TaqMan PCR Research Articles

Early detection of rare and elusive endangered species using environmental DNA: a case study for the Eurasian otter and the white-clawed crayfish in northwestern Italy

Monitoring, management and conservation of rare and elusive species often requires early detection of individuals, especially for re-introduced and endangered taxa. Environmental DNA (eDNA) approaches can enhance the detection power of traditional biomonitoring methods for low-density, newly-established populations. In this study, we used species-specific Real Time PCR TaqMan assays to assess the presence of two endangered freshwater species, the white-clawed crayfish Austropotamobius pallipes and the Eurasian otter Lutra lutra at eight sites in four river catchments in Liguria (northwestern Italy). The Eurasian otter was considered extinct in the study area since the 1980s. However, recent, although scattered sightings indicated a recolonisation by a few individuals. The white-clawed crayfish populations declined drastically and became increasingly dispersed in the western part of Liguria. Our eDNA analysis confirmed the presence of both species in some of the selected rivers and detected Eurasian otter DNA where the species was not recorded through traditional monitoring methods. This study confirms eDNA-based monitoring approaches as valuable tools to assess the presence of rare and elusive species and help implement protection plans at a local scale.

Application of wastewater-based epidemiology for monitoring COVID-19 in hospital and housing wastewaters

An alternative and complementary diagnostic method of surveillance is provided by wastewater-based surveillance (WBS), particularly in low-income nations like Nepal with scant wastewater treatment facilities and clinical testing infrastructure. In this study, a total of 146 water samples collected from two hospitals (n = 63) and three housing wastewaters (n = 83) from the Kathmandu Valley over the period of March 2021–Febraury 2022 were investigated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using quantitative reverse transcription TaqMan PCR assays targeting the N and E genes. Of the total, 67 % (98/146) samples were positive for SARS-CoV-2 RNA either by using N- or E-gene assay, with concentrations ranging from 3.6 to 9.1 log10 copies/L. There was a significant difference found between positive ratio (Chi-square test, p < 0.05) and concentration (t-test, p = 0.009) of SARS-CoV-2 RNA detected from hospital wastewater and housing waters. Wastewater data are correlated with COVID-19 active cases, indicating significance in specific areas like the Hospital (APFH) (p < 0.05). According to the application of a bivariate linear regression model (p < 0.05), the concentrations of N gene may be used to predict the COVID-19 cases in the APFH. Remarkably, SARS-CoV-2 RNA was detected prior to, during, and following clinical case surges, implying that wastewater surveillance could serve as an early warning system for public health decisions. The significance of WBS in tracking and managing pandemics is emphasized by this study, especially in resource-constrained settings.

Abstract 7295: A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR

Abstract The Janus kinase 2 (JAK2) gene is located on chromosome 9p24.1 and encodes a non-receptor tyrosine kinase that plays a central role in cytokine and growth factor signaling. The JAK2 protein is especially important for controlling the production of blood cells from hematopoietic stem cells. In fact, the JAK2 mutation at amino acid 617 (Valine 617 to Phenylalanine, V617F) is the most frequently detected mutation in myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). This dominant gain-of-function V617F mutation results in constitutive tyrosine phosphorylation activity, leading to uncontrolled blood cell production, including increased numbers of erythrocytes, neutrophils, and platelets. To detect the JAK2 V617F mutation in DNA from patient blood samples, we developed a unique real-time PCR assay using our proprietary XNA technology that enables the assay to selectively amplify the mutant sequence by using a synthetic DNA analog XNA (Xenonucleic Acid). The JAK2 V617F mutation detection assay is designed to detect a valine-to-phenylalanine mutation at amino acid 617 (V617F). PCR primers, TaqMan probes, and XNA were designed for the JAK2 V617F mutation, along with primers and probes for an internal control gene in the duplex setting. The analytical performance of the assay was verified and validated using a variety of control samples. The results revealed that the XNA completely suppressed the amplification of wildtype sequence while only the V617F mutant sequence was amplified. The JAK2 V617F mutation detection assay is highly reproducible with intra- and inter-assay coefficients of variation of less than 10%. Results for the assay’s analytical sensitivity indicated that V617F could be detected with about 0.25% mutant allele frequency in 10 ng DNA input. In addition, no significant cross-amplification between V617F and V617I was observed. Thus, the JAK2 V617F mutation detection assay is specific for the V617F mutation. The JAK2 V617F mutation detection assay, using XNA-based technology, provides high sensitivity and specificity to detect the JAK2 V617F mutation. Thus, this assay would be a useful tool for clinical decision-making in determining the presence of the JAK2 V617F mutation in DNA from patient blood samples. Citation Format: Shuo Shen, Robert Brown, Daniel Kim, Larry Pastor, Andrew Y. Fu, Pushpinder Kaur, Alisha Babu, Wei Liu, Aiguo Zhang, Hiromi Tanaka, Michael Y. Sha. A novel XNA technology-based assay to detect JAK2 V617F mutation by real-time PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7295.

