TaqMan Real-time PCR Research Articles

Progress in understanding the genetic underpinning of coronary artery disease: from genotype to phenotype

Abstract Background The functional dissection of genetic risk opens the door to strategies for pharmacological manipulation of molecular and cellular processes implicated in human diseases, including coronary artery disease (CAD). Aim To assess the relationship between a set of single nucleotide polymorphisms previously associated with CAD and coronary artery calcium (CAC) score in an asymptomatic population from Portugal. Methods Prospective study in a cohort of 1,284 subjects aged 59.3±8.9 years, 73.6% males without apparent CAD. The CAC score was performed by cardiac computed tomography and reported as Agatston units according to the Hoff nomogram. Anthropometric, conventional, and biochemical risk factors were evaluated. Thirty-three single nucleotide polymorphisms were genotyped by TaqMan real-time PCR. Bivariate and multivariate logistic regression analysis estimated variables associated with the CAC score. Results After bivariate analysis, PHACTR1 rs1332844 C>T, a downstream regulator of the endothelin-1 gene, showed a significant association with CAC score (p=0.015), together with CDKN2B-AS1 variants rs4977574 A>G in the locus 9p21.3 (p=0.002) and rs1333049 G>C (p=0.010). MTHFD1L gene encodes a mitochondrial enzyme responsible for homocysteine remethylating in methionine, and rs6922269 G>A showed protection against artery calcification (p=0.013). After multivariate logistic regression, PHACTR1 rs1332844 (CT+TT) (p=0.009) and CDKN2B-AS1 rs4977574 A>G (GG) (p=0.002) remained in the equation as independently associated with arterial calcification, together with male sex (p=0.039), age (p<0.0001), hypertension (p=0.001), diabetes (p<0.0001), smoking status (p<0.0001) and obesity (p=0.020). MTHFD1L rs6922269 AA also remained as significant protection from arterial calcification (p=0.027). Conclusion The present work showed two genetic variants, PHACTR1 and CDKN2B-AS1, linked to arterial wall calcification, herewith clinical and environmental risk factors. MTHFD1L showed protection. Understanding the genetic basis of vessel calcification can enable lifestyle changes or pharmacologic therapy to attenuate risk before the disease becomes clinic. More research in this field is critical to understanding the genetic basis of CAD through an intermediate phenotype, coronary arterial wall calcification.

Gene-gene and gene-environment interaction in coronary artery disease risk

Abstract Coronary Artery Disease (CAD) is a lethal illness that kills millions of individuals each year worldwide. This disorder is multifactorial, resulting in the complex interplay of genetic, epigenetic, and environmental factors. Exploring gene-gene and gene-environment interactions is essential to understanding the aetiology of common complex diseases like CAD. Aim We proposed investigating the interaction between two genetic variants robustly associated with CAD by GWAS (MTHFR rs1801133 and APOE rs7412/rs429358) in cardiovascular susceptibility. After that, consider whether there was a new interaction between this genetic association and environment (hypertension, dyslipidemia and smoking). Methods A case-control study enrolled 3,157 individuals, 1,721 coronary patients (with at least 70% stenosis of one or more major coronary arteries or its primary branches on the coronary angiography) and 1,436 controls without CAD. Traditional risk factors were investigated, and the two polymorphisms were performed by TaqMan real-time PCR. Bivariate analysis was used to study genotypic proportions between CAD and non-CAD patients with respective odds ratios (OR). Logistic regression analysis displayed the interaction between the two genetic variants each other and with some important environmental risk factors. Results In bivariate analysis, the genetic models were studied for both genes, being the dominant model the one which showed significance for CAD. In MTHFR, 57.8% of the patients and 53.4% of the controls presented the CT+TT genotype (OR=1.20; p=0.013). In APOE, 26.5% of the patients and 22.3% of the controls had E2E4;E3E4+E4E4 (OR=1.26; p=0.006). The first multivariate logistic regression showed a synergistic interaction between the two genetic variants (OR=1.50; p=0.001). After adjusting for conventional risk factors, the logistic regression analysis entered the MTHFR rs1801133 and APOE rs7412/rs429358, showing a combined interaction effect between those genes and smoking status with an OR of 3.44 (p<0.0001), with hypertension (OR=1.55; p=0.002) and with dyslipidemia (OR=1.71; p<0.0001). Conclusion According to our results, the genetic interaction between APOE and MTHFR synergistically affected the susceptibility to CAD. This interaction, linked to conventional risk factors, consistently increased the risk of CAD in our population, particularly in the group of genetic carriers who did not quit smoking.

