Real-time Relative PCR Research Articles

Detection of Mycobacterium avium Subspecies Paratuberculosis in Pooled Fecal Samples by Fecal Culture and Real-Time PCR in Relation to Bacterial Density.

Simple SummaryParatuberculosis is a worldwide disease causing serious impacts to the dairy industry. Within the context of paratuberculosis control programs, dairy herds have to be classified as either paratuberculosis-positive or paratuberculosis-free with minimum effort but with sufficient reliability. We aimed to estimate the detection rate of positive herds using a combination of random sampling and pooling of five or ten fecal samples. The pooled samples were analyzed with two different laboratory methods (bacterial culture and polymerase chain reaction). Pools of size 10 can be used without significant decrease of detection probability compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of paratuberculosis-positive herds, but the detection probability in herds with only few infected animals (<5.0%) is not high enough to recommend this approach for one-time testing in such herds.Within paratuberculosis control programs Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds have to be detected with minimum effort but with sufficient reliability. We aimed to evaluate a combination of random sampling (RS) and pooling for the detection of MAP-infected herds, simulating repeated RS in imitated dairy herds (within-herd prevalence 1.0%, 2.0%, 4.3%). Each RS consisted of taking 80 out of 300 pretested fecal samples, and five or ten samples were repeatedly and randomly pooled. All pools containing at least one MAP-positive sample were analyzed by culture and real-time quantitative PCR (qPCR). The pool detection probability was 47.0% or 45.9% for pools of size 5 or 10 applying qPCR and slightly lower using culture. Combining these methods increased the pool detection probability. A positive association between bacterial density in pools and pool detection probability was identified by logistic regression. The herd-level detection probability ranged from 67.3% to 84.8% for pools of size 10 analyzed by both qPCR and culture. Pools of size 10 can be used without significant loss of sensitivity compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of MAP-infected herds, but is not recommended for one-time testing in low prevalence herds.

Multicentric prospettive study of validation of angiogenesis-related gene polymorphisms in hepatocellular carcinoma patients treated with sorafenib: Interim analysis of INNOVATE study.

4075 Background: In the ePHAS study we analyzed three eNOSpolymorphisms and at univariate analysis, patients with eNOS-786 -TTgenotype had significantly shorter median Progression Free Survival (PFS) and Overall Survival (OS) compared to those with other genotypes. On the basis of these preliminary results, our aim is to validate in a prospective study this data in patients with HCC treated with sorafenib. Methods: This is a prospective Italian multicenter study, that includes 141 HCC patients receiving sorafenib. We analyzed eNOS-786and itwas analyzed by Real Time PCR in relation to the primary end point (OS). Event-time distributions were estimated using the Kaplan-Meier method and survival curves were compared using the log-rank test. Results: 141 HCC patients (122 males and 19 females), prospectively treated with sorafenib from May 2015 to September 2018 were included. Median age was 69 years (range 28-88 years). 120 patients had Child-Pugh A and 21 had Child-Pugh B7. 43 had BCLC-B and 98 patients had BCLC-C. Atunivariate analysis, we confirmed that eNOS-786 TT genotype were significantly associated with a lower median OS than the other genotypes (8.8 vs 15.7 months, HR 1.69, 95% CI 1.02-2.83 p=0.0424). Following adjustment for clinical covariates (age, gender, etiology, BCLC stage, serum α-FP level, MELD score), multivariate analysis confirmed eNOS- 786 and BCLC stage as the independentsprognostic factors predicting OS (TTvsTC+CC; HR: 2.39, 95% CI 1.14-5.03 p=0.0211; C vs B;2.23, 95% CI 1.44-4.77 p=0.039). Conclusions: Our prospective study confirms the prognostic role of eNOS-786 in advanced HCC patients treated with sorafenib. Clinical trial information: NCT02786342.

ENOS polymorphisms and clinical outcome in advanced HCC patients receiving sorafenib: final results of the ePHAS study.

