Real-time PCR Detection System Research Articles

PSIII-11 Impacts of late gestational in utero heat stress on small intestine dynamics of neonatal dairy calves

Abstract Late gestation heat stress in dairy cows has been shown to negatively affect offspring growth and development. In utero heat stressed calves have altered small intestine morphology and an impaired ability to absorb colostral immunoglobulin G (IgG). Thus, the objective of this study was to characterize potential cellular and molecular alterations in the small intestine of neonatal in utero heat stressed calves. In the last ~56 d of gestation pregnant dams were either heat stressed (shade; n = 41) or cooled (shade, fans, soakers; n = 41) resulting in heifers that were in utero heat stressed (IUHS, n = 15) or in utero cooled (IUCL, n = 15). Heifers were fed 3.78 L of high-quality colostrum within 2 h of birth. Apparent efficiency of absorption (AEA) of IgG was calculated by measuring IgG content fed in colostrum and in 24 h serum. Within 4.6 ± 2.3 h of birth a subset of calves (n = 8/treatment) not fed colostrum were euthanized to collect samples from the ileum. Tissue for histological analysis was placed in 10% neutral buffered formalin before being dehydrated, embedded in paraffin, cut into 5 μm thicknesses, and mounted to glass slides. The TUNEL method was used to identify apoptotic cells using the Keyence BZ-X800 Microscope. Tissue for gene expression analysis was snap-frozen and kept at -80°C before RNA extraction and cDNA synthesis. The transcript abundance of endosomal (FCGRT, DAB2, MAMDC4), tight junction (ZO1, ZO2, ZO3, OCLN, CLDN1), and heat shock (HSP90, HSF1) related genes were measured by qPCR using the CFX96 Real-Time PCR detection System (BIO-RAD). Data were analyzed using PROC MIXED or PROC GLIMMIX depending on the response variable. TUNEL results are expressed as the proportion of positive cells to total cells. Quantitative PCR data were analyzed by assessing the estimates of the model relative to the geometric mean of 3 reference genes (ACTB, GADPH, RSP9; ΔΔCT). The concentration of colostrum IgG fed to calves was not different (136.1 vs. 122.5 g/L; IUHS vs. IUCL, respectively; P = 0.32), but AEA of IgG was reduced in IUHS relative to IUCL calves (23.5 vs. 33.3%; P < 0.01). The proportion of apoptotic cells within the villi of the ileum was increased in IUHS calves compared with IUCL (2.11 vs. 1.57%; P < 0.01). Ileum gene transcript abundance of DAB2 and MAMDC4 were increased (P < 0.01), while FCGRT transcripts tended to increase in IUHS calves (P = 0.08). All tight junction and heat shock-related gene transcripts were increased within the ileum of IUHS calves (P ≤ 0.02) with the exceptions of CLDN1 and HSP90, which were not different between treatments (P ≥ 0.22).These results suggest that exposure to heat stress in utero impact small intestine integrity after birth and affect key IgG transporters, providing insight into underlying mechanisms responsible for reduced IgG absorption in IUHS calves.

Development of a multiplex real-time PCR assay for the simultaneous detection of mpox virus and orthopoxvirus infections

Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.

Study of head and neck squamous cell carcinoma transcriptome after proton therapy