GJA4 gene inhibition may represent a potential target for novel antithrombotic therapies

Abstract The GJA4 gene encodes a protein called connexin 37, a component of gap junctions in vascular endothelial cells, crucial in regulating endothelial function and influencing inflammation, platelet adhesion and thrombus formation. This gene deletion in mice diminished thrombus formation in vitro in human blood. In these circumstances, connexin inhibition with pharmacological agents may represent potential avenues for developing novel antithrombotic agents. Aim To investigate whether the GJA4 rs618675 T>C variant is a risk factor for progressing atherosclerosis and cardiovascular (CV) events in an asymptomatic free of apparent CAD from a Portuguese population. Methods A prospective study was performed with 1421 individuals without apparent CV disease (73.6% male, aged 52.2±8.3 years) followed during an extended period of 7.2±5.1 years). GJA4 rs618675 T>C variant was genotyped by TaqMan real-time PCR technique (Applied Biosystems). Conventional, anthropometric, biochemical and clinical risk factors were studied. Bivariate analysis and multivariate Cox regression adjusted for age, gender, traditional risk factors, biochemical markers, and the genotypes under study was performed to determine which variables were, significantly and independently, associated with CV events. Kaplan-Meier's estimate assessed the prognosis. Results Bivariate analysis showed that 50.6% of wild genotype (TT) carriers had CV events vs. 66.2% without events. Of the risk genotype (CC) carriers, 10.1% had CV events, and 3.4% did not. After the Kaplan-Meier analysis, the TT carriers had 90% CV event-free survival time and the CC risk carriers had only 64% of event-free survival. Finally, after adjustment, CC genotype remained in the equation with an HR of 3.1 (p=0.003) and CT heterozygous with HR of 1.6 (p=0.043), together with Hypertension (HR 3.0; p<0.0001), Smoking habits (HR 2.3; p=0.001) and Diabetes (HR 1.9; p=0.015). Conclusion: The CC risk genotype of the GJA4 rs618675 represented an independent risk factor for progressing atherosclerosis and CV events in a Portuguese Population. Thrombus formation often begins with the adhesion of platelets to the endothelium, and connexin 37 may influence this process. The inhibition of connexin 37 emerges as a promising candidate for future cardiovascular prevention as well as for the development of new antithrombotic drugs.

TRP36-ELISA for E. canis detection: Concordance with TaqMan real-time PCR and point-of-care testing

Ehrlichia canis, a widely distributed tick-borne pathogen, requires prompt and accurate diagnosis for effective management. Real-time PCR serves as the reference standard for diagnosing E. canis infection, whereas serological tests are crucial for detecting antibody responses indicative of infection or post-exposure status. Early detection during the acute phase of the disease is essential for optimal treatment and recovery. E. canis Tandem Repeat Protein 36 (TRP36) elicits early acute phase responses. We developed an enzyme-linked immunosorbent assay (ELISA) using recombinant TRP36 to detect IgM and IgG antibodies against E. canis. We assessed the diagnostic agreement, sensitivity, and specificity of the rTRP36-ELISA and a commercial test kit (SNAP 4Dx Plus Test) with TaqMan Real-time PCR (qPCR) using the NCSS 2023 program version 23.0.1. Leftover serum samples from 32 dogs at the Veterinary Teaching Hospital were randomly selected and examined for E. canis infection using qPCR, rTRP36-ELISA, and SNAP 4Dx Plus Test. qPCR detected positive results in 12 (37.5 %), whereas ELISA and SNAP 4Dx Plus detected positive results in 10 (31.25 %) and 13 (40.62 %), respectively. Moderate agreement (κ = 0.545) was observed between rTRP36-ELISA and qPCR, and fair agreement (κ = 0.379) between SNAP 4Dx Plus and qPCR. Substantial agreement (κ = 0.79) was found between rTRP36-ELISA and SNAP 4Dx Plus. Compared to qPCR, the sensitivity and specificity of rTRP36-ELISA were 70 % and 77.27 %, respectively, compared to 53.85 % and 73.86 %, respectively, for SNAP 4Dx Plus. Our findings suggest that TRP36 is a promising antigen for E. canis serodiagnosis, potentially improving sensitivity and specificity.

