to salt stress, and the close evolutionary relationship between Charophytes and land plants can provide broader insights. We investigated the response to salt stress of previously identified transport mechanisms in two species of Chara, Chara longifolia (salt-tolerant), and Chara australis (salt-sensitive): a cation transporter (HKT), a Na+ /H+ antiport (NHX), H+ -ATPase (AHA), and a Na+ -ATPase (ENA). The presence of these candidate genes has been confirmed in both species of Chara, with the exception of the Na+ -ATPase, which is present only in salt-tolerant Charalongifolia. Time-course Illumina transcriptomes were created using RNA from multiple time points (0, 6, 12, 24 and 48h) after freshwater cultures for each species were exposed to salt stress. These transcriptomes verified our hypotheses of these mechanisms conferring salt tolerance in the two species examined and also aided in identification of specific transcripts representing our genes of interest in both species. The expression of these transcripts was validated through use of qPCR, in a similar experimental set-up used for the RNAseq data described above. The RNAseq and qPCR data showed significant changes of expression mechanisms in C.longifolia (respectively), a down-regulation of HKT and a substantial up-regulation of ENA. Significant responses to salt stress in salt-sensitive C.australis show up-regulation of NHX and AHA.

Accurate and fast identification of Campylobacter fetus in bulls by real-time PCR targeting a 16S rRNA gene sequence

Campylobacter fetus is an important animal pathogen that causes infectious infertility, embryonic mortality and abortions in cattle and sheep flocks. There are two recognized subspecies related with reproductive disorders in livestock: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). Rapid and reliable detection of this pathogenic species in bulls is of upmost importance for disease control in dairy and beef herds as they are asymptomatic carriers. The aim of the present work was to assess the performance a real-time PCR (qPCR) method for the diagnosis of Campylobacter fetus in samples from bulls, comparing it with culture and isolation methods. 520 preputial samples were both cultured in Skirrow's medium and analyzed by qPCR. The estimated sensitivity of qPCR was 90.9% (95% CI, 69.4%–100%), and the specificity was 99.4% (95% CI, 98.6% - 100%). The proportion of C. fetus positive individuals was 2.1% by isolation and 2.5% by qPCR. Isolates were identified by biochemical tests as Cfv (n = 9) and Cff (n = 2). Our findings support the use of qPCR for fast and accurate detection of C. fetus directly from field samples of preputial smegma of bulls. The qPCR method showed to be suitable for massive screenings because it can be performed in pooled samples without losing accuracy and sensitivity.

Use of qPCR for the analysis of population heterogeneity and dynamics during Lactobacillus delbrueckii spp. bulgaricus batch culture

Direct molecular methods such as real-time polymerase chain reaction (qPCR) and propidium monoazide (PMA)-qPCR have been successfully used for quantifying viable microorganisms in the food industry. This study attempted to use qPCR and PMA-qPCR for quantifying Lactobacillus delbrueckii spp. bulgaricus sp1.1 physiological states. The qPCR standards of the 16S rRNA gene were employed to calibrate the qPCR assay, which contributed to an amplification efficiency of 98.42%. The number of copies of the 16S rRNA gene was linearly related to cell density, and this linear relationship was used to construct a quantitative curve (R2 =0.9981) with a detection limit of 15.1 colony-forming units mL−1·reaction−1. qPCR in combination with an optimal PMA concentration (60 μM) helped in discriminating and quantifying the viable cells, without any interference by heat-killed cells. Compared with the conventional methods, the population heterogeneity of viable, culturable, dormant-like and membrane-permeabilized cells were well identified and quantified using qPCR during L. delbrueckii spp. bulgaricus sp1.1 batch culture. Despite the restriction in the enumeration of lysed cells, qPCR-based methods facilitated reliable identification and quantification of bacterial physiological states and provided additional knowledge on the dynamics of L. delbrueckii spp. bulgaricus sp1.1 physiological states.

Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication

Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14 CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration

BackgroundThe most commonly used diagnostic tool for soil-transmitted helminths (STH) is the Kato-Katz (KK) thick smear technique. However, numerous studies have suggested that the sensitivity of KK can be problematic, especially in low prevalence and low intensity settings. An emerging alternative is quantitative polymerase chain reaction (qPCR).MethodsIn this study, both KK and qPCR were conducted on stool samples from 648 participants in an STH epidemiology study conducted in the delta region of Myanmar in June 2016.ResultsPrevalence of any STH was 20.68% by KK and 45.06% by qPCR. Prevalence of each individual STH was also higher by qPCR than KK, the biggest difference was for hookworm with an approximately 4-fold increase between the two diagnostic techniques. Prevalence of Ancylostoma ceylanicum, a parasite predominately found in dogs, was 4.63%, indicating that there is the possibility of zoonotic transmission in the study setting. In individuals with moderate to high intensity infections there is evidence for a linear relationship between eggs per gram (EPG) of faeces, derived from KK, and DNA copy number, derived from qPCR which is particularly strong for Ascaris lumbricoides.ConclusionsThe use of qPCR in low prevalence settings is important to accurately assess the epidemiological situation and plan control strategies for the ‘end game’. However, more work is required to accurately assess STH intensity from qPCR results and to reduce the cost of qPCR so that is widely accessible in STH endemic countries.

Re-evaluation of Streptococcus pneumoniae carriage in Portuguese elderly by qPCR increases carriage estimates and unveils an expanded pool of serotypes

Streptococcus pneumoniae (pneumococcus) is a leading cause of infections worldwide. Disease is preceded by asymptomatic colonization of the upper respiratory tract. Classical culture-based methods (CCBM) suggest that colonization in the elderly is <5%. Recently, use of qPCR has challenged these observations. We estimated pneumococcal carriage prevalence and serotypes among Portuguese elderly using qPCR and compared results with those obtained by CCBM. Nasopharyngeal and oropharyngeal paired samples (599 each) of individuals over 60 years living in nursing (n = 299) or family (n = 300) homes were screened for the presence of pneumococci by qPCR targeting lytA and piaB. Positive samples were molecular serotyped. Use of qPCR improved detection of pneumococci in oropharyngeal samples compared to CCBM: from 0.7% to 10.4% (p < 0.001) in the nursing home collection, and from 0.3% to 5.0% (p < 0.001) in the family home collection. No significant differences were observed between both methods in nasopharyngeal samples (5.4% vs. 5.4% in the nursing homes; and 4.3% vs. 4.7% in the family homes). Twenty-one serotypes/serogroups were detected by qPCR compared to 14 by CCBM. In conclusion, use of qPCR suggests that pneumococcal carriage in Portuguese elderly is approximately 10%, and unveiled a large pool of serotypes. These results are important to understand progression to disease and impact of pneumococcal vaccines in the elderly.

Detection of enteric parasite DNA in household and bed dust samples: potential for infection transmission

BackgroundEnteric parasites are transmitted in households but few studies have sampled inside households for parasites and none have used sensitive molecular methods.MethodsWe collected bed and living room dust samples from households of children participating in a clinical trial of anthelmintic treatment in rural coastal Ecuador. Dust was examined for presence of DNA specific for 11 enteric parasites (Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Toxocara canis and T. cati, Giardia lamblia, Blastocystis hominis, Cryptosporidium spp., and Entamoeba histolytica) by quantitative PCR (qPCR).ResultsOf the 38 households sampled, 37 had positive dust for at least one parasite and up to 8 parasites were detected in single samples. Positivity was greatest for B. hominis (79% of household samples) indicating a high level of environmental fecal contamination. Dust positivity rates for individual pathogens were: S. stercoralis (52%), A. lumbricoides (39%), G. lamblia (39%), Toxocara spp. (42%), hookworm (18%) and T. trichiura (8%). DNA for Cryptosporidium spp. and E. histolytica was not detected. Bed dust was more frequently positive than floor samples for all parasites detected. Positivity for A. lumbricoides DNA in bed (adjusted OR: 10.0, 95% CI: 2.0–50.1) but not floor dust (adjusted OR: 3.6, 95% CI: 0.3–37.9) was significantly associated with active infections in children.ConclusionsTo our knowledge, this is the first use of qPCR on environmental samples to detect a wide range of enteric pathogen DNA. Our results indicate widespread contamination of households with parasite DNA and raise the possibility that beds, under conditions of overcrowding in a humid tropical setting, may be a source of transmission.