Aim. To evaluate changes in the transcriptome of head and neck squamous cell carcinoma (HNSCC) tissue cells in patients after proton therapy.Materials and methods. Biopsy material obtained from 3 HNSCC patients before and after proton therapy at a total dose of 10 isoGy was homogenized, purified, and concentrated. Then total RNA was isolated with further purification and concentration with the RNA Clean & Concentrator kit (Zymo Research). Library quantitation was assessed using the Qubit 2.0 instrument (Invitrogen, Life Technologies). After isolation of 1 μg total RNA for sequencing, libraries were prepared on the Illumina platform using the TruSeq RNA Sample Prep Kit v2 with a 10-cycle enrichment step according to the manufacturer’s recommendations. The quality of RNA and the resulting libraries was checked using the Agilent 2100 Bioanalyzer system (Agilent Tec. Inc., USA). The RIN parameter for RNA was at least 7. The library concentration was assessed by real-time PCR on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). Final libraries were pooled in equimolar ratios before sequencing on the Illumina HiSeq 2500 platform using 50 base-pair paired-end reads. The Q20 parameter for all samples was > 97%, and the number of reads averaged 60.2 million per sample. Raw reads were processed using the RTA 1.17.21.3 and Casava 1.8.2 (Illumina). The enrichment analysis was performed using the PANTHER 17.0 software.Results. The transcriptome analysis of HNSCC after proton radiation therapy (5 x 2 isoGy) at a total dose of 10 isoGy revealed 1,414 significantly differentially expressed genes. The 10 most and least expressed genes and their associated signaling pathways were identified. A number of signaling pathways associated with the underexpressed genes were detected in HNSCC after proton therapy, such as: STAT5; PD-1 signaling pathway; marked MET-mediated activation of PTK2 signaling pathway, PDGF signaling; CD22-mediated regulation of BCR; and FCERI-mediated MAPK activation. In addition to the above signaling pathways, activation of collagen degradation, FCGR3A-mediated phagocytosis, and FCGR3A-mediated interleukin (IL)-10 synthesis are of interest. In the enrichment analysis among highly expressed genes, keratinization and biological oxidation processes were activated in HNSCC tissues after proton therapy.Conclusion. Proton therapy in HNSCC leads to overexpression of genes involved in the regulation of keratinization and biological oxidation processes as well as to underexpression of genes associated with suppression of signaling pathways: STAT5, PD-1, MET-mediated activation of PTK2 signaling pathway, PDGF signaling; CD22-mediated regulation of BCR; FCERI-mediated MAPK activation, collagen degradation, FCGR3A-mediated phagocytosis activation, and FCGR3A-mediated IL-10 synthesis. All signaling pathways of underexpressed genes function in HNSCC cells if there is no negative influence on the tumor from outside (irradiation or delivery of antitumor drugs). The predominance of suppressed signaling pathways over activated ones most likely indicates a decrease in the functional potential of cells after proton therapy. The dose-dependence of PT effects necessitates further study of changes in cellular and molecular-genetic signatures of HNSCC after proton irradiation with different doses.

Development of an ARMS multiplex real-time PCR assay for the detection of HLA-B*13:01 genotype by detecting highly specific SNPs.

HLA-B*13:01 was strongly associated with Dapsone Hypersensitivity Syndrome (DHS). This study aimed to develop and validate a rapid and economical method for HLA-B*13:01 genotyping. Two tubes multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*13:01 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. The accuracy of the assay was 100% for HLA-B*13:01 genotyping. The detection limit of the new method was 0.025 ng DNA. The positive rate of HLA-B*13:01 in the Bouyei (20%, n = 50) populations was significantly higher than that in the Uighur population (4%, n = 100), Han (4.5%, n = 200), and Tibetan (1%, n = 100) ( P < 0.05). The proposed method is rapid and reliable for HLA-B*13:01 screening in a clinical setting.

Inhibition of Citrus Huanglongbing Disease by Paenibacillus polymyx KN-03 and Analysis with Transcriptome and Microflora

Soil drench treatment using Paenibacillus polymyxa strain KN-03 was applied to citrus plants infected with Candidatus Liberibacter asiaticus (CLas). The infection status was assessed using PCR and a real-time quantitative PCR detection system (qPCR). The application of KN-03 resulted in a notable reduction in CLas levels in citrus plants. Specifically, by the 257th day post treatment commencement, following 24 KN-03 applications, the negative rates of CLas in the vein, root tip, and shoot tip were 50%, 0%, and 50%, respectively. After 24 cycles, KN-03 application significantly enhanced plant growth and stimulated reactive oxygen production in citrus leaves compared to control plants. Transcriptome analysis identified specific upregulated pathways. Furthermore, flora analysis revealed an increased abundance of microorganisms possessing potential utilization value, including Burkholderia-Caballeronia-Paraburkholderia, uncultured_bacterium_o_Acidobacteriales, uncultured_bacterium_f_Gemmatimonadaceae, and Rhodanobacter, in the root zone. Moreover, the BugBase analysis indicated that KN-03 treatment increased the abundance of beneficial rhizosphere bacteria associated with biofilm formation, element mobilization, and stress tolerance. These findings support the utility of Paenibacillus polymyxa KN-03 as an effective plant-growth-promoting bacterium for CLas management, with additional benefits for plant growth and soil health, specifically offering detoxification resources for shoot tip grafting.