Sorafenib may reduce endothelial nitric oxide synthase (eNOS) activity by inhibiting vascular endothelial growth factor receptors (VEGF-R), leading to a decrease in nitric oxide production. In the Italian multicenter ePHAS (eNOS polymorphisms in HCC and sorafenib) study, we analyzed the role of eNOS polymorphisms in relation to clinical outcome in patients with hepatocellular carcinoma (HCC) receiving sorafenib. Our retrospective study included a training cohort of 41 HCC patients and a validation cohort of 87 HCC patients, all undergoing sorafenib treatment. Three eNOS polymorphisms (eNOS -786T>C, eNOS VNTR 27bp 4a/b and eNOS+894G>T) were analyzed by direct sequencing or Real Time PCR in relation to progression-free survival (PFS) and overall survival (OS) (log-rank test). In univariate analysis, training cohort patients homozygous for eNOS haplotype (HT1:T-4b at eNOS-786/eNOS VNTR) had a lower median PFS (2.6 vs. 5.8 months, P < 0.0001) and OS (3.2 vs.14.6 months, P = 0.024) than those with other haplotypes. In the validation set, patients homozygous for HT1 had a lower median PFS (2.0 vs. 6.7 months, P < 0.0001) and OS (6.4 vs.18.0 months, P < 0.0001) than those with other haplotypes. Multivariate analysis confirmed this haplotype as the only independent prognostic factor. Our results suggest that haplotype HT1 in the eNOS gene may be capable of identifying a subset of HCC patients who are resistant to sorafenib.

Expression and Influence of Galectin-3 on Missed Abortion

Objective To explore the influence of galectin-3 on missed abortion. Methods Forty cases of normal intrauterine early pregnancy were randomly divided into 2 groups: surgical abortion group (group A, n=20) and medical abortion group (group B, n=20). The third group was missed abortion group (group C, n=20) with the gestational age less than 13 weeks. Serum was isolated from the blood samples, collected and used for ELISA quantification of galectin-3. Villus and decidua tissues were collected from the abortus for immunohistochemical examination and real-time fluorescence relative quantitative PCR. Results The level of galectin-3 in the serum was the lowest in missed abortion group (P Conclusion Galectin-3 is related to the development of villus and decidua during early pregnancy. The decreased expression of galectin-3 may promote the occurrence of missed abortion.

Liver microRNA hsa-miR-125a-5p in HBV Chronic Infection: Correlation with HBV Replication and Disease Progression

To study in HBsAg chronic carriers the expression of liver hsa-miR-125a-5p and its correlation with liver HBV-DNA values and clinical presentation, 27 consecutive Caucasian, HBsAg/anti-HBe/HBV-DNA-positive patients who were naive to nucleos(t)ide analogues and interferon therapy and had no marker of HCV, HDV or HIV infection and no history of alcohol intake were enrolled. For each patient, liver HBV DNA and liver hsa-miR-125a-5p were quantified by real-time PCR in relation to β-globin DNA or RNU6B, respectively. Liver fibrosis and necroinflammation were graded by applying Ishak's scoring system. Liver hsa-miR-125a-5p was detected in all patients enrolled and a correlation between its concentration and liver HBV DNA was demonstrated (p<0.0001). Higher liver hsa-miR-125a-5p concentrations were observed in patients with HBV-DNA plasma level >103 IU/ml (p<0.02), in those with HAI >6 (p = 0.02) and those with fibrosis score >2 (p<0.02) than in patients with lower scores. Higher HBV-DNA liver concentrations were found in patients with abnormal AST (p = 0.005) and ALT serum levels (p = 0.05), in those with serum HBV DNA higher than 10E3 IU/mL (p = 0.001) and those with fibrosis score >2 (p = 0.02) than in patients with a lower load. By multivariate logistic regression analysis, liver hsa-miR-125a-5p was identified as an independent predictor of disease progression: O.R. = 4.21, C.I. 95% = 1.08–16.43, p<0.05, for HAI >6; O.R. = 3.12, C.I. 95% = 1.17–8.27, p<0.05, for fibrosis score >2. In conclusion, in HBsAg/anti-HBe-positive patients, the liver hsa-miR-125a-5p level correlated with liver and plasma HBV-DNA values and was associated to a more severe disease progression.