Diagnostic Uncertainty and the Epidemiology of Feline Foamy Virus in Pumas (Puma concolor)

Feline foamy virus (FFV) is a contact-dependent retrovirus forming chronic, largely apathogenic, infections in domestic and wild felid populations worldwide. Given there is no current ‘gold standard’ diagnostic test for FFV, efforts to elucidate the ecology and epidemiology of the virus may be complicated by unknown sensitivity and specificity of diagnostic tests. Using Bayesian Latent Class Analysis, we estimated the sensitivity and specificity of the only two FFV diagnostic tests available—ELISA and qPCR—as well as the prevalence of FFV in a large cohort of pumas from Colorado. We evaluated the diagnostic agreement of ELISA and qPCR, and whether differences in their diagnostic accuracy impacted risk factor analyses for FFV infection. Our results suggest ELISA and qPCR did not have strong diagnostic agreement, despite FFV causing a persistent infection. While both tests had similar sensitivity, ELISA had higher specificity. ELISA, but not qPCR, identified age to be a significant risk factor, whereas neither qPCR nor ELISA identified sex to be a risk factor. This suggests FFV transmission in pumas may primarily be via non-antagonistic, social interactions between adult conspecifics. Our study highlights that combined use of qPCR and ELISA for FFV may enhance estimates of the true prevalence of FFV and epidemiological inferences.

MiR-1291 Functions as a Potential Serum Biomarker for Bullous Pemphigoid.

Background Bullous pemphigoid (BP) is a common T helper 2- (Th2-) dominated autoimmune blistering skin disease with significant mortality. MicroRNAs (miRNAs), which are endogenous noncoding RNA molecules, have been reported to be potential biomarkers for some autoimmune diseases; however, to date, there exist no reports on serum expression profiles of miRNAs in BP patients. Methods A RNA quantitative PCR- (qPCR-) based array was conducted on sera from 20 active BP patients and 20 healthy controls for screening of miRNAs. Significantly dysregulated miRNAs were validated with use of qPCR as performed on sera samples of 45 active BP patients and 60 healthy controls. Serum CCL17, anti-BP180, and anti-BP230 levels were measured with use of ELISA. Results Relative baseline expression levels of serum miR-1291 were significantly upregulated in the 45 BP patients as compared with the 60 healthy controls (P < 0.001) and significantly decreased in the disease control stage (n = 13, P = 0.006). In addition, these baseline miR-1291 levels showed a significant positive correlation with the baseline levels of serum CCL17 (P < 0.001) and anti-BP180 (n = 38, P = 0.024). Like that observed for miR-1291, baseline levels of serum CCL17 were also significantly elevated in the 45 BP patients compared with the 60 healthy controls (P < 0.001) and significantly decreased in the disease control stage (n = 13, P = 0.002). However, for anti-BP180, baseline serum levels were significantly elevated in only 38 of the 45 BP patients and significantly decreased in the disease control stage (n = 10, P = 0.004). Conclusions Relative expression levels of serum miR-1291 can reflect disease activity of BP. miR-1291 may function as an important new serum biomarker for BP.

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR

BackgroundAlthough more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA.MethodsWe assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106–1); (ii) diluted template DNA from pure oocysts (×10–×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105–1 and 5 × 104–50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10–×1000).ResultsSerial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations.ConclusionsFor many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

Use of qPCR and RT-qPCR for monitoring variations of microcystin producers and as an early warning system to predict toxin production in an Ohio inland lake