A multiplex direct PCR method for the rapid and accurate discrimination of three species of spider mites (Acari: Tetranychidae) in fruit orchards in Beijing.

Spider mites (Acari: Tetranychidae) are polyphagous pests of economic importance in agriculture, among which the two-spotted spider mite Tetranychus urticae Koch has spread widely worldwide as an invasive species, posing a serious threat to fruit tree production in China, including Beijing. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is also a worldwide pest of fruit trees and woody ornamental plants. The cassava mite, Tetranychus truncatus Ehara, is mainly found in Asian countries, including China, Korea and Japan, and mainly affects fruit trees and agricultural crops. These three species of spider mites are widespread and serious fruit tree pests in Beijing. Rapid and accurate identification of spider mites is essential for effective pest and plant quarantine in Beijing orchard fields. The identification of spider mite species is difficult due to their limited morphological characteristics. Although the identification of insect and mite species based on PCR and real-time polymerase chain reaction TaqMan is becoming increasingly common, DNA extraction is difficult, expensive and time-consuming due to the minute size of spider mites. Therefore, the objective of this study was to establish a direct multiplex PCR method for the simultaneous identification of three common species of spider mites in orchards, A. viennensis, T. truncatus and T. urticae, to provide technical support for the differentiation of spider mite species and phytosanitary measures in orchards in Beijing. Based on the mitochondrial cytochrome c oxidase subunit I (COI) of the two-spotted spider mite and the cassava mite and the 18S gene sequence of the hawthorn spider mite as the amplification target, three pairs of specific primers were designed, and the primer concentrations were optimized to establish a direct multiplex PCR system for the rapid and accurate discrimination of the three spider mites without the need for DNA extraction and purification. The method showed a high sensitivity of 0.047ng for T. truncatus and T. urticae DNA and 0.0002ng for A. viennensis. This method eliminates the DNA extraction and sequencing procedures of spider mite samples, offers a possibility for rapid monitoring of multiple spider mites in an integrated microarray laboratory system, reducing the time and cost of leaf mite identification and quarantine monitoring in the field.

DNA extraction using FTA Elute cards for detection and enumeration of fecal indicator bacteria in the Chao Phraya River, Thailand

Utilizing molecular methods for monitoring and determining water quality in developing countries can be problematic. Effective extraction and storage of DNA from environmental samples is especially difficult due to lack of access to sufficient laboratory facilities for molecular work. Use of a commercial product for the extraction of DNA, as well as its application in determining the microbial quality of surface waters, was evaluated and compared. Surface water samples collected from the Chao Phraya River in Thailand were processed to enumerate Escherichia coli. Quantification was done through culture methods (Colilert Quanti-tray) and quantitative real-time PCR (SYBR green and Taqman assays). Processing of surface water samples and culture enumeration of E. coli was done in Thailand, while DNA extraction and enumeration using real-time PCR was done in Korea. E. coli MPN counts were mean log 2.89/100ml. Quantitative real-time PCR assays counts ranged from 2.38 to 3.59 mean log CFU/100 ml. Taqman PCR assays had no significant difference in mean log counts compared to conventional culture methods. However mean log counts of SYBR green PCR assays were significantly higher than counts obtained by culture methods and Taqman PCR assays (P&lt;0.05). Results demonstrated FTA Elute cards can be employed for the extraction of microbial DNA from surface waters and have potential use in detection of fecal indicator bacteria from environmental water samples.