TRPM8 Contributes to Early Temperature-Induced Platelet Activation

Background: Like sensory neurons, platelets are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Platelet activation by temperature could contribute to numerous clinical observations, most importantly to the well-known reduced lifespan of ex vivo-stored platelets for transfusion. In this study, we sought to dissect the molecular basis of the cold-induced platelet activation response and specifically, evaluate the role of the thermosensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in platelets and their precursor cells. Methods: We tested for the presence of TRPM8 on the megakaryocytic cell line MEG-01 and human platelets by immunoblotting, confocal microscopy, and imaging flow cytometry. The effect of TRPM8 on platelets was evaluated using the commercially available TRPM8 inhibitor PF-05105679. In addition, we used TRPM8 agonists such as menthol, WS-12, and icilin. Platelet shape change and αIIbβ3 integrin activation were assessed by imaging flow cytometry and conventional flow cytometry. We tested for platelet aggregation using light transmission aggregometry and for intracellular calcium by reading fluorescence on a plate reader after loading platelets with Fura-2-AM. Calcium release in response to temperature decrease was assessed after loading platelets or TRPM8-transfected HEK293T/17 and reading fluorescence in a Real-Time PCR Detection System. Results: We detected TRPM8 mRNA and protein in MEG-01 cells and platelets but there was a remarkable variability among donors. Further evidence for TRPM8's presence in platelets came from the fact that inhibitors of TRPM8 prevented early temperature-induced platelet activation events, such as αIIbβ3 integrin activation and shape change. Unlike temperature changes, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. Platelets exposed to below-normal body temperature displayed a cytosolic calcium increase, but this was independent of TRPM8 and entirely dependent on the calcium release from the endoplasmic reticulum. Conclusion: We found evidence for TRPM8 expression in platelets but the expression varies greatly between individuals and is of unclear physiologic significance. Our study suggests that the cold response of platelets is complex, and TRPM8 appears to play a calcium-independent role in the early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies.

Dynamic of Puccinia striiformis f. sp. tritici Urediniospore in Longnan, a Critical Oversummering Region of China.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici Erikss (Pst), is a devastating disease resulting in yield reduction. Because the temperature limits the overwintering and oversummering of Pst, and it cannot complete the whole year cycle in most areas of China. Longnan located in the southeast of Gansu province is one of the annual cycle areas of Pst, which can supply urediniospores in autumn to eastern wheat-growing areas in China. In this study, a TaqMan real-time quantitative PCR (TaqMan-qPCR) detection system for Pst urediniospores was established, and the detection limit was a single urediniospore. The dynamics of Pst urediniospores in Longnan were monitored by spore trapping and TaqMan-qPCR for 3 years. Meanwhile, the meteorological conditions including air temperature, relative humidity and precipitation were recorded. Results showed that Pst urediniospores can be captured from March to December, and two peaks of urediniospore number appeared in May and June, respectively. The number of urediniospore is closely related to temperature and precipitation from March to June. In addition, we found that the number of Pst urediniospore had the peak value when the average air temperature was 10~21℃ and the relative humidity was 60%~85% from May to June. The exponential model could describe the variation of Pst urediniospore number based on average temperature and precipitation from March to June. It is worth mentioning that the exponential model based on average temperature 7 days before spore capture has a great advantage in predicting the urediniospore density in the air. This study laid a foundation for establishment of a prediction model for wheat stripe rust based on the number of urediniospores and meteorological factors.

B-272 Interference Assays: How Different Substances can Affect the Detection of Respiratory Viruses Using a Quick Molecular Solution

Abstract Background Some substances present in clinical samples could affect the performance of qPCR and significantly impact its results. If nucleic acids are obtained using quick lysis reagents, the inhibition effect could be even worse since no washing steps are included. Thence, the aim of this study was to evaluate some potentially interfering substances that may be present in respiratory samples and their effect on the diagnosis using a quick molecular solution. Methods Ten negative nasopharyngeal swab samples in Viral Transport Medium (VTM, Vircell) were pooled and enriched with a known concentration of viral cultures from ATCC (Influenza A: VR-95PQ™ A/Puerto Rico/8/34; Human Respiratory Syncytial virus (RSV): VR26PQ™; and SARS-CoV-2: VR-1986HK™ SARS-Related Coronavirus 2, Isolate USA-WA1/2020) to obtain positive samples around the limit of detection. The pool was divided into seven aliquots. Six of them were spiked (each) with a known concentration of potentially respiratory interfering substances, according to FDA recommendations (Table 1). Another aliquot did not contain any substance and was considered as the positive control. Each aliquot was processed in triplicate using VIASURE Resp. viruses Quick Lysis Reagent (Certest Biotec S. L), following the manufacturer’s instructions. Nucleic acids were analysed with VIASURE SARS-CoV-2, Flu &amp; RSV Real Time PCR Detection Kit (Certest Biotec S.L) in triplicate on CFX96™ Real-Time PCR Detection System (Bio-Rad). Results Almost all substances did not interfere throughout the workflow at the concentrations tested. Only benzocaine had a negative effect on the three pathogens analysed (SARS-CoV-2, Influenza A and RSV), detecting less than 8/9 replicates. This problem was resolved when the substance was tested at 0.15 mg/ml. Conclusion Evaluation of potentially interfering substances in quick molecular solutions is important, especially if nucleic acids processing methods do not have many purification steps. Thus, the study confirmed that the diagnosis using this solution is not easily compromised.

