TaqMan PCR Research Articles

Next-generation molecular diagnostics (TaqMan qPCR and ddPCR) for monitoring insecticide resistance in Bemisia tabaci.

Insecticide resistance has developed in several populations of the whitefly Bemisia tabaci worldwide and threatens to compromise the efficacy of chemical control. The molecular mechanisms underpinning resistance have been characterized and markers associated with the trait have been identified, allowing the development of diagnostics for individual insects. TaqMan and Droplet Digital PCR (ddPCR) assays were developed and validated, in individual and pooled whitefly samples, respectively, for the following target-site mutations: the acetylcholinesterase (ace1) F331W mutation conferring organophosphate-resistance; the voltage-gated sodium channel (vgsc) mutations L925I and T929V conferring pyrethroid-resistance; and the acetyl-CoA carboxylase (acc) A2083V mutation conferring ketoenol-resistance. The ddPCR's limit of detection (LoD) was <0.2% (i.e. detection of one heterozygote whitefly in a pool of 249 wild-type individuals). The assays were applied in 11 B.tabaci field populations from four locations in Crete, Greece. The F331W mutation was detected to be fixed or close to fixation in eight of 11 B.tabaci populations, and at lower frequency in the remaining ones. The pyrethroid-resistance mutations were detected at very high frequencies. The A2083V spiromesifen resistance mutation was detected in eight of 11 populations (frequencies=6.16-89.56%). Spiromesifen phenotypic resistance monitoring showed that the populations tested had variable levels of resistance, ranging from full susceptibility to high resistance. A strong spiromesifen-resistance phenotype-genotype (A2083V) correlation (rs =-0.839, P=0.002) was observed confirming the ddPCR diagnostic value. The ddPCR diagnostics developed in this study are a valuable tool to support evidence-based rational use of insecticides and resistance management strategies. © 2022 Society of Chemical Industry.

Synthesis and characterization of N-rich fluorescent bio-dots as a reporter in the design of dual-labeled FRET probe for TaqMan PCR: A feasibility study.

DNA-based analytical techniques have provided an advantageous sensing assay in the realm of biotechnology. Bio-inspired fluorescent nanodots are a novel type of biological staining agents with excellent optical properties widely used for cellular imaging and diagnostics. In the present research, we successfully synthesized bio-dots with excellent optical properties and high-quantum yield from DNA sodium salt through the hydrothermal method. We conjugated the bio-dots with 3' Eclipse Dark Quencher (Eclipse)-labeled single-strand oligodeoxyribonucleotide according to carbodiimide chemistry, to design a fluorescence resonance energy transfer (FRET) probe. The results confirmed the prosperous synthesis and surface functionalization of the bio-dot. Analysis of size, zeta potential, and FTIR spectroscopy verified successful bioconjugation of the bio-dots with probes. UV-visibility analysis and fluorescence intensity profile of the bio-dot and bio-dot@probes represented a concentration-dependent quenching of fluorescent signal of bio-dot by Eclipse after probe conjugation. The results demonstrated that TaqMan PCR was not feasible using the designed bio-dot@probes. Our results indicated that bio-dot can be used as an efficient fluorescent tag in the design of fluorescently labeled oligonucleotides with high biocompatibility and optical features.

Feasibility of quantification based on novel evaluation with stool DNA and fecal immunochemical test for colorectal cancer detection