Differential Expression of Down-regulated MicroRNA-126 and Micro RNA-22 in Ovarian Carcinogenesis

Background: Non-coding RNA i.e. MicroRNA plays an important role in the progression and pathogenesis of different types of tumours including ovarian cancer. Deregulated microRNA expression could transform the passivity of cancer, disease progression, chemoresistance, and mobility. The miRNA profile of healthy and cancerous samples was observed to be different. MicroRNA expression analysis and profiling have a bright future in improving the effectiveness of ovarian cancer treatment that is customized to the patient's specific requirements. Expression analysis of serum microRNA-126 and microRNA-22 in ovarian carcinogenesis has been done in the present study. Methods: Present study has been done in the serum of eighty- two epithelial ovarian cancer patients and eighty- two their age-matched healthy women to investigate +the expression of miR-126 and miR22 was TaqMan PCR microRNA assay. The correlation of expression miR-126 and miR-22 with epidemiological and clinicopathological factors was determined statistically. Results: Results showed a 3.37-fold decrease in miR-126 expression and a 2.67-fold reduction in miR-22 expression in ovarian cancer patients. Decreased serum level of miR-22 and miR-126 in ovarian cancer patients has been correlated with the FIGO stage. Results revealed 87.46 sensitivity and 91.12 specificities for down-regulated microRNA 22 with FIGO stage of ovarian cancer and 71.26 sensitivity and 81.12 specificities respectively. More sensitivity and specificity of microRNA 22 with FIGO stage of ovarian cancer was revealed as compared to microRNA 126. Conclusion: Present study revealed downregulated expression of miR-126 and miR-22 in the serum of epithelial ovarian cancer patients. Among all epidemiological and clinical parameters, the FIGO stage of ovarian cancer revealed a substantial association with microRNA level. Results revealed that the sensitivity and specificity of down-regulated microRNA 22 in ovarian cancer were 87.46 and 91.12 and for microRNA 126 with 71.26 and 81.12 respectively. This may be investigated and verified as a durable diagnostic biomarker for the pre-diagnosis of ovarian carcinogenesis.

TaqMan Real-Time PCR Assay for Specific Detection and Differentiation of Xanthomonas translucens pv. undulosa from Other Pathovars Targeting a Recombination Mediator Gene, recF.

Bacterial leaf streak and black chaff diseases of wheat caused by Xanthomonas translucens pv. undulosa is becoming a major constraint to growers and trade since it is seedborne. Molecular tools for specific detection/differentiation of pv. undulosa are lacking. We report the development of a TaqMan real-time PCR for specific detection/identification of pv. undulosa targeting the recombination mediator gene (recF). Analysis of the complete recF (1,117 bp) sequences identified the gene as a reliable phylogenetic marker for identification of pv. undulosa, differentiating it from the other pathovars; recF-based sequence homology values among the 11 pathovars correlated well with genome-based DNA-DNA hybridization values. The discriminatory power of recF to differentiate pv. undulosa from the other pathovars is due to nucleotide polymorphic positions. We used these nucleotide polymorphisms to develop a TaqMan PCR for specific detection of pv. undulosa. The specificity of the assay was validated using 67 bacterial and fungal/oomycete strains. The selected primers and the double-quenched FAM-labeled TaqMan probe were specific for the detection of 11 pv. undulosa/secalis strains. The 56 strains of other X. translucens pathovars (n = 39) and non-Xanthomonas spp. (n = 17) did not exhibit any detectable fluorescence. Also, greenhouse-inoculated and naturally infected wheat leaf samples showed positive reactions for the presence of pv. undulosa DNA but not healthy control plants. The TaqMan assay reliably detected as low as 1-pg DNA amount and 10 colony forming units of the target pathogen per reaction. This TaqMan assay could be useful to regulatory agencies with economic benefits to wheat growers.

An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells.

This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells. Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR. C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05). Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.

Определение аналитической чувствительности и специфичности методов ПЦР для диагностики черной бактериальной пятнистости томата

The work is devoted to molecular genetic methods for the diagnosis of pathogens of bacterial spot of tomato – a harmful bacterial disease of tomatoes and peppers, predominantly spread in the open ground in the southern regions. The research was conducted on the basis of the All- Russian Plant Quarantine Center (FGBU “VNIIKR”) in 2022–2023. The objective of the work was to determine the analytical sensitivity and specificity of molecular genetic methods for the detection and identification of three species of different pathological types of bacteria of the genus Xanthomonas – pathogens of bacterial spot of tomato. Due to the necessity of import substitution of expensive reagents, the tests were carried out with domestic reagents produced by OOO “Evrogen” and ZAO “Dialat”. The results of the effectiveness of real-time PCR (qPCR) are presented, the analysis of which showed that all tested primer systems can be recommended for the detection of bacteriosis pathogens. At the same time, qPCR AFLP derived Taqman PCR and XopD Taqman PCR were found to have high analytical sensitivity (102–103 CFU/ml). It has been shown that three out of four primer systems according to to the methodology of Koenraadt et al. (2009) also have a high sensitivity of 102 CFU/ml. The analytical sensitivity of the Bs-XpF/R primers for the detection of X. euvesicatoria pv. perforans was 104 CFU/ml, which is sufficient for samples with a high concentration of the pathogen and for the identification of a pure bacterial culture. All primers are 100% specific for the target strains of pathogens; no cross-reactions with other strains have been observed. The proposed reliable operational method for detecting pathogens of bacterial spot in tomato and pepper seeds will significantly reduce yield losses and increase the economic efficiency of domestic vegetable production.