B-269 Plasmodium Detection: Sera, Blood, Do They All Work?

Abstract Background When diagnosing malaria, testing employs blood samples using either microscopy, the current gold standard or molecular diagnostic tests (more sensitive but significantly more expensive). In Plasmodium spp. screening programs in at-risk populations where there are not many resources available, dried blood on filter paper is used to ease transport, storage and preservation of the sample. Although Plasmodium detection studies using blood serum as a sample are available, it isn´t always as sensitive as using whole blood-based sampling, as the concentration of DNA in plasma is proportional to the level of parasitaemia. This study aims to confirm the differences in sensitivity using both samples, demonstrating the importance of choosing the right type of sample to use for diagnosis. Methods The study was carried out using the VIASURE Malaria Real Time PCR Detection Kit with sera and blood samples from 94 inhabitants from Nueva Esperanza community in the Peruvian Amazon collected between 2007 and 2020, establishing a comparative analysis between matrixes. Nucleic acid extraction was performed using the QIAamp DNA Mini Kit from QIAGEN and the VIASURE assay was performed using QuantStudio®6 Flex Real-Time PCR System thermocycler (Thermo Fisher Scientific) and the BioRad CFX96TM Real-Time PCR Detection System for analysing peripheral blood samples and the BioRad CFX96TM Real-Time PCR Detection System for analysing sera. Results From the total of samples, only 5 sera (5.31%) showed amplification of Plasmodium spp., whereas from blood samples, 30 resulted to be Plasmodium spp-positive. Therefore, 26.59% of detection is lost when serum is used as a sample, as opposed to blood. Conclusion After this analysis it could be concluded that malaria detection sensitivity is appreciably higher for blood samples than for sera samples. Thus, serum samples aren´t the best choice for Plasmodium spp. testing and whole blood-based matrixes are preferable for this diagnosis. These results reinforce the idea of the importance of the sample type when diagnosing different pathogens, as well as the relevance of a confirming comparative analysis when similar sample types are involved.

B-271 Molecular EQA Programs Verify the Use of a Quick Lysis Reagent in a Wide Range of Matrices and Pathogens

Abstract Background External Quality Assessment (EQA) programmes, like Quality Control for Molecular Diagnostics (QCMD), allow laboratories to verify the correct functionality of the molecular workflows used in the diagnosis of different pathogens. The aim of this study was to evaluate whether a quick molecular workflow had a good functionality for the diagnosis of different pathogens. Methods A total of 90 frozen samples from 2022 QCMD programmes were analysed. 20 transport medium (TM), 9 TM or saline and 1 saline samples from respiratory panels (QAV164188, QAV164189, QAM174193), 20 synthetic faecal matrix samples from gastrointestinal panels (QAP124154, QAB124153), 13 TM, 4 simulated swabs and 3 urines from sexual transmitted infections (STI) panels (QAB154177, QAM174201), and 18 TM and 2 synthetic cerebrospinal fluid from central nervous system (CNS) panels (QAV174195, QAM174196). Samples were processed using an in-house processing method called quick lysis reagent (QLR) (Certest Biotec S. L.). In parallel, samples were also extracted using the automatic robot KingFisher (Thermo Fisher Scientific). Nucleic acids were analysed at least in duplicate with qPCRs from VIASURE using the CFX96™ Real-Time PCR Detection System (Bio-Rad). Results were compared to the final diagnosis provided by QCMD and with the automatic extractor results. Results The in-house processing method QLR, in combination with VIASURE qPCRs, could process and identify bacteria, parasites and viruses from respiratory, gastrointestinal, STI and CNS panels. Negative samples were correctly detected. This workflow obtained a 100% of sensitive and specificity, same as the automatic extractor. Conclusion These EQA programmes tested the correct functionality of a quick molecular workflow, based on the VIASURE diagnostic products, that could detect different pathogens (bacteria, viruses, and parasites) from different origins (respiratory, gastrointestinal, STI and CNS).