BackgroundStool DNA (sDNA) tests and fecal immunochemical test (FIT) are used for the detection of colorectal cancer (CRC). Here we performed a novel evaluation using sDNA and FIT to assess their performance in CRC screening and monitoring in Hubei, China.MethodsStool samples were collected from a high-risk population in Hubei, China (n = 359). sDNA tests and FIT were performed to test for KRAS mutations, NDRG4 and BMP3 methylation, and check hemoglobin levels. The methylation in BMP3 and NDRG4 genes was detected by TaqMan PCR method from human fecal samples. KRAS gene mutation in human fecal DNA was tested using TaqMan probe and amplification-refractory mutation system method. The colloid gold method was used for detection of hemoglobin in fecal samples. Finally, a novel evaluation by software was used to calculate the comprehensive value of the combined results for CRC detection and monitoring.ResultsThe sensitivity and specificity of the novel evaluation for early CRC (stage I and II), advanced adenoma (AA), and non-colon cancer neoplasm (NA) detection were 95.45% and 81.6%, 29.63% and 75.9%, and 23.08% and 75.17%, respectively. The receiver operating characteristic (ROC) curves of the combined value for the above diseases were 0.945 ± 0.015, 0.543 ± 0.055, and 0.547 ± 0.038, respectively. The levels of the novel evaluation were not significantly associated with the pathology and stage (P > 0.05). In 20 out of 22 CRC patients, the novel evaluation of sDNA and FIT had decreased below threshold (< 165) at after surgery.DiscussionThe novel evaluation with sDNA test and FIT has increased sensitivity for screening of CRC and AA. The novel evaluation may have potential importance as an indicator of early CRC. Additionally, the dynamic changes of the comprehensive value after surgery were correlated with CRC treatment.

Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy.

Aberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the blood cell surface, we studied the concentration, representation, and fragmentation of tumor-derived methylated DNA in cell-free and cell-surface-associated DNA. We found that long DNA fragments (more than 10 kb) are mainly associated with the surface of blood cells. However, in plasma short DNA fragments (100–1000 bp) were also found along with long DNA fragments. Isolation of short fragments after separation of cirDNA in 6% PAGE followed by quantitative PCR (L1 element) has shown that short DNA fragments in healthy females represent 22% versus 0.5–4.4% in breast cancer patients. The methylated form of the RARβ2 gene was detected only in long DNA fragments by Real-time TaqMan PCR of bisulfite-converted DNA. The methylation index of cirDNA from healthy women was estimated at 0%, 9%, and 7% in plasma, PBS-EDTA, and trypsin eluates from the surface of blood cells, respectively. The methylation index of breast cancer patients’ DNA was found to be 33%, 15%, and 61% in the same fractions confirming the overrepresentation of methylated DNA in csbDNA.

MicroRNA expression profiling of endocrine sensitive and resistant breast cancer cell lines

BackgroundMicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion. MethodsMicroRNA extracted from MDA-MB-231 (de novo ER-) and ER-silenced (acquired ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR. ResultsEach cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50–60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes. ConclusionsThese data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.

Simultaneous presence of Mycoplasma salivarium and Tannerella forsythia in the implant sulcus after lateral augmentation with autogenous root grafts is associated with increased sulcus probing depth.

To characterize a potential pathogenic role of Mycoplasma salivarium and bacterial co-detection patterns on different implant augmentation types. 36 patients were non-randomly assigned to autogenous lateral alveolar ridge augmentation with either cortical autogenous bone blocks, or healthy autogenous tooth roots or non-preservable teeth. Mucosal inflammation was assessed by probing pocket depth (PD) at all sampling sites and by bleeding on probing (BOP) in a subset of sampling sites, and standardized biofilm samples were obtained from the submucosal peri-implant sulcus and sulcus of a contralateral tooth at two times (t1 after implant placement; t2 after six months). Seven bacterial species were quantified using Taqman PCR. Mucosal inflammation did not differ between augmentation groups, but peri-implant sulci showed increased abundance of M. salivarium after augmentation with autogenous tooth roots lasting for at least six months (t1 p = 0.05, t2 p = 0.011). In M. salivarium-positive samples, Tannerella forsythia was correlated with PD (R = 0.25, p = 0.035) This correlation was not observed in M. salivarium-negative samples. Compared to all other samples, PD was deeper in co-detection (i.e., simultaneous M. salivarium and T. forsythia) positive samples (p = 0.022). No association of single or co-detection of bacteria with BOP was observed. Presence of M. salivarium in peri-implant sulci varies with augmentation method and is associated with increased PD but not BOP. A potential causal role of M. salivarium in inflammation through a mechanism involving co-presence of T. forsythia requires further study.

Pharmacogenetics of Metformin Transporters Suggests No Association with Therapeutic Inefficacy among Diabetes Type 2 Mexican Patients.