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers.

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

Real-time PCR assays that detect genes for botulinum neurotoxin A-G subtypes.

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories.

Genetic Polymorphism in Alcohol Metabolism and Drinking Behavior are Associated with Gastric Cancer Risk in Men.

Objective In recent years, there has been a growing focus on health risks associated with alcohol consumption. The present study investigated whether or not the genetic variant of aldehyde dehydrogenase 2 (ALDH2) influences the risk of gastric cancer among individuals identified as hazardous drinkers using the Alcohol Use Disorders Identification Test (AUDIT), which provides a comprehensive assessment of hazardous drinking behavior. Patients We enrolled men with hazardous drinking behavior (AUDIT score ≥ 8) who had undergone gastric cancer screening (either endoscopy or a barium X-ray examination of the upper gastrointestinal tract) between April 2013 and March 2020 within 1 year from entry and who had subsequently undergone at least one more gastric cancer screening up to March 2021. Functional single-nucleotide polymorphisms of ALDH2 (rs671) were measured using a direct TaqMan PCR method with unprocessed saliva. Results A total of 246 men were enrolled, comprising 193 individuals with active ALDH2 (ALDH2*1/*1) and 53 with less-active ALDH2 (ALDH2*1/*2). The cumulative incidence of gastric cancer in the less-active group was higher than in the active ALDH2 group (p=0.01, hazard ratio: 4.6, 95% confidence interval: 1.2-16.7). Alcohol consumption was lower in the less-active ALDH2 group than in the active ALDH2 group, although no marked difference was observed in the AUDIT score. Conclusion In individuals with hazardous drinking behavior, a heightened risk of gastric cancer was observed among those with less-active ALDH2 variants, even when their alcohol consumption was comparatively lower than in those with active ALDH2 variants.

The relationship between polymorphic variants of the endothelial NO-synthase gene and the course of diabetic nephropathy in patients with type 2 diabetes mellitus

Objective — to identify a possible association between the endothelial nitric oxide synthase (eNOS) gene G894T (rs1799983) polymorphism and the markers of renal function and glucose metabolism in patients with type 2 diabetes mellitus (DM2) with diabetic nephropathy (DN).&#x0D; Materials and methods. Examinations 126 patients with diabetic nephropathy (DN). The control group included 20 healthy individuals. Patients with DM2 were divided into two groups depending on the eNOS rs1799983 gene polymorphism: group I — patients with DN and G/G genotype eNOS G894T gene (n=80); group II — patients with DN and G/T and T/T genotypes eNOS G894T gene (n=46). Genotyping of the of the eNOS rs1799983 gene polymorphism was performed by the Taq‑Man® Fast Universal PCR Master Mix (Applied Biosystems, USA) and TaqMan® SNP Assay (Applied Biosystems, USA).&#x0D; Results. It has been established that in patients with DN, the distribution of genotypes of the G894T polymorphism of the eNOS gene corresponded to the Hardy‑Weinberg equilibrium in all groups and does not differ significantly from European populations. In diabetic patients with DN the 3 times higher total frequency of G/T and T/T genotypes eNOS G894T gene was established when compared to the control group. These findings prove the undoubted influence of the T allele on the development of DN in this group of patients. DN patients with genotypes G/T and T/T had significantly higher blood glucose and HOMA index (p &lt;0.05) than G homozygotes (genotype G/G). Patients with DN and G/G genotype eNOS G894T gene had better glomerular filtration rate and lower albuminuria compared to patients with G/T and T/T genotypes (p &lt;0.05).&#x0D; Conclusions. The establishing of association of eNOS gene polymorphism with the disease and further assessment of individual genetic risk is important for the development of a differentiated approach to the prevention and treatment of DN in patients with DM2 depending on the hereditary predisposition of an individual patient. Therefore, currently one of the most progressive approaches is to develop a strategy for early diagnosis, prognosis and preventive therapy of the disease using genetic markers.