Protective Effect of EBF Transcription Factor 1 (EBF1) Polymorphism in Sporadic and Familial Spontaneous Preterm Birth: Insights from a Case-Control Study.

This study investigated the potential role of specific single-nucleotide polymorphisms (SNPs) in the genes Astrotactin 1 (ASTN1), EBF Transcription Factor 1 (EBF1), Eukaryotic Elongation Factor, Selenocysteine-tRNA Specific (EEFSEC), Microtubule-Associated Serine/Threonine Kinase 1 (MAST1), and Tumor Necrosis Factor Alpha (TNF-α) to assess whether these genetic variants contribute to the risk of spontaneous preterm birth (sPTB). A case-control study was conducted involving 573 women from Croatia and Slovenia: 248 with sporadic sPTB (positive personal and negative family history of sPTB before 37 weeks' gestation), 44 with familial sPTB (positive personal and family history of sPTB before 37 weeks' gestation), and 281 control women. The analysis of ASTN1 rs146756455, EBF1 rs2963463, EBF1 rs2946169, EEFSEC rs201450565, MAST1 rs188343966, and TNF-α rs1800629 SNPs was performed using TaqMan real-time PCR. p-values were Bonferroni-adjusted for multiple comparisons. EBF1 SNP rs2963463 was significantly associated with sPTB (p adj = 0.03). Women carrying the CC genotype had a 3-4-times lower risk of sPTB (p adj < 0.0001). In addition, a significant difference in the frequency of the minor C allele was observed when comparing familial sPTB cases with controls (p adj < 0.0001). All other associations were based on unadjusted p-values. The minor T allele of EBF1 SNP rs2946169 was more frequent in sPTB cases overall than in controls, especially in sporadic sPTB (p = 0.045). Similarly, the CC genotype of ASTN1 SNP rs146756455 was more frequent in sporadic sPTB cases compared to controls (p = 0.019). Finally, the TNF-α SNP rs1800629 minor A allele and AA genotype were more common in the familial sPTB group compared to sporadic sPTB and controls (p < 0.05). The EBF1 SNP rs2963463 polymorphism showed a protective effect in the pathogenesis of sPTB, particularly in women carrying the CC genotype. Moreover, EBF1 SNP rs2946169 and ASTN1 SNP rs146756455, as well as TNF-α SNP rs1800629, were associated with an increased risk of sPTB, representing suggestive potential risk factors for sporadic and familial sPTB, respectively.

A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria

The transfer of biocide and antibiotic resistance genes by mobile genetic elements is the most common mechanism for rapidly acquiring and spreading resistance among bacteria. The qacEΔ1 gene confers the resistance to quaternary ammonium compounds (QACs). It has also been considered a genetic marker for the presence of class 1 integrons associated with multidrug-resistant (MDR) phenotypes in Gram-negative bacteria. In this study, a TaqMan real-time PCR assay was developed to detect the qacEΔ1 gene in Gram-negative bacteria. The assay has a detection limit of 80 copies of the qacEΔ1 gene per reaction. No false-positive or false-negative results have been observed. Simultaneous amplification and detection of the 16S rRNA gene is performed as an endogenous internal amplification control (IAC). The TaqMan real-time PCR assay developed is a rapid, sensitive, and specific method that could be used to monitor resistance to QACs, the spread of class 1 integrons, and the prediction of associated MDR phenotypes in Gram-negative bacteria.