Mexico has been under official epidemiological alert due to diabetes since 2016. This study presents new information on the frequency and variants of metformin transporters OCT1, OCT2, OCT3, ABCB1, and CYP2C9 variants as well. It also reports the association with HbA1c control on 103 DMT2 patients. They were genotyped through real-time PCR (TaqMan assays) and grouped according to treatment: metformin and metformin + glibenclamide. Metformin plasmatic levels were determined through mass spectrometry. The analysis of HbA1c showed statistical significance across genotypes in polymorphisms rs72552763 (p = 0.022), rs622342 (p = 0.009), rs1128503 (p = 0.021), and rs2032582 (p = 0.009) within the monotherapy group. Bivariate analysis found no association between any polymorphism and HbA1c control. Two logistic regression models accounted for two diplotypes in OCT1 and ABCB1, including statistically significant covariates. The first model yielded significance in age (p = 0.026), treatment period [p = 0.001], BMI ≥ 25 kg/m2 (p = 0.043), and combined therapy (p < 0.001). There was no association with GAT/GAT of rs72552763 or A/A rs622342 in OCT1. The second model yielded significance in age (p = 0.017), treatment period (p = 0.001), BMI ≥ 25 kg/m2 (p = 0.042), and combined therapy (p < 0.001), finding no association with C/C of rs1128503 or G/G of rs2032582 in ABCB1. Our multinomial logistic regression results may benefit future predictive analyses in diabetic populations.

Abstract 3127: Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod

Abstract Background: Taxanes are extensively used in the chemotherapy of breast cancer in the neoadjuvant, adjuvant and metastatic settings. Paclitaxel is known to have both immunostimulatory and immunosuppressive effects, but its impact on the chemokine system within the tumor microenvironment (TME) remains unclear. In the breast TME treated with neoadjuvant taxane chemotherapy, high levels of the cytotoxic T cell (CTL) attractants CXCL9, CXCL10 and their common receptor CXCR3 predict higher probability of pathological complete response (pCR) and improved long term outcomes. We previously developed a chemokine modulatory regimen (CKM) combining interferon-α with TLR3 agonist rintatolimod to selectively increase CTL-attracting chemokines in the TME but reduce the production of chemokines attracting myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) in the TME. Here we evaluated paclitaxel’s impact on chemokine production in the human breast TME and CKM’s ability to eliminate undesirable aspects of taxane chemotherapy. Methods: Fresh breast cancer tissues obtained during routine surgeries, breast cancer cell lines BT-549 and MDA-MD-231 and peripheral blood monocyte-derived macrophages were analyzed immediately or cultured ex vivo for 24 hours in the absence or presence of paclitaxel and/or CKM components. The expression of chemokine genes and secretion of chemokines by freshly harvested and ex vivo-treated tumor explants, cancer cell lines and macrophages were analyzed by quantitative PCR (Taqman) and ELISA. Paired student t-test was used for statistical analyses. Results: Breast cancer explants spontaneously expressed high levels of MDSC/Treg-attractants CXCL12 and CCL22, but only marginal levels of CTL and natural killer (NK) cell attractants CXCL9, CXCL10, CXCL11 and CCL5. Unexpectedly, paclitaxel treatment resulted in further elevation of granulocyte/MDSC-attractant CXCL8 and CCL22, which was reversed by the combination of paclitaxel with CKM. At the same time, while paclitaxel alone did not induce any of the CTL attractants tested, its combination with the CKM was highly effective in inducing CXCL9, CXCL10, CXCL11 and CCL5. Conclusions: Our results identify an undesirable aspect of paclitaxel’s impact on breast cancer. The ability of CKM to enhance the expression of CTL/NK cell attractants and suppress the production of Treg/MDSC attractants produced by paclitaxel provides a strong rationale for combined use of CKM in taxane-based chemo-immunotherapy of breast cancer and potentially other diseases. Citation Format: Shipra Gandhi, Melissa Grimm, Ronald Slomba, Kazuaki Takabe, Pawel Kalinski. Negative impact of paclitaxel on human breast tumor microenvironment and its reversal by the combination of interferon-α with TLR3 agonist rintatolimod [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3127.