Public concern over cyanobacterial blooms has increased due to their higher frequency of occurrence and their potential ecological and health impacts. Detection of microcystin (MC) producers (MCPs) using qPCR and RT-qPCR allows for the rapid identification of blooms by combining specificity and sensitivity with a relatively high throughput capability. Investigation of MCP population composition (correlation, dominance), toxin gene expression, and relationship to MC concentration was conducted using a panel of qPCR assays targeting mcyA, E and G on weekly and daily water samples collected from an Ohio inland reservoir lake. Further, these data were used to develop early warning thresholds for prediction of MC concentrations exceeding the US EPA Health Advisory cutoff value (>0.3 μg L−1) using receiver operating characteristic curves and tobit regression. MCP Microcystis genomic copy number made up approximately 35% of the total Microcystis spp. and was the dominant toxic subpopulation of MCPs. The expressed MCPs were 0.2% of the extant genomic copy numbers, while toxic Microcystis had higher expressed proportion (0.5%) than that of toxic Planktothrix (0.04%). Microcystis toxin genes increased in June and July but decreased in August and September along with similar trends of cell replication. Quantities of both RT-qPCR and qPCR followed the same trend and were highly correlated with MC-ADDA, while RT-qPCR not only reflected the active toxin genes or toxic species, but also indicated the beginning and ending of toxin production. A one-week early warning of MC exceedance over the EPA Health Advisory was based on signaling of qPCR and RT-qPCR using receiver operating characteristic curves. This study illustrates the potential use of qPCR or RT-qPCR as an early warning system of extant and MC producing potentials during a toxic algal bloom, with predictive powers of 50%–60% and 30%–40% (p < 0.001), respectively, and false positive rates of about 70% for both LC-MS/MS or ELISA.

Assessment of the use of compost stability as an indicator of alkane and aromatic hydrocarbon degrader abundance in green waste composting materials and finished composts for soil bioremediation application

Green waste composting materials and finished composts were collected from different commercial ex situ composting sites all treating source segregated green waste feedstocks. Stability of each material was determined using the standard ORG0020 dynamic respiration test. To assess whether stability could be used as an indicator for the potential suitability of green waste composting materials and finished composts as amendments for soil bioremediation, comparison was made with alkane and aromatic hydrocarbon degrader abundance determined using a quantitative PCR (qPCR) approach. Specifically, primers targeting alkB and, polyaromatic hydrocarbon ring-hydroxylating dioxygenases genes (PAH-RHD) of Gram positive (GP) and Gram negative (GN) populations were used for qPCR analysis. The results showed no direct correction between compost stability and gene abundance. Further, increase in alkB gene abundance was not linked to PAH-RHD gene abundance. The results support the use of qPCR as a tool for screening organic amendments on a site by site basis for soil bioremediation treatment.

An in vivo comparative evaluation of antimicrobial efficacy of chitosan, chlorhexidine gluconate gel and their combination as an intracanal medicament against Enterococcus faecalis in failed endodontic cases using real time polymerase chain reaction (qPCR)

AimTo evaluate the in vivo antibacterial efficacy of 2% chlorhexidine gel (CHX), 2% chitosan (CS) gel and their combination as an intracanal medicament against Enterococcus faecalis during endodontic retreatment procedure, with the use of qPCR. MethodA total of 45 single rooted permanent teeth were selected from 28 systemically healthy patients (mean age of 43 years). After complete disinfection and access opening as well as gutta-percha (GP) removal, the first microbiological pretreatment sample (S1) was collected. After completion of instrumentation, a post-instrumentation sample (S2) was taken and the teeth were randomly divided into three experimental groups: 2% CHX gel; 2% CS gel; 2% CHX with CS; Fuji IX glass ionomer cement was used to seal the access; after 7 days, the post medication sample (S3) was collected and E. faecalis was quantified using qPCR. ResultsMaximum reduction in bacterial mean CFU (×106) counts was observed between S1 and S2; S2 and S3; S1 and S3 in 2% CHX with chitosan group compared to other groups. Percentage reduction in CFU (×106) counts at different time intervals (S1-S2; S2-S3; S1-S3) was maximum for the 2% CHX with the chitosan group. Comparison of the mean CFU (×106) count within the 2% CHX gel group and the 2% CHX with chitosan group at different time intervals (S1,S2,S3) was found to be highly significant (P = 0.001); whereas in group II, it was significant (P = 0.002). Conclusion2% CHX with chitosan group showed the highest microbial reduction against E. faecalis during retreatment of failed endodontic cases.