Establishment and Application of a Multiplex PCR NGS Method for the Genotyping of HLA-Class I and HPA.

Selecting compatible HLA-Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA-Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA-Class I and HPA genotypes. Ten pairs of specific primers for HLA-A, HLA-B, HLA-C, HPA-1 through HPA-6w, HPA-15 and HPA-21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA-A, HLA-B and HLA-C and the TaqMan real-time PCR method in-house for the genotyping of HPA-1 through HPA-6w, HPA-15 and HPA-21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA-A, HLA-B, HLA-C and HPA-1 through HPA-6w, HPA-15 and HPA-21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA-A*11:451, HLA-A*30:01:26, HLA-B*39:201 and HLA-B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA-Class I and HPA simultaneously, which is high-throughput, rapid and accurate and could be applied to build a platelet donor bank.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG+GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

Real-time quantitative reverse transcription PCR assay for the detection of Nuomin virus – An emerging tick-borne virus

Nuomin virus (NOMV), an emerging tick-borne virus (TBVs) identified in 2020, has been associated with fever, headache, and potential liver dysfunction in infected individuals. This study presents a novel TaqMan real-time quantitative PCR method designed for the rapid, sensitive, and specific detection of NOMV, facilitating early diagnosis. Utilizing Beacon Designer software 8.0, we optimized the PCR assay including the development of primers and probes to precisely target the conserved region of the NOMV genome, followed by optimization of primer and probe concentrations and annealing temperature. The resulting assay demonstrated robust performance, with standard curve represented by the equation y=−3.29x+39.42, a high correlation coefficient (R2 = 0.995) and an efficiency 99.53 %. Importantly, the method exhibited exceptional specificity, which did not yield cross-reacting signals from other TBVs, including Songling virus (SGLV), Beiji virus (BJNV), tick-borne encephalitis virus (TBEV), Yezo virus (YEZV), Alongshan virus (ALSV), and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). The assay’s detection limit was remarkably low, reaching 10 copies/μL, representing a 100-fold increase compared to semi-nested RT-PCR. Additionally, it demonstrated excellent repeatability, with coefficients of variation for intra- and inter-group tests consistently below 3 %. Clinical evaluations confirmed the assay’s superior performance, highlighting its high specificity, sensitivity, and reproducibility for NOMV detection. In conclusion, the method developed in this study provides a valuable tool to support timely management of NOMV infections, with significant implications for clinical practice.

First Molecular Evidence of Leishmania Infantum in Domestic Cats and Associated Risk Factors from the Black Sea Region of Türkiye.

The objectives of the present study are to determine the molecular prevalence of Leishmania spp. in the owned domestic cats in the Black Sea Region of Türkiye and analyze the associated risk factors in FeL. Conjunctival swabs (CS), blood, demographic, and clinical data were collected from 150 owned cats brought to the Veterinary Teaching Hospital during 2020-2022. Leishmania kinetoplast DNA (kDNA) from CS was screened by TaqMan Real-Time PCR (qPCR) with the genus-specific primers and a probe. All qPCR positive products were also amplified and sequenced to identify Leishmania species by ITS1 primers. Molecular prevalence of L. infantum found as 12.6% (19/150) in the observed cats in the Black Sea Region of Türkiye. There was a significant difference (p < 0.05) between neutered and intact cats with regarding to L. infantum positivity. Intact cats found to be 0.368 times more prone to be L. infantum-positive (L+). Dermatological lesions were found the most common (26.3%) problems in the L + cats. The median leucocyte count was the only parameter that was found statistically (p < 0.05) lower in the L + group (6.60) than the negative group (L-) (8.96), when comparing the WBC, NEU/LYM, MONO/LYM, EOS/LYM and PLT/LYM values. This study presented the molecular occurrence of FeL in the Black Sea Region of Türkiye for the first time indicating that the carrier status of the cats makes them alternative reservoirs for possible zoonotic transmission of L. infantum in this zone.