Platelet-derived growth factor D expression in adrenal cells is modulated by corticosteroids: putative role in adrenal suppression

BackgroundAdrenal suppression is a clinically concerning side effect of inhaled corticosteroid (ICS) treatment in patients with asthma. Increased susceptibility to ICS-induced adrenal suppression has previously been identified in those with the rs591118 polymorphism in platelet-derived growth factor D (PDGFD). The mechanism underpinning this relationship is not known.MethodsH295R cells were genotyped for rs591118 using a validated Taqman PCR allelic discrimination assay. H295R cell viability was determined after treatment with beclometasone and fluticasone (range 0–330 μM). Cortisol was measured in cell culture medium using competitive enzyme immunoassay.ResultsPDGFD protein expression in H295R cells was confirmed using Western blotting. When ACTH and forskolin were added to H295R cells, a reduction in PDGFD expression was seen, which was then restored by incubation with prochloraz, a known inhibitor of steroidogenesis. A dose-dependent, decrease in PDGFD expression was observed with beclometasone (over a 24 h incubation period) but not with beclometasone incubations beyond 24 h nor with fluticasone (at 24 or 48 h).ConclusionsH295R cells express PDGFD protein, which can be modulated by incubation with steroidogenesis agonists and antagonists and additionally with exogenous beclometasone.ImpactPDGFD is expressed in the human adrenal cell line, H295R, and expression can be modulated by beclometasone as well as agonists/antagonists of steroidogenesis.This builds on previous research that identified a SNP in PDGFD (rs591118) as an independent risk factor for adrenal suppression in adults and children with obstructive airway disease treated with inhaled corticosteroids.First in vitro experiments to support a link between the PDGF and cortisol production pathways, supporting the hypothesis that PDGFD variants can affect an individual’s sensitivity to corticosteroid-induced adrenal suppression.

Plasma Torquetenovirus (TTV) microRNAs and severity of COVID-19

BackgroundTorquetenovirus (TTV), a widespread anellovirus recognized as the main component of the healthy human virome, displays viremia that is highly susceptible to variations in immune competence. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Among TTV-encoded miRNAs, miRNA t1a, t3b, and tth8 have been found in biological fluids. Here, the presence of TTV DNA and TTV miRNAs in the plasma of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected subjects was investigated to monitor the possible association with coronavirus disease 2019 (COVID-19) severity.MethodsDetection of TTV DNA and miRNA t1a, t3b, and tth8 was investigated in plasma samples of 56 SARS-CoV-2-infected subjects with a spectrum of different COVID-19 outcomes. TTV DNA and TTV miRNAs were assessed with a universal single step real-time TaqMan PCR assay and miRNA quantitative RT-PCR miRNA assay, respectively.ResultsThe TTV DNA prevalence was 59%, whereas at least one TTV miRNA was found in 94% of the patients tested. miRNA tth8 was detected in 91% of subjects, followed by miRNAs t3b (64%) and miRNAt1a (30%). Remarkably, although TTV DNA was unrelated to COVID-19 severity, miRNA tth8 was significantly associated with the degree of disease (adjusted incidence rate ratio (IRR) 2.04, 95% CI 1.14–3.63, for the subjects in the high severity group compared to those in the low severity group).ConclusionsOur findings encourage further investigation to understand the potential role of TTV miRNAs in the different outcomes of COVID-19 at early and late stages.