BRAF V600E Mutation Test in Fine Needle Aspiration (FNA) Specimens of Thyroid Nodules by Competitive Allele-Specific TaqMan PCR (castPCR)

Background: BRAF V600E mutation has been confirmed as a significant molecular indicator for the diagnosis and prognosis of papillary thyroid carcinoma (PTC). Sanger sequencing is the recognized approach for BRAF V600E mutation analysis, yet it is not proficient at detecting mutations that occur at low frequencies, especially in thyroid fine needle aspiration (FNA) specimens. This study aimed to evaluate the diagnostic value of Competitive Allele-Specific TaqMan PCR (castPCR) as a new reliable method for BRAF V600E mutation test in thyroid FNA specimens compared to Sanger sequencing. Methods: Thirty patients with thyroid nodules were enrolled prospectively at Dharmais Cancer Hospital Jakarta from 2013–2014. The BRAF V600E mutation was analyzed from FNA specimens using castPCR and Sanger Sequencing methods. The sensitivity and specificity of those methods were compared with routine diagnostic histopathological examination as a gold standard for PTC assessment.Results: A total of 66.6% (20/30) cases were confirmed as PTC by postsurgical histopathological examination. Among these, the BRAF V600E mutation was found in 60.0% (12/20) by castPCR and 40.0% (8/20) by Sanger sequencing. Neither castPCR nor Sanger sequencing detected BRAF V600E mutation in other nodular thyroid diseases. The sensitivity and specificity of castPCR were 60.0% and 100.0%, while Sanger sequencing was 40.0% and 100.0%, respectively. Conclusions: This study indicated that castPCR has a higher sensitivity for the BRAF V600E mutation test than Sanger Sequencing, especially in samples with a low abundance of mutant alleles. A sensitive, rapid, and easy castPCR method is reliable for evaluating BRAF V600E mutation and can improve PTC preoperative diagnosis in clinical settings.

A multiplex Taqman PCR assay for MRSA detection from whole blood.

Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of hospital and community-acquired infections worldwide. MRSA is associated with worse clinical outcomes that can lead to multiple organ failure, septic shock, and death, making timely diagnosis of MRSA infections very crucial. In the present work, we develop a method that enables the positive enrichment of bacteria from spiked whole blood using protein coated magnetic beads, followed by their lysis, and detection by a real-time multiplex PCR directly. The assay targeted bacterial 16S rRNA, S. aureus (spa) and methicillin resistance (mecA). In addition, an internal control (lambda phage) was added to determine the assay's true negative. To validate this assay, staphylococcal and non-staphylococcal bacterial strains were used. The three-markers used in this study were detected as expected by monomicrobial and poly-microbial models of the S. aureus and coagulase-negative staphylococci (CoNS). The thermal cycling completed within 30 mins, delivering 100% specificity. The detection LoD of the pre-processing step was ∼ 1 CFU/mL from 2-5mL of whole blood and that of PCR was ∼ 1pg of NA. However, the combined protocol led to a lower detection limit of 100-1000 MRSA CFUs/mL. The main issue with the method developed is in the pre-processing of blood which will be the subject of our future study.

Comparison of Polymerase Chain Reaction (PCR) assay performance in detecting Decapod penstylhamaparvovirus 1 in penaeid shrimp

Decapod Penstylhamaparvovirus 1, commonly known as infectious hypodermal and hematopoietic necrosis virus (IHHNV), remains an economically important viral pathogen for penaeid shrimp aquaculture due to its effects on growth performance. The World Organization for Animal Health (WOAH, Paris, France) recommended methods for the detection of IHHNV include both conventional and real-time PCR. However, published reports and anecdotal evidence suggest the occurrence of non-specific amplifications when testing for IHHNV using the WOAH protocols. Studies were designed to develop a sensitive, robust TaqMan PCR method for detection of IHHNV in the three commercially important penaeid shrimp: Penaeus vannamei, P. monodon and P. stylirostris. We compared the performance of the WOAH-recommended real-time PCR method to several published as well as in-house designed primer/probe sets spanning the entire genome of IHHNV. Our results show that (1) more than one primer/ probe set is needed when testing for the infectious form of IHHNV in all three species of shrimp and (2) primer pairs qIH-Fw/qIH-Rv and 3144F/ 3232R have diagnostic characteristics that would enable IHHNV detection in all three shrimp species. These findings are valuable for a large-scale screening of shrimp using a TaqMan real-time PCR assay.