Association of FOXO3 (rs17069665) gene polymorphism and childhood acute lymphoblastic leukemia in Egypt

BackgroundAcute lymphoblastic leukemia (ALL) is the most common malignancy in children. Genetic variations, particularly gene polymorphisms, have been closely linked to increased susceptibility to ALL. One of those genes is Forkhead box O3 (FOXO3), which is considered a potential tumor suppressor gene. AimThis study intended to examine the potential significance of the FOXO3 (rs17069665) single nucleotide polymorphism (SNP) as a risk factor for childhood ALL, in addition to its effect on the laboratory results, clinical manifestations and the clinical outcome after induction of chemotherapy. Subjects and methodsSixty-six newly diagnosed ALL children and 70 healthy children of matched age and sex as controls were recruited. FOXO3 (rs17069665) polymorphism was detected using TaqMan real time PCR. ResultsHigher frequencies of the (AG) genotype and G-allele of FOXO3 (rs17069665) variant were present in ALL patients in comparing with the controls (16.7 % vs. 4.3 %, p = 0.017 and 11.4 % vs. 2.1 %, p = 0.003, respectively). The frequencies of the FOXO3 (rs17069665) SNP reflected a noticeably higher risk of ALL under diverse genetic models, including the co-dominant model (AG vs. AA, OR = 2.55), dominant (AG + GG vs. AA, OR = 2.81), and allelic (G-allele vs. A-allele, OR = 2.9) models. The single case of c-MYC mutation was observed with the (GG) genotype. No significant association between FOXO3 (rs17069665) SNP polymorphism and response to chemotherapy was found. ConclusionOur findings showed that the FOXO3 (rs17069665) polymorphism was associated with a greater incidence of ALL in Egyptian children, which might be a potential biomarker for ALL susceptibility.

Association of glutamine synthetase polymorphisms rs2296521, rs10911021 and rs12136955 with plasma ammonia concentration in valproic acid-treated Egyptian epilepsy patients.

The use of valproic acid (VPA) in the treatment of some psychiatric and neurological disorders such as bipolar disorder, migraines, and epilepsy is associated with hyperammonemia. However, the mechanism of this negative effect of VPA is unclear. In this study, we investigate gene glutamate-ammonia ligase (GLUL) polymorphisms for the glutamine synthetase (GS) enzyme, a key enzyme that catalyzes the removal of ammonia by incorporating it with glutamate to form glutamine, and we investigate whether it has a relationship with the emergence of hyperammonemia during VPA-based therapy. We enrolled 180 Egyptian epilepsy patients in this study. Patient history, general and neurological examination and blood samples from arm veins were taken. Real time TaqMan PCR polymorphism for three polymorphism SNPs (rs2296521, rs10911021 and rs12136955) of GLUL was done. We assessed the relationship between the patient features, including three GLUL polymorphisms, and the development of hyperammonemia during VPA-based therapy. We found that the ammonia levels showed a positive correlation with VPA treatment duration (p = 0.015) and a negative correlation with carbamazepine total dose per day (p = 0.027) and with WBCs count (p = 0.026). Also, female patients having rs2296521 SNPs with the A allele and patients having rs10911021 SNPs with the C allele were at high risk for elevated plasma ammonia levels. Moreover, patients having rs12136955 SNPs with the A allele or associated hypertension as a co-morbidity were at high risk for elevated plasma ammonia levels. Female patients who have rs2296521 with the A allele, rs10911021 with the C allele, or rs12136955 with the A allele, are independent risk factors for elevated plasma ammonia levels during VPA-based therapy. Moreover, carbamazepine combined therapy may protect against the development of hyperammonemia in VPA-treated patients.