Association of the rs4646994 in ACE gene with susceptibility to tuberculosis in a region of the Brazilian Amazon

BackgroundTuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis and represents an important global public health issue. Single-nucleotide polymorphisms and INDELs are common genetic variations that can be located in genes associated with immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections like tuberculosis. This study aimed to investigate the association between the 17 genetic polymorphisms and susceptibility to tuberculosis in a Brazilian population.MethodsThis case-control study enrolled 283 individuals with active tuberculosis and 145 health care workers. Four INDELs and 13 single nucleotide polymorphisms and were genotyped using Multiplex PCR method and TaqMan SNP Genotyping Assays. Group comparisons for categorical variables were performed using the chi-squared test, whilst the t-Student test was used to analyze the continuous variables. Multiple logistic regression analyses were performed to estimate the odds ratio (OR) with 95% confidence intervals (CI). Deviation from Hardy-Weinberg equilibrium was assessed using chi-squared tests with Bonferroni correction. The results were analyzed comparing the genotypic distributions adopting the dominant model and the estimated values ​​of p corrected for multiple tests through FDR (False Discovery Rate) test.ResultsThe HWE test confirmed that the genotypic frequencies for polymorphisms were balanced. The frequency of Del allele was 73 and 75%, in cases and controls respectively. Frequency of Del allele was significantly higher in the control group than TB group. The homozygous Del/Del genotype was present in 51.6% of cases and 58.6% of controls. The rare Ins/Ins genotype was present in only 7.6% of controls and 6% of cases. The ACE Del/Del genotype was significantly higher in the cases than in controls revealing significant protection for TB in the domain model (OR = 0.465; p < 0.005).ConclusionsThe Del/Del genotype of the rs4646994 in ACE gene was associated with susceptibility to tuberculosis. The identification of genetic variants responsible for susceptibility to tuberculosis will allow the development of new diagnostic tools for tuberculosis infection. These studies will help improve control and the future eradication of this disease.

MO1008: The Role of IL-27/IL-27R in BKPYV Reactivation and Graft Rejection in Kidney Transplant Recipients

Abstract BACKGROUND AND AIMS Kidney transplantation is the only cure for patients with end-stage renal disorders. Therefore, protecting these patients from transplant rejection is a critical lifesaving project after transplantation. Graft rejection and BK polyoma virus (BKPyV) associated nephropathy (BKVAN) is one of the common causes of graft loss among kidney transplant recipients (KTRs) through activating innate and adaptive immunity. In between, the role of cytokines is very perilous in handling the immune responses against rejection, such as IL-27, and in this study, it is tried to investigate the role of IL-27 and its receptor (IL-27R; WSX-1) in KTRs. METHOD EDTA-treated blood samples were collected from 90 KTRs that were divided into three equal groups of BKV-inactive/non-reject (G1), BKV-active/non-reject (G2) and BKV-inactive/reject (G3), after being monitored by real-time PCR (Taq-Man) in plasma. A total of 30 normal individuals were considered as healthy control group (G0). A real-time PCR (SYBR Green) technique is used to determine the expression level of studied genes. RESULTS The results of gene expression comparisons showed that the expression level of IL-27 was significantly higher in G1 compared with the G2 group. Analysis also showed that the difference in the expression level of genes between the G3 and G0 groups was statistically significant. Also, the same comparison between the G1 and G0 groups showed a significant difference. The area under the ROC curve (AUC) showed that IL-27 is a significant discriminating molecule between all three studied groups of patients. The analyses revealed that the expression level of studied genes has a significant correlation with viral load. Finally, the ROC analysis showed that IL-27 and IL-27R seem to be important molecules for discrimination between G2 and G3 patient groups. CONCLUSION It is not easy to determine the IL-27 pro-inflammatory function as an endogenous regulatory mechanism, which might be one of the reasons for the immune system’s inability to inhibit the BKPyV reactivation in KTRs. Therefore, knowing the fact that in vivo loss of IL-27 signalling would not make drastic defects in immune development, turns IL-27 an attractive candidate for therapeutic research. It is important to consider the role of cytokines in different rejection conditions and the role of some cytokines should be more studied, such as IL-27 and IL-27R in course of kidney rejections, which happens especially between 2 weeks and 2 months after transplantation.

Prognostic Value of LINC-ROR (rs1942347) Variant in Patients with Colon Cancer Harboring BRAF Mutation: A Propensity Score-Matched Analysis.