A-298 Performance Analysis of Three Mpox Real Time PCR Kits With Lesion Swab Clinical Samples

Abstract Background The appearance of skin lesions can be a symptom of infection or reactivation of the HSV-1, HSV-2, VZV or T. pallidum or even monkeypox virus infection. Due to the contact-bound ease of spread of these pathogens, an accurate and fast diagnosis, such as PCR-based testing, is key to a good epidemiological management. The aim of this study was to determine the clinical performance of three qPCR assays. Methods A total of 334 cutaneous swabs collected from patients attended at the Hospital Universitario Miguel Servet were analysed using the three assays under study: VIASURE Herpes virus 1, Herpes virus 2 &amp; Varicella Zoster Virus Real Time PCR Detection Kit, VIASURE Treponema pallidum Real Time PCR Detection Kit and VIASURE Mpox Real Time PCR Detection Kit. Samples were collected between December 2021 and November 2022. These samples were requested through the Biobanco del Sistema de Salud de Aragón (BSSA) and have the approval of the Aragón Ethics Committee (PI22/409). DNA/RNA extraction was performed using the automated extraction method magLEAD® 12gC instrument with the MagDEA Dx SV kit and amplification was performed using the thermocycler CFX96™ Real-Time PCR Detection System (BioRad). The obtained results were compared with the reference assays RealStar® Orthopoxvirus PCR Kit 1.0 (Altona) and Allplex™ Genital ulcer assay (Seegene®). Results After data analysis a very good overall agreement was obtained for all the studied targets. Conclusion The assays under study demonstrated to be a good tool for differential analysis in skin lesions compatible with HSV-1, HSV-2, VZV, T. pallidum or mpox virus infection. In addition, the shared thermal protocol of the assays allowed for the simultaneous detection in the sample, creating a personalized panel option for the user, as opposed to the reference assays employed, which required two separate analyses, taking longer to reach the full assessment of the sample.

A-300 Evaluation of Different Storage Conditions of Nasopharyngeal Swabs for the Detection of Influenza A, Influenza B and RSV

Abstract Background Every year, Influenza A (FluA), Influenza B (FluB) and Respiratory Syncytial Virus (RSV) affect our communities and increase the number of patients in our health systems. Molecular techniques, such as real-time PCR have become essential, not only for the diagnostic but also for the epidemiological studies. High amounts of samples or retrospective analysis need the use of samples stored at different conditions. For this reason, a stability study of nasopharyngeal swabs (NP) in Viral Transport Medium (VTM) for FluA, FluB and RSV was performed. Methods Twenty-eight negative FluA, FluB and RSV NP samples in VTM (Vircell, Spain) were used in this study. All samples were mixed into one pool and divided into 7 aliquots. One of them served as a negative control. The others were enriched (each) with different concentrations of viral cultures from ATCC (VR-95PQ™, VR1804PQ™, VR26PQ™) to obtain two positive samples for each pathogen. Samples were stored under specific conditions of time and temperature before being processed: 1 and 2 days stored at 25°C, 3 and 6 days stored at 4°C, and 8, 30 and 180 days stored at −20°C. Each condition was extracted in triplicate with the MagDEA Dx SV kit, using the magLEAD® 12gC instrument (Precision System Science Co.) and analysed with VIASURE SARS-CoV-2, Flu &amp; RSV Real Time PCR Detection Kit (Certest Biotec S.L) in triplicate on CFX96™ Real-Time PCR Detection System (Bio-Rad). The results were compared with the data obtained on the first day (0 h). Results All positive samples were detected in all conditions and matrices. No amplifications were observed in negative samples. There were no major differences compared with the data obtained on the first day. However, a Cq delay of around 1 unit was observed in all pathogens when samples were stored 2 days at 25°C. For this reason, this storage condition would not be recommended. Conclusion NP samples in VTM for the detection of FluA, FluB and RSV can be stored at 25°C for up to 1 day, 4°C for up to 6 days, or frozen at −20°C for up to 180 days.