Gene Polymorphisms of the antioxidant enzymes NOX, GSTP, and GPX and diabetic nephropathy risk in Saudi patients with type 2 diabetes

Oxidative stress has been hypothesized to play a crucial role in the complications of type 2 diabetes (T2D). Hyperglycaemia-mediated increases in free radicals have a deleterious effect on cellular compartments and nucleic acids, leading to imbalances between free radicals and antioxidant enzymes. This case–control study comprised two groups with 100 participants (50 T2D and 50 DN patients) and aimed to examine the association between single-nucleotide polymorphisms (SNPs) in the genes encoding nicotinamide adenine dinucleotide phosphate oxidase (NOX), glutathione S-transferase P (GSTP1), and glutathione peroxidase 1(GPX1) and diabetic nephropathy (DN) risk in patients with T2D. An SNP genotyping assay was performed using TaqMan assay and real-time PCR to identify the SNPs (NOX rs4673, GSTP rs1695, GPX1 rs1050450). The Sanger method was used to validate our findings. Fisher chi-square analyses revealed no significant differences in these genes when comparing T2D patients with and without DN. Our findings suggest no association between the rs4673, rs1695, and rs1050450 SNPs and DN in Saudi patients with T2D.

Lack of association between TRPV1 gene polymorphisms and risk of migraine chronification: a case-control study and meta-analysis.

To confirm a previously reported association of TRPV1 rs8065080 with the risk of transformation from episodic (EM) to chronic migraine (CM) and to extend knowledge about the role of other TRPV1 single nucleotide polymorphisms (SNPs), we first investigated the impact of three TRPV1 SNPs (rs8065080, rs222747 and rs222749) on the risk of migraine chronification in a case-control study. A systematic review and meta-analysis were then conducted to summarize the accumulated findings. Genotyping of the selected TRPV1 SNPs was performed using TaqMan real-time PCR in 167 EM and 182 CM participants. Crude and adjusted odds ratios with associated 95% confidence intervals were calculated in the log-additive, dominant, and recessive genetic models. A comprehensive literature search was performed in PubMed, Web of Knowledge, Cochrane Library, and OpenGrey until February 2024. In our case-control study, no association was found between TRPV1 SNPs and the risk of migraine chronification, both in the unadjusted logistic regression models and after adjustment for confounding clinical variables. The results of the meta-analysis with a total of 241 participants with EM and 223 with CM confirmed no association between TRPV1 SNPs and the risk of migraine chronification in any of the genetic models tested. The results of the present case-control study and meta-analysis exclude a major role of TRPV1 rs8065080, rs222747, and rs222749 as risk factors for migraine chronification. However, further research is needed to investigate the gene-gene and gene-environment interactions of TRPV1 SNPs on the risk of transformation from episodic to chronic migraine.

Dynamic of Puccinia striiformis f. sp. tritici urediniospore and its correlation with wheat stripe rust epidemics in Xiangyang of China

Stripe rust is one of the main diseases of wheat, which seriously threatens wheat production and food security all over the world. Xiangyang located in the Northwest of Hubei province in China is one of the main winter propagation and spring epidemic regions of Puccinia striiformis f. sp. tritici (Pst), which can provide urediniospores to the major wheat-growing regions in eastern and northeastern China. Understanding the dynamic of Pst urediniospore is important for giving prediction of wheat stripe rust epidemic for eastern and northeastern China and controlling the epidemic of wheat stripe rust. In this study, spore trapper and TaqMan real-time quantitative PCR (TaqMan-qPCR) detection system were employed to monitor Pst urediniospore from December 2018 to December 2022 in Xiangyang. Weather variables including air temperature, relative humidity, sunshine duration and rainfall were collected to clarify the relationship with urediniospore density in the air. In addition, the relationship between disease index of wheat stripe rust and urediniospore density in the air was analyzed. Results showed that Pst urediniospore could be captured in the air all year round. The order of the density of urediniospore from most to least was from April to June, October to December, January to March, and July to September except 2022. The urediniospore density reached the peak when the air temperature was 10–22 °C and the relative humidity was 70%∼85% from April to May in 2019, 2020 and 2021. The density of Pst urediniospores from February to April was linearly related to the total precipitation of 25 days prior to the final day of a 7-day trapping period. There was a significant positive correlation between the disease index of wheat stripe rust and the cumulative urediniospore density 2–4 weeks before the investigation date of wheat stripe rust from March to May (P < 0.05). There was no significant correlation between the disease index and the cumulative urediniospore density from 1–4 weeks after the investigation date of stripe rust from March to May (P > 0.05). This study laid a foundation for the establishment of wheat stripe rust prediction model based on urediniospore density and meteorological factors.