Emerging studies show that long intergenic non-protein coding RNA, regulator of reprogramming (LINC-ROR) is aberrantly expressed in several types of cancer, including colon cancer (CC). LINC-ROR intronic variant rs1942347 may impact gene regulation and disease phenotype. We aimed to explore the potential association of LINC-ROR (rs1942347) with the clinicopathological features and outcome of CC cases. Archived FFPE (n = 180) CC samples were enrolled. Taq-Man allelic discrimination PCR was used for genotyping in propensity-matched cohorts with/without positive staining for mutant BRAF protein after eliminating confounders bias. The rs1942347*A allele variant was associated with high pathological grade, larger tumor size, distant metastasis, and mortality. Multiple logistic regression analysis adjusted by sex and BRAF mutation showed A/A genotype carriers to have 3 times more risk of early onset of cancer (OR = 3.13, 95%CI = 1.28–7.69, p = 0.034) than T/T genotype carriers. Overall analysis showed that rs1942347*A allele carriers had higher risk of mortality under heterozygote (OR = 2.13, 95%CI = 1.08–4.35, p = 0.003), homozygote (OR = 5.0, 95%CI = 1.69–14.29, p = 0.003), dominant (OR = 3.33, 95%CI = 1.20–9.09, p = 0.003), and recessive (OR = 2.63, 95%CI = 1.37–5.0, p = 0.011) models compared to T/T allele carriers. Stratified analysis by BRAF status revealed that the ancestor T/T allele conferred protection in BRAF mutant CC patients and was associated with a 73–93% reduced risk of mortality under heterozygote/homozygote comparison models. Using Kaplan–Meier curves, carriers of the A/A genotype had shorter survival times than T/T cohorts. The univariate Cox regression model revealed that the A/A genotype was associated with a 3.5 times greater mortality risk than the T/T genotype. However, after adjustment by multiple Cox regression analysis, the risk was insignificant. In conclusion, this is the first study identifying the potential association of the LINC-ROR (rs1942347) variant with CC prognosis.

EXAMINING AN ASSOCIATION BETWEEN FUT2 GENE AND HELICOBACTER PYLORI INFECTION RELATED TO CLINICAL MANIFESTATIONS

The aim of the study was to examine an association of the rs602662 FUT2 genetic locus with the status of H. pylori infection and development of related diseases (chronic gastritis, gastric ulcer and 12 duodenal ulcer).Methods: The study included 91 patients, divided into two groups - "case" and "control". Criteria for the “case” group enrollment: diagnosis of gastric or duodenal ulcer, chronic non-atrophic gastritis; positive test for H. pylori.The “control” group included patients with episodic complaints of dyspepsia while undergoing a comprehensive examination, with negative test for H. pylori, as well as having no history of former therapy on H. pylori elimination.The study for the presence of the polymorphic locus rs602662 of the FUT2 gene was carried out by the standard TaqMan PCR method on a Real-Time CFX96 Touch amplifier. The follow-up period was 6 months.Results: The main group included 50 patients aged 21 to 50 years, the control group – 41 patients. Patients infected with H. pylori more often noticed symptoms of dyspepsia - in 36%, compared with the control group - 9.7%. A family history of associated diseases in the main group was significantly differed, χ2 = 4.97, p 0.05.To assess the contribution of the rs602662 locus genotype in FUT2 gene to the risk of clinically manifested H. pylori infection, the main group was divided into subgroups. In the distribution of alleles in these groups, significant differences were revealed. Allele "A" has a protective effect regarding the onset of clinical symptoms of dyspepsia. The odds ratio (OR) with the carriage of allele "A" (genotypes A / A and G / A versus G / G) to have clinical symptoms with a positive H. pylori status was 0.175 (CI = [0.049-0.625] chi2 = 7.79 p = 0.0053 ).Conclusion:1. No association of alleles and genotypes of the rs602662 locus of the FUT2 gene with the status of H. pylori infection was revealed.2. Carriage of allele "A" have a significant association with the absence of clinical symptoms in patients with a positive status of H. pylori infection, OR 0.175 (CI = [0.049-0.625] chi2 = 7.79 p = 0.0053).

Development of a Real-time TaqMan PCR Assay for The Detection of Porcine Circovirus 4.

IntroductionPorcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims.Material and MethodsFifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus’ genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the ORF2 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established.ResultsThe results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R2) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%).ConclusionThis study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4.

Clinical significance of studying the genetic polymorphism of adhesion factors in Helicobacter pylori-associated diseases

The aim of the work was to study the clinical significance of the genetic polymorphism of the adhesion factor - the genetic locus rs602662 FUT2 in Helicobacter pylori-associated diseases. Methods: The study included 91 patients. The study for the presence of the polymorphic locus rs602662 of the FUT2 gene was carried out by the standard TaqMan PCR method on a “Real-Time CFX96 Touch” amplifier. The duration of the study was 6 months. Results: When assessing the contribution of the genotype of the rs602662 locus of the FUT2 gene as a risk factor for the occurrence of clinical manifestations in H. pylori infection, it was found that the A allele has a protective effect on the occurrence of clinical symptoms of dyspepsia. The odds ratio (OR) for allele “A” carriers (genotypes A/A and G/A versus G/G) to have clinical symptoms with a positive H. pylori status was 0.175 (CI=[0.049-0.625] chi2=7.79 p=0.0053). Conclusion. As a result of the study, it was revealed that the carriage of the “A” allele has a significant associative relationship with the absence of clinical symptoms in patients with H. pylori infection 0.175 (C.I.= [0.049-0.625] chi2=7.79 p=0.0053).

The prevalence and distribution of Anaplasma phagocytophilum genotypes in Ixodes ricinus nymphs collected from farm- and woodland sites in Ireland

Despite the economic importance of grass-based livestock production in Ireland and the fact that many veterinarians and farmers regard tickborne fever (TBF) as an increasingly important disease, especially in sheep, little is currently known about the prevalence and genetic diversity of its causative agent, Anaplasma phagocytophilum. In the present study, 1376 nymphal Ixodes ricinus ticks collected from woodland, farmland, bog and limestone pavement habitats were screened for A. phagocytophilum using TaqMan PCR. Positive samples were further analysed by conventional nested PCR targeting the 16S rRNA, msp4 and groEL loci.Overall 4.5% I. ricinus nymphs were found to be infected. The genetic heterogeneity was comparable to that reported elsewhere in Europe, with greater genetic diversity of 16S variants in ticks collected from farmland than from woodland. All isolates belonged to groEL ecotype I indicating that rodents and birds do not contribute to the epidemiology of tickborne fever in Ireland. In the 16S and groEL loci, a number of the Irish isolates matched European sequences from humans, horses and dogs. The 16S sequences that were identical to human isolates from Europe also matched Ap-ha, the most common human pathogenic strain in the USA. Three isolates also matched published sequences from horses in the msp4 locus. No isolate matched human, equine and canine sequences in all 3 loci. Our results represent the first molecular characterization of Irish A. phagocytophilum isolates.

The effect of BKV reactivation on cytokines behavior in kidney transplanted patients

BackgroundBK virus associated nephropathy (BKVAN) is one of the common causes of graft loss among kidney transplanted recipients (KTRs). The current treatment for BKV nephropathy is decreasing the immunosuppressive regimen in KTRs. Interleukin-27 (IL-27) is a multifunctional cytokine that might be the front-runner of an important pathway in this regard. Therefore, in current study it is tried to evaluate the changes in the expression level of IL-27 and some related molecules, resulting from BKV reactivation in KTR patients.MethodsEDTA-treated blood samples were collected from all participants. Patients were divided into two groups, 31 kidney transplant recipients with active and 32 inactive BKV infection, after being monitored by Real time PCR (Taq-Man) in plasma. Total of 30 normal individuals were considered as healthy control group. Real time PCR (SYBR Green) technique is used to determine the expression level of studied genes.ResultsThe results of gene expression comparisons showed that the expression level of IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 genes was significantly higher in inactive group in comparison to active group. The expression level of TLR4 was lower in both active and inactive groups in comparison to control group. ROC curve analysis showed that IL-27 and IRF7 are significantly different amongst other studied genes. Finally, the analyses revealed that the expression level of most of the studied genes (except for TNF-α and TLR4) have significant correlation with viral load.ConclusionsOur findings revealed that IL-27, IFN-γ, TNF-α, TNFR2 and IRF7 expression level is higher in inactive group and TLR4 expression level is lower in patients’ groups in comparison to control group. Also, ROC curve analysis showed IL-27 and IRF7 can significantly differentiate studied groups (BKV active vs. inactive). Therefore, these results might help elucidating the pattern in charge of BKV reactivation in kidney transplanted patients.