B-267 Validation of a New Molecular Test to Quantify Plasma Cytomegalovirus Load

Abstract Background Cytomegalovirus (CMV) is a DNA virus that often causes severe disease in immunosuppressed or immunocompromised patients. With the expanding indications for immune-modulating agents and transplant patients, the number of patients at risk for developing CMV disease is increasing. Test results are used to determine active CMV disease, risk of developing active disease, response to therapy, risk of relapsed infection, and/or appropriate time to discontinue therapy. For this purpose, molecular assays are attractive because of their broad linear range and low limits of detection. CerTest Biotec has developed a quantitative real time PCR assay for the detection and quantification of cytomegalovirus in plasma samples. Methods Plasma-EDTA samples characterized as positive or negative and quantified by IU/ml were selected. The selected samples were collected from both male and female adults (except 1 sample of an infant) between July 2016 and December 2020. Two CE-marked molecular reference assays were used for the comparative analysis: RealStar® CMV PCR Kit 1.0 (Altona), using the CFX96™ Real-Time PCR Detection System (Bio-Rad) and extracted with the QIAamp® MinElute® Virus Spin Kit (QIAGEN); and cobas® CMV (Roche Diagnostics), using the cobas® 8800 system. VIASURE analysis was performed using the following workflow: the automated extraction method MagDEA® Dx SV with magLEAD® 12gC and CFX96™ Real-Time PCR Detection System (Bio-Rad). Results A total of 273 EDTA-plasma clinical samples were analysed, 120 samples were reported as CMV positive samples and quantified and 153 samples were reported as CMV negative samples. Comparative analysis determined &amp;gt;99% sensitivity and specificity. Regarding quantification, all methods obtained a correlation value of R2 ≥ 0.77. Conclusion With this study it was possible to observe that the VIASURE CMV q Real Time PCR Detection Kit present good clinical sensitivity and specificity compared to reference molecular assays results. No false positive results were observed. However, all variables employed on the workflow contribute to final quantification results and therefore should be taken into account as accumulative error in the calculation.

B-270 Validation of a New Molecular Assay for the Detection and Quantification of Hepatitis B Load in Plasma

Abstract Background Hepatitis B virus (HBV) is an enveloped DNA virus and is the most common worldwide cause of viral hepatitis in humans. It is primarily transmitted through blood or blood products. Although the diagnosis of hepatitis viral infections is usually performed with serological markers in blood, this technique has limitations and therefore the diagnosis of the disease by qPCR allows an improvement in the diagnosis of the infection, therapeutic decision making and evaluation of the response to treatment. Therefore, CerTest Biotec has developed a quantitative real-time PCR assay for the detection and quantification of hepatitis B virus in plasma samples. Methods Plasma-EDTA samples characterized as positive or negative and quantified in IU/ml were analyzed. The selected samples were collected from infants and adults of both sexes between September 2019 and August 2020. The origin of the population is diverse, with the majority being European. For comparative analysis, the cobas® HBV molecular assay (Roche Diagnostics) using the cobas® 8800 system was used. VIASURE analysis was performed using the following workflow: the MagDEA® Dx SV automated extraction method with magLEAD® 12gC and CFX96™ Opus Real-Time PCR Detection System (Bio-Rad). Results A total of 210 clinical EDTA plasma samples were tested, of which 110 were positive for hepatitis B virus and 100 were negative. The comparative analysis determined a sensitivity and specificity &amp;gt;99%. In addition, the positive predictive value (PPV) and negative predictive value were &amp;gt;99%. As for quantification, an R2 correlation value of 0.9095 was obtained. Conclusion This clinical evaluation demonstrated that the VIASURE HBV q Real Time PCR Detection Kit is a good tool for the clinical diagnosis of the virus as it has good sensitivity and specificity. No false positive or false negative results were observed. Moreover, the correlation of the quantification with the reference method gives a value of R2 &amp;gt; 0.9. Therefore, clinical diagnosis of hepatitis B virus by real-time PCR can encompass the limitations of the current diagnostic technique by serology.

DYSLIPIDEMIA AND HYPERGLYCEMIA IN HYPERTENSIVE PATIENTS ASSOCIATE WITH GNB3 (RS5443) AND NOS3 (RS2070744) GENES’ ALLELIC STATE