Load of the ash dieback pathogen hymenoscyphus fraxineus differs in soil

AbstractThe ascomycete Hymenoscyphus fraxineus causes the devastating ash dieback disease of European ash (Fraxinus excelsior L.). Spore traps are often used to measure the amount of ascospores in the environment, but the pathogen-load of the soil in ash stands has not been recorded so far. This is of particular interest with regard to the occurrence of ash stem necrosis, a decisive factor for the severe course of the disease. In order to gain a more differentiated insight into the pathogen-load in ash stands, we analysed soil samples from four ash tree sites in southern Germany, covering a clone plantation, two seed orchards and a forest. The pathogen-load was determined using a quantitative TaqMan real-time PCR assay for ten to twenty plots per stand. Results obtained by the species-specific assay highlighted that the pathogen-load is heterogeneously distributed in the ash stands. H. fraxineus DNA targets were detected in 17% of the soil samples. The pathogen-load differed according to soil depth, with the highest pathogen abundance in the top 5 cm, followed by 5–10 cm and finally 10–15 cm. Pathogen-load and thereby infection pressure were found to be highly variable for the individual trees in one stand. Overall, the study discovered detectable levels of H. fraxineus in the soil of all four study sites, which supports the hypothesis that H. fraxineus can be found in the soil of ash stands. The qPCR approach was found to be an effective method for monitoring the load of H. fraxineus in soil and for demonstrating the successful application of the method on the sample type of custom-made spore traps. Results suggest the implication of site-specific pathogen-load determination in future H. fraxineus-monitoring and selection of less susceptible ash trees for breeding and seed production.

Development of TaqMan-based real-time PCR based on ψ gene for quantitative detection of CAR-T cells

Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = −3.3841x + 41.402 (R2 = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/μL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.

P-266 Profiling redox effects induced by micro-nanoplastics in the female reproductive system: insights from human granulosa cells

Abstract Study question Are nano-microplastics (NMPs) taken up into human granulosa cells causing redox alterations? Summary answer NMPs enter into human granulosa cells and influence viability, energy production and antioxidant response. What is known already In recent decades, anthropogenic activities have increased the production of global plastic with millions of tons produced every year. Under the action of different physical, chemical or biological agents, plastic waste breaks down into (NMP) particles that propagate in the environment posing threats for human health. NMPs translocate from digestive tract to circulatory systems reaching organs and cells, including female gonads. Nevertheless, there is insufficient knowledge of their effects on reproductive functions in mammals to allow for an accurate risk assessment to be conducted and any risks managed in animal and human being contexts. Study design, size, duration Human ovarian granulosa cells (KGN cell line) were exposed to NMPs of different sizes (40 nm, 70 nm, 100 nm, and 200 nm) at concentrations ranging from 5 to 1000 μg/ml for 24 h and then processed for cellular and biochemical analyses Participants/materials, setting, methods NMPs uptake by granulosa cells was tested by the employment of fluorescent NMPs and observation under confocal laser scanning microscopy. Cell viability was assed by Cell Counting Kit8. The effects of NMPs on mitochondrial bioenergetics was evaluated by MitoStress kit (Seahorse Xfe96, Agilent). ATP production was evaluated by Cell Titer-Glo ATP assay kit (Promega). Key enzymes of antioxidant response were analysed at transcript and protein level by using real-time Taqman PCR and Western blotting, respectively. Main results and the role of chance We demonstrated that NMPs are taken up by granulosa cells at all tested concentrions along with significant decrease of cell vitality with all sizes and concentrations. Seahorse analysis revealed an altered bioenergetic profiles. ATP levels increase at 70, 100 and 200 nm (one way ANOVA p = 0.001) at all tested concentrations (one way ANOVA p &amp;lt; 0.001). The gene and protein expression of catalase (CAT) superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) increased at 5 and 100 µg/ml with all sizes except 100 nm, whereas the level of sirtuins (SIRT1 and SIRT3) transcripts and protein decreased. Reduced levels of phosphorylated NRF2, the transcription factor that activates antioxidant responsive elements (ARE)-mediated gene expression were observed. Limitations, reasons for caution The use of human cell lines requires careful consideration when transferring to human testing. Wider implications of the findings Our results contribute to understanding the effects of NMPs on mammalian fertility in order to find possible protective approaches. Considering that the level of susceptibility of mammalian female germ cells to NMPs is still unknown, present results contribute to evidence-based strategies allowing living and working in a health promoting environment. Trial registration number Effects of combined exposure to nano/microplastics and plastic additives on mammalian female fertility (CUP E53D2301100006)