Association of a genetic variant in Interleukin-10 gene with increased risk and inflammation associated with cervical cancer.

Cervical-cancer is among the most commonly diagnosed cancers in women, and infection with human papillomavirus (HPV) is associated with an increased risk of cervical cancer and altered serum concentrations of inflammatory cytokines. We have explored the association between a genetic variation in the Interleukin-10 (IL-10) gene (rs1800896) and cervical cancer risk and its relationship with tissue Interferon gamma (IFN-γ), Transforming growth factor beta (TGF-β), Tumor necrosis factor alpha (TNF-α) concentrations in women with cervical cancer. A total of 315 women with, or without cervical cancer, were recruited into the study. DNA was extracted from cervical cells, and genotyping was undertaken using Taq-man real-time PCR. The genotype frequency and allele distribution were analyzed together with their association with pathological data. The association of the rs1800896 gene variation with tissue levels of the inflammatory cytokines was also investigated. Our data showed a significant association between the A allele of the rs1800896 gene variant and the presence of cervical cancer. In particular, patients with AG/AA genotypes had an increased risk of cervical cancer with an odds ratio of 1.929 (95% confidence interval [CI]: 0.879-4.23, P<0.001) in a recessive model, compared with the GG genotype. Also, the tissue concentrations of IFN-γ, TGF-β, and TNF-α in cervical tissues were significantly higher in women with cervical cancer (P<0.001) and were associated with the AA genotype. We have found an association between the polymorphism rs1800896 in the IL-10 gene and an increased risk of cervical cancer as well as a higher level of tissue inflammatory cytokines. Further investigations are necessary on the value of emerging biomarkers for the risk stratification for the management of cervical cancer patients.

Performance characteristics of two real-time TaqMan polymerase chain reaction assays for the detection of WSSV in clinically diseased and apparently healthy prawns.

This study aimed to generate data on performance characteristics for 2 real-time TaqMan PCR assays (CSIRO and WOAH WSSV qPCRs) for the purposes of (1) detection of white spot syndrome virus (WSSV) in clinically diseased prawns and (2) detection of WSSV in apparently healthy prawns. Analytical sensitivity of both assays was 2 to 20 genome copies per reaction, and analytical specificity was 100% after testing nucleic acid from 9 heterologous prawn pathogens and 4 prawn species. Results obtained after testing more than 20 000 samples in up to 559 runs with the CSIRO WSSV qPCR and up to 293 runs with the WOAH WSSV qPCR demonstrated satisfactory repeatability for both assays. Both assays demonstrated median diagnostic sensitivity (DSe) 100% (95% CI: 94.9-100%) when testing clinically diseased prawns. When 1591 test results from apparently healthy prawns were analysed by Bayesian latent class analysis, median DSe and diagnostic specificity (DSp) were 82.9% (95% probability interval [PI]: 75.0-90.2%) and 99.7% (95% PI: 98.6-99.99%) for the CSIRO WSSV qPCR and 76.8% (95% PI: 68.9-84.9%) and 99.7% (95% PI: 98.7-99.99%) for the WOAH WSSV qPCR. When both assays were interpreted in parallel, median DSe increased to 98.3 (95% PI: 91.6-99.99%), and median DSp decreased slightly to 99.4% (95% PI: 97.9-99.99%). Routine testing of quantified positive controls by laboratories in the Australian laboratory network demonstrated satisfactory reproducibility of the CSIRO WSSV qPCR assay. Both assays demonstrated comparable performance characteristics, and the results contribute to the validation data required in the WOAH validation pathway for the purposes of detection of WSSV in clinically diseased and apparently healthy prawns.