Evaluating monitoring options for conservation: comparing traditional and environmental DNA tools for a critically endangered mammal

While conservation management has made tremendous strides to date, deciding where, when and how to invest limited monitoring budgets is a central concern for impactful decision-making. New analytical tools, such as environmental DNA (eDNA), are now facilitating broader biodiversity monitoring at unprecedented scales, in part, due to time, and presumably cost, of methodological efficiency. Genetic approaches vary from conventional PCR (cPCR; species presence), to metabarcoding (community structure), and qPCR (relative DNA abundance, detection sensitivity). Knowing when to employ these techniques over traditional protocols could enable practitioners to make more informed choices concerning data collection. Using 12 species-specific primers designed for cPCR, eDNA analysis of the Yangtze finless porpoise (YFP; Neophocaena asiaeorientalis asiaeorientalis), a critically endangered aquatic mammal within the Yangtze River, we validated and optimized these primers for use in qPCR. We tested repeatability and sensitivity to detect YFP eDNA and subsequently compared the cost of traditional (visual and capture) sampling to eDNA tools. Our results suggest cPCR as the least expensive sampling option but the lack of PCR sensitivity suggests it may not be the most robust method for this taxon, predominately useful as a supplementary tool or with large expected populations. Alternatively, qPCR remained less expensive than traditional surveys, representing a highly repeatable and sensitive method for this behaviorally elusive species. Cost comparisons of surveying practices have scarcely been discussed; however, given budgetary constraints particularly for developing countries with limited local oversight but high endemism, we encourage managers to carefully consider the trade-offs among accuracy, cost, coverage, and speed for biodiversity monitoring.

Quantitative Real-Time PCR to Measure YAP/TAZ Activity in Human Cells.

Transcription coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ, also known as WWTR1) are homologs of the Drosophila Yorkie (Yki) protein and are major downstream effectors of the evolutionarily conserved Hippo pathway. YAP/TAZ play critical roles in regulation of cell proliferation, apoptosis, and stemness, thus mediate functions of the Hippo pathway in organ size control and tumorigenesis. The Hippo pathway inhibits YAP/TAZ through phosphorylation, which leads to YAP/TAZ cytoplasmic retention and degradation. Dephosphorylated and nuclear-localized YAP/TAZ bind to transcription factors, especially the TEAD family proteins, thus transactivate the expression of specific genes. Therefore, measuring the expression level of YAP/TAZ target genes is a critical approach to assess Hippo pathway activity. Through gene expression profiling in different tissues and cells using techniques such as microarray and RNA-seq, many target genes of YAP/TAZ have been identified. Some of these genes were confirmed to be direct YAP/TAZ targets by chromatin immunoprecipitation (ChIP)-PCR or ChIP-seq. These works made it possible to quickly determine YAP/TAZ activity by measuring the mRNA levels of several YAP/TAZ target genes, such as CTGF, CYR61, and miR-130a by quantitative real-time PCR (qPCR). In this chapter, we demonstrate the use of qPCR to measure YAP/TAZ activity in MCF10A cells.

Temporal patterns of airborne Phytophthora spp. in a woody plant nursery area detected using real-time PCR

In this study, spore trap monitoring was applied to provide a proof of concept for the use of qPCR to detect Phytophthora in aerial samples and provide valuable information for epidemiological studies in nurseries. Two qPCR TaqMan assays were developed to detect pathogen DNA: the first used a generic probe to detect Phytophthora spp., and the second was based on a specific probe for detecting P. ramorum and P. lateralis. All samples tested positive for the genus Phytophthora, although P. ramorum and P. lateralis were not detected. In late spring and in autumn, two main peaks of Phytophthora sporulation were observed. Peaks were preceded by rainfall, high relative humidity, and mild temperature. From mid-May to the end of August, Phytophthora DNA detected in the air increased with relative humidity, while it decreased with increasing mean temperature. There was also a positive correlation between Phytophthora DNA detected and rainfall in the same period. No significant correlations between Phytophthora DNA and temperature or rainfall were found from the end of August to December. Our results are in agreement with those obtained with classical diagnostic methods based on microscopy, but the approach used here enabled rapid detection and relative quantification of the target organisms, thus assisting in the implementation of disease management strategies.