USE OF TAQMAN ARRAY TO INVESTIGATE INFECTION IN DIABETIC FOOT WOUNDS

IntroductionDiabetic foot disease is a major public health problem with an annual NHS expenditure in excess of £1 billion. Infection increases risk of major amputation fivefold. Due to the polymicrobial nature of diabetic foot infections, it is often difficult to isolate the correct organism with conventional culture techniques, to deliver appropriate narrow spectrum antibiotics. Rapid DNA-based technology using multi-channel arrays presents a quicker alternative and has previously been used effectively in intensive care and respiratory medicine.MethodsWe gained institutional and Local Ethics Committee approval for a prospective cohort study of patients with clinically infected diabetic foot wounds. They all had deep tissue samples taken in clinic processed with conventional culture and real-time PCR TaqMan array.Results50 samples were taken from 39 patients between October 2020 and March 2022. 84% of patient were male, 88% had type 2 diabetes. The ulcers were of variable chronicity prior to sampling (range 1–113 weeks) and mean HbA1c was 67.2mmol/mol. Ulcers were on the heel (3), midfoot (6) and forefoot (41). Minimum follow up was 3 months. 6 ulcers healed, 24 patients were admitted due to foot disease, there were 2 major amputations and 4 deaths. TaqMan array results were available a mean of 4.3 days earlier than culture results. 9 patients had negative conventional cultures and 8 were negative onarray testing. 17 patients had the same organisms detected on culture and array. 16 of these 17 had additional organisms detected by array. The most frequent organisms detected on array that were not detected by culture were Staphylococcus spp., Enterobacter, Pseudomonas and fungi.ConclusionTaqMan array shows promise in detecting infecting organisms from diabetic foot wounds and providing earlier results than standard culture, which may enable appropriate and timely antibiotic therapy.

Impact of IL-6 and IL-6r variants on HIV-1 susceptibility and progression to AIDS: a case-control study in a Moroccan population

Interleukin-6 (IL-6), a pro-inflammatory cytokine, is an important regulator of the inflammatory immune response. We aimed to assess the association of common single nucleotide polymorphisms (SNPs) in IL-6 (rs1800795 G > C, rs1800797 A > G) and interleukin-6 receptor (IL-6R) (rs2228145 A > C) genes with HIV-1 infection, AIDS progression, and response to treatment. In this case-control study involving 199 individuals living with HIV-1 and 200 HIV-uninfected controls, we conducted genotyping of IL-6/IL-6R SNPs using TaqMan real-time PCR assays. Soluble IL-6 levels were measured using ELISA. No associations were found between the investigated SNPs and HIV infection. However, a significant association was noted between the C-G and G-A haplotypes and susceptibility to HIV-1 infection. Additionally, a significant association was revealed between HIV-1 RNA viral loads and IL-6 SNP G > C in the post-treatment HIV group. Interestingly, we observed a significant association between the investigated SNPs and protection against progression to AIDS, namely the IL-6 G > A SNP in its recessive model and the IL-6R A > C SNP in its codominant and dominant models. Nevertheless, we found no significant differences between IL-6 levels and the different genotypes and alleles of the IL-6 gene either before or after combination antiretroviral therapy. IL-6 promoter haplotypes are associated with susceptibility to HIV-1 infection. Furthermore, IL-6 A > G and IL-6R A > C polymorphisms have been associated with protection against AIDS progression. Interestingly, the IL-6 G > C SNP may affect the response to treatment in people living with HIV-1.