Studies of the pathogenicity of WSSV to the cultured shrimp Litopenaeus vannamei under chlorpyrifos stress

In order to evaluate the hazard of chlorpyrifos to the cultured shrimp Litopenaeus vannamei,the lethal effect of the shrimp infected with white spot syndrome virus(WSSV)under the chlorpyrifos stress was tested,while the quantity of WSSV in the gill tissue and acetylcholinesterase acticity in the muscle tissue were analyzed.The 96 h median lethal concentration(LC50)of chlorpyrifos to the shrimp was determined.The results showed that the 96 h LC50 is 0.758 μg/L(0.521-0.987 μg/L),and as time prolonged,the values of LC50 significantly declined,which showed a positive concentration-response relationship.On this basis,the stress concentration of chlorpyrifos of 0.2 μg/L was determined.After the shrimp were exposed to 0.2 μg/L chlorpyrifos for 4 days,they were infected with WSSV by injection.It showed that the cumulate mortality of chlorpyrifos-WSSV treatment(83.33%±4.7%)was significantly higher than that of ethanol-WSSV group(40.00%±0.9%).Meanwhile,the amount of WSSV was also determined by Real-time Relative PCR at 48,72 and 96 h post-injection.WSSV quantity of chlorpyrifos-WSSV treatment was about 4 times that of ethanol-WSSV control at 72 h.WSSV quantity of chlorpyrifos-WSSV treatment was significantly increased at 96 h,which was 4.9 times that of chlorpyrifos-WSSV treatment at 72 h,and 5.9 times that of ethanol-WSSV treatment at 96 h.The acetylcholinesterase(AchE)activity in shrimp muscle tissue under chlorpyrifos stress was 20% lower than that in control group.This showed that the proliferation rate of WSSV in shrimp increased rapidly under chlorpyrifos stress,which raised the mortality rate of the shrimp.

The balance of expression of PTPN22 splice forms is significantly different in rheumatoid arthritis patients compared with controls

BackgroundThe R620W variant in protein tyrosine phosphatase non-receptor 22 (PTPN22) is associated with rheumatoid arthritis (RA). The PTPN22 gene has alternatively spliced transcripts and at least two of the splice forms have been confirmed to encode different PTPN22 (LYP) proteins, but detailed information regarding expression of these is lacking, especially with regard to autoimmune diseases.MethodsWe have investigated the mRNA expression of known PTPN22 splice forms with TaqMan real-time PCR in relation to ZNF592 as an endogenous reference in peripheral blood cells from three independent cohorts with RA patients (n = 139) and controls (n = 111) of Caucasian origin. Polymorphisms in the PTPN22 locus (25 SNPs) and phenotypic data (gender, disease activity, ACPA and RF status) were used for analysis. Additionally, we addressed possible effects of methotrexate treatment on PTPN22 expression.ResultsWe found consistent differences in the expression of the PTPN22 splice forms in unstimulated peripheral blood mononuclear cells between RA patients and normal controls. This difference was more pronounced when comparing the ratio of splice forms and was not affected by methotrexate treatment.ConclusionsOur data show that RA patients and healthy controls have a shift in balance of expression of splice forms derived from the PTPN22 gene. This balance seems not to be caused by treatment and may be of importance during immune response due to great structural differences in the encoded PTPN22 proteins.

Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels

A relative quantification system (RQ-PCR) was used to monitor the correlations between the activity of the nor-1 (=aflD) gene of Aspergillus flavus using real-time PCR in relation to phenotypic aflatoxin B(1) (AFB(1) ) production and populations of A. flavus in stored peanuts at three water activity levels (a(w) , 0·95, 0·90 and 0·85) for 6 weeks. Real-time PCR was used to amplify the nor-1 gene (target gene), and benA56 (β-tubulin gene) used as a control gene. Expression of three structural genes, nor-1 (=aflD), ver-1 (=aflM), and omtA (=aflP), and the regulatory gene aflR of the aflatoxin biosynthetic pathway were also assayed. There were significant differences between nor-1 gene expression at the three a(w) levels; higher expression at 0·90 a(w) in weeks 1-3, when compared to 0·95. In contrast, in the driest treatment (0·85 a(w) ) none or very low nor-1 expression occurred. The populations of A. flavus colony-forming units (CFUs g(-1) ) increased over time with the highest at 0·95 a(w) . Highest AFB(1) production was at 0·90 and 0·95 a(w) from weeks 3-6. A(w) had a significant effect on aflR transcription at 0·95 a(w) over the 6-week period, while at 0·90 a(w) , only in the last 2 weeks. Correlations between different factors showed that log AFB(1) × log CFUs, log AFB(1) × a(w) , and log CFUs × a(w) were statistically significant, while log CFUs × RQ-PCR and RQ-PCR × a(w) were not. The AflR gene may not have an important role in the regulation of nor-1 expression in food matrices (e.g. peanuts). Determination of correlations between nor-1 expression and aflatoxin production by A. flavus in raw peanuts under different a(w) levels could be helpful to predict potential risk of aflatoxin production during storage of this hygroscopic food product and minimize contamination with the AFB(1) .

Stress- and Growth Rate-Related Differences between Plate Count and Real-Time PCR Data during Growth of Listeria monocytogenes

To assess the overestimation of bacterial cell counts in real-time PCR in relation to stress and growth phase, four different strains of L. monocytogenes were exposed to combinations of osmotic stress (0.5 to 8% [vol/vol] NaCl) and acid stress (pH 5 to 7) in a culture model at a growth temperature of 10 degrees C or were grown under optimal conditions. Growth curves obtained from real-time PCR, optical density, and viable count data were compared. As expected, optical density data revealed entirely different growth curves. Good to moderate growth conditions yielded good correlation of real-time PCR data and plate count data (r(2) = 0.96 and 0.99) with similar cell counts. When growth conditions became worse, the numbers of CFU decreased during the stationary phase, whereas real-time-PCR-derived bacterial cell equivalents differed in this regard; the correlation worsened (r(2) = 0.84). However, fitted growth curves revealed that maximum growth rates calculated from real-time PCR data were not significantly different from those derived from plate count data. The overestimation of bacterial cell counts by real-time PCR observed in the stationary phase under higher-stress conditions might be explained by the accumulation of viable but nonculturable bacteria or dead bacteria and extracellular DNA. Considering these results, real-time PCR data collected from naturally contaminated samples should be viewed with caution.

Gene expression as an indication for ochratoxin A biosynthesis inPenicillium nordicum

The expression level of theotapksPN gene ofPenicillium nordicum was determined by Reverse Transcription Real Time PCR in relation to several growth parameters known to have an influence on ochratoxin A biosynthesis, like pH, medium composition and temperature. Generally there was a good correlation between the expression level of this key gene and ochratoxin A biosynthesis. The results, which were obtained by Real Time PCR for a single gene, could generally be confirmed by microarray analysis with a whole set of genes related to ochratoxin A biosynthesis. Growth conditions which favour the expression of the ochratoxin A biosynthetic genes also resulted in the biosynthesis of ochratoxin A by the fungal culture, indicating that the influence of abiotic parameters is mediated via the regulation of transcription of the ochratoxin A biosynthetic genes. The influence of several parameters, like pH, salt concentration and temperature on the expression of the ochratoxin A biosynthetic genes has been analysed. Optimum conditions for the activation of the ochratoxin A biosynthesis genes were: pH 6.0-8.0, 25 °C and a sodium chloride concentration of 20 g/l. This was paralleled by the capacity ofP. nordicum to produce ochratoxin A under the same conditions. After combining the most inhibitory conditions (15 °C, sodium chloride 10 g/l, pH 5.0) nearly a complete deactivation of the ochratoxin A biosynthetic genes could be observed resulting in a complete abolishment of ochratoxin A biosynthesis. In contrast, a high expression and a high production of ochratoxin A could be observed after growth ofP. nordicum under more optimal conditions (25 °C, 20 g/1 sodium chloride, pH 8.0).