Objective: More than 700 million people worldwide live with untreated essential arterial hypertension (EAH). The objective of the study was to evaluate the risks of metabolic disorders in EAH patients depending on guanine nucleotide-binding protein-↓3 subunit (GNB3, rs5443) and endothelial nitric oxide synthase (NOS3, rs2070744) genes’ allelic state. Design and method: The cohort case-control study involved 100 EAH patients (moderate-very high cardiovascular risks, aged 45-65 years) and 48 practically healthy (control). Metabolic disorders were evaluated by glucose blood value and lipids panel: total cholesterol (TC), triglycerides (TG), Low-, and High- density lipoprotein cholesterol (LDL-C, HDL-C) levels. The atherogenic index (AI) was calculated by the equation: (TC-HDL-C)/HDL-C. GNB3 (rs5443) and NOS3 (rs2070744) genotyping perfrmed by TaqMan probes in CFX96 Real-Time PCR Detection System. Results: Hyperglycemia (&gt;6.1 mmol/l), hypertriglyceridemia (TG&gt;1.7 mmol/l) and decreased HDL-C (&lt;1.2 mmol/l) were relatively more common in EAH patients than in the control group by 36.11% (⇙2 = 17.88; p&lt;0.001), 23.61% (⇙2 = 6.43; p = 0.011) and 25.0% (⇙2 = 8.32; p = 0.004) respectively. Therefore, the fasting hyperglycemia increases the EAH risk in the examined population ninefold [OR95%CI:2.86-27.08; p&lt;0.001], hypertriglyceridemia and decreased HDL-C elevates the risk almost 3 and 3.5 times as well [OR95%CI:1.23-5.56; p = 0.009 and OR95%CI:1.46-8.71; p = 0.003], respectively. The risk of metabolic disorders (dyslipidemia and hyperglycemia) in EAH patients does not depend on NOS3 gene polymorphism (rs2070744). Contrary, in T-allele patients of the GNB3 gene (rs5443) prevailed subjects with elevated LDL-C (&gt;3.0 mmol/l) over those with CC-genotype by 13.89% (p = 0.05). Other parameters of lipid metabolism and hyperglycemia did not differ significantly between GNB3 (rs5443) genes allelic state. However, the mutational T-allele of the GNB3 gene (825C&gt;T) increases the risk of hyperlipidemia 8.5 times [OR 95%CI:0.99-72.70; p = 0.05] due to atherogenic LDL-C, with the protective role of CC-genotype [OR = 0.12; OR 95%CI:0.01-1.0; p = 0.048]. Conclusions: Fasting hyperglycemia, hypertriglyceridemia and lowered HDL-C, enhance the arterial hypertension risk 3-9 times (p&lt;0.01). The polymorphic site of GNB3 (rs5443) gene, but not NOS3 (rs2070744) gene associate with hyperlipidemia in hypertensive patients.

DNA methyltransferase expression (DNMT1, DNMT3a and DNMT3b) as a potential biomarker for anti-VEGF diabetic macular edema response.

DNA methylation is involved in Diabetic Retinopathy progression showing a metabolic memory mechanism. However, the association of DNA methyltransferase with diabetic macular edema is still unknown. We aimed to describe the differences in DNA methyltransferase gene expression in patients with different diabetic macular edema responses. A total of 27 diabetic patients, aged 59-90 years, were prospectively enrolled in this cross-sectional study. The participants were classified into control group (CG, n = 11), diabetic macular edema responders (rDME, n = 9) and non-responder diabetic macular edema (nrDME, n = 7) after anti-vascular endothelial growth factor (anti-VEGF) treatment. Only cases with a complete ophthalmological examination, digital 133° color fundus, and SD-OCT assessments were used. After RNA extraction and first-strand cDNA synthesis, quantitative real-time PCR was performed with specific primers on the CFX Connect™ Real-Time PCR Detection System to assess differential transcriptional expression patterns. The DNMT1 gene showed a positive correlation (r = 0.617; p = 0.043) with Best Corrected Visual Acuity (BCVA) in CG, a positive correlation (r = 0.917; p = 0.010) with HbA1c in nrDME and a negative correlation (r = -0.659; p = 0.049) with GCL-IPL thickness in rDME. DNMT3A gene showed a positive correlation (r = -0.890; p = 0.001) with Sub-foveal Choroidal thickness in rDME whereas DNMT3b gene showed a negative correlation (r = -0.815; p = 0.007) with HbA1c and RNFL (r = -0.664; p = 0.026) in CG. Patients with similar metabolic profile risk factors showed associated DNA methyltransferase transcriptional expression patterns differences fitting with the anti-VEGF diabetic macular edema response. Further studies are needed to clarify if these results (1) reflect disease evolution, (2) translate the therapeutic impact, (3) or can help to predict the therapeutic resistance profile.

Hemotransmissive infections in blood donor cats

The paper provides information about studies conducted on 490 cats, aged from 1 to 8 years, in females and males belonging to different breeds. All animals tested for infection passed the stage of clinical examination and had no contraindications to donation, and accordingly had no visible clinical signs of hemotransmissive infections (GTI). A detailed clinical examination of the patient and competent collection of anamnesis allows you to minimize the risks of detecting GTI in an animal. GTI diagnostics was performed by PCR on Bio-Rad CFX Connect Real-Time PCR Detection System and BioRad CFX 96 Real-Time PCR Detection System, ELISA – on Thermo Scientific Multiskan FC Microplate Photometer. According to the results of the study, feline immunodeficiency virus (VIC) was detected in 11 cats (2.2%), feline leukemia virus (VLC) in 5 cats (1%), hemoplasmosis (Candidatus Mycoplasma haemominutum) in 7 cats (1.4%), Bartonella henselae in 5 cats (1%). Despite the low percentage of animals in which GTI was detected, it is worth noting the need for blood banks for animals in which all potential donors undergo mandatory diagnostics. Many of the animals that turned out to be carriers of GTI had previously donated blood privately, thereby not helping recipients, but transmitting diseases to them, often incurable. Animals in contact with GTI carriers should be monitored regularly and in more depth at the GTI. Even a positive patient history and the absence of clinical signs in the animal require additional monitoring in the form of blood tests, which is very rarely carried out privately when owners donate animals as donors outside of blood banks. This fact contributes to the spread of GTI during hemotransfusion.

The complex influence of the combination of the BDNF (rs6265), VDR (rs2228570), and NMDA (rs4880213) genotypes on the development of cognitive disorders in patients with autoimmune thyroiditis andhypothyroidism

Background. Numerous studies have demonstrated that thyroid conditions can affect cognitive function. Gene polymorphisms associated with pathology of the endocrine and nervous system have ethnic and population specificity, which determines the need to study them in a certain region. The purpose of the study was to investigate the combined impact of the BDNF (rs6265), VDR (rs2228570), and NMDA (rs4880213) gene polymorphisms on cognitive impairment in patients with autoimmune thyroiditis and hypothyroidism among the population of Western regions of Ukraine, and to predict the onset of cognitive disorders. Materials and methods. The study involved a total of 153 patients with autoimmune thyroiditis and hypothyroidism. Genotyping of the VDR (rs2228570), BDNF (rs6265), and NMDA (rs4880213) gene polymorphism using TaqMan probes and TaqMan Genotyping Master Mix (4371355) was performed on CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., USA). Polymerase chain reaction for TaqMan genotyping was carried out according to the kit instructions (Applied Biosystems, USA). We detect a decline in cognitive function using the Mini-Mental State Examination. Results. Carrying a combination of CC/AG/CC genotypes significantly reduces the risk of developing cognitive impairment (odds ratio (OR)=0.1410; 95% confidence interval (CI) 0.0181–1.0965; p=0.0416). At the same time, carrying a combination of CT/AG/CT genotypes increases the risk of cognitive impairment by more than 5 times (OR=5.1915; 95% CI 1.2471–21.6107; p=0.0214) and a combination of CT/AG/TT genotypes — by 10 times (OR=10.1224; 95% CI 1.1037–92.8401; p=0.0281). Carriers of the CT/AA/CT genotype combination have a 6.4-fold increased risk of cognitive impairment (OR=6.4062; 95% CI 1.2019–34.1471; p=0.0253). Conclusions. Among patients with autoimmune thyroiditis and hypothyroidism, carriers of the CC/AG/CC genotype combination of the BDNF (rs6265), VDR (rs2228570) and NMDA (rs4880213) genes have a reduced risk of developing cognitive disorders, while carriers of the CT/AG/CT, CT/AG/TT and CT/AA/CT have an increased risk of cognitive impairment.

Development of a one-run real-time PCR detection system for pathogens associated with poultry infectious diseases.

Infectious diseases are an important issue in the poultry industry, requiring early diagnosis and countermeasures. To address this, we present a system based on TaqMan real-time PCR to detect pathogen genome in specimens collected from chickens. We designed 12 primer-probe sets for pathogens causing respiratory or systemic symptoms. In field samples, we detected three viruses, including DNA and RNA viruses, and three bacteria. The chicken anemia virus and Avibacterium paragallinarum were detected only in young and laying hens, respectively. Bacteria were detected only in throat swabs, and gallid alphaherpesvirus 2 was detected in different specimens at each developmental stage. Our novel TaqMan real-time PCR system effectively detects pathogen's gene in chickens, while taking age into account.

A single-tube multiplex real-time PCR for HLA-B*38:02 genotype by detecting highly specific SNPs.

Purpose: HLA-B*38:02 is closely related to carbimazole/methimazole-induced agranulocytosis. This study aimed to develop and validate a rapid and economical method for HLA-B*38:02 genotyping. Methods: A single-tube multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*38:02 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. Results: The accuracy of the assay was 100% for HLA-B*38:02 genotyping. The detection limit of the new method was 0.05ng DNA. The positive rate of HLA-B*38:02 in the Han (8%, n=100), Bouyei (17.8%, n=90)and Tibetan (12.7%, n=110) populations was significantly higher than that in the Uighur population (1%, n=100) (p<0.05). Conclusion: The proposed method is rapid and reliable for HLA-B*38:02 screening in a clinical setting.