ZNF259 rs964184 genetic variant is associated with metabolic syndrome in a Portuguese population

Abstract Background Zinc finger protein (ZPR1) encoded by the ZNF259 gene has been associated with defects in transcription and cell cycle progression. Additionally, the promoter site of ZPR1 binds to peroxisome proliferator-activated receptor gamma (PPARG), which plays a crucial role in insulin sensitivity and obesity. It's not consensual in its association with Metabolic Syndrom (MetS). Aim To estimate the influence of the ZNF259 rs964184 variant in MetS appearance. Methods A case-control study was performed with 3134 subjects (mean age 52.8 ± 8.1 years, 76.4% male) recruited from the Research Unit database, a regional quality clinical registry of hospital admissions. 1756 were patients with MetS and 1378 controls without MetS. MetS was diagnosed according to the International Diabetes Federation (IDF) criteria. The ZNF259 rs964184 C&amp;gt;G was genotyped with the TaqMan PCR assay (Applied Biosystems 7300 Real-Time). The bivariate analysis evaluated the genotypic and allelic distribution in the two groups, with and without MetS. Multivariate Logistic Regression assessed the variables independently associated with MetS. Results There were significant differences in genotype and allele distributions for the ZNF259 C&amp;gt;G variant between patients with MetS and without MetS. Wild-type genotype CC was increased in the non-MetS group, whereas the risk GG was higher in patients with MetS (P=0.008). Similarly, C allele frequencies were significantly higher in the non-MetS group, while the G allele was highly present MetS population (P=0.002). After multivariate logistic regression, GC genotype (OR=1.23; 95%CI:1.05–1.45; p=0.011) and GG genotype (OR=1.59; 95%CI:1.01–2.50; p=0.047) remained as independent risk factors for Metabolic Syndrome. Conclusions This study presents novel data and findings that may have important implications for assessing MetS risk in our population. For the first time in a Portuguese population, we demonstrated that ZNF259 genetic changes are significantly associated with more than 60% increased probability of having Metabolic Syndrome.

Time trends in human papillomavirus prevalence and genotype distribution in vulvar carcinoma in Norway.

Approximately 25%-43% of all vulvar carcinomas are associated with human papillomavirus (HPV). In many countries, vulvar carcinoma incidence rates are increasing, possibly due to greater HPV exposure. However, studies exploring changes in HPV prevalence and genotype distribution in vulvar carcinoma over time are scarce. Our aim was to evaluate time trends in HPV prevalence and genotype distribution in vulvar squamous cell carcinoma in an unselected, nationwide sample of Norwegian women. Further, we explored clinical and histopathological aspects in relation to HPV status and investigated whether HPV status was associated with survival. All vulvar squamous cell carcinoma cases from 1970-1975 and 2000-2005 were extracted from the Cancer Registry of Norway and corresponding tissue blocks were retrieved. After detailed histology review, HPV testing was conducted using real-time TaqMan PCR. Overall survival rates were calculated using the Kaplan-Meier method. Multivariable Cox regression analysis was performed to estimate hazard ratios adjusted for age at diagnosis, stage and diagnostic period. Histological review was performed on 352 vulvar squamous cell carcinoma cases. We were able to obtain valid HPV analysis results for 282 cases, Overall, 29.8% (95% CI 24.5%-35.5%) of cases were high-risk HPV (hrHPV)-positive. When comparing the two periods, we found that the percentage of hrHPV-positive tumors increased significantly from 23% (95% CI 16.0%-31.4%) in 1970-1975 to 35.3% (95% CI 27.8%-43.3%) in 2000-2005 (P = 0.025). The predominant genotypes were HPV 16 (73%), HPV 33 (21%), and HPV 18 (6%), with similar distributions in both periods. In the more recent cohort, several additional genotypes were detected: HPV 6, 11, 39, 45, 52, 58 and 66 were found in smaller percentages, ranging from 1.8% to 3.6%. In univariate analysis, patients with HPV-positive tumors showed improved overall survival compared with patients with HPV-negative tumors (hazard ratio [HR] 0.65, 95% CI 0.48-0.86). The prevalence of HPV in vulvar squamous cell carcinomas in Norway was significantly higher in 2000-2005 than in 1970-1975. The three predominant genotypes were HPV 16, 33 and 18 in both time periods. However, several other HPV genotypes have emerged over the last decades. HPV-positivity was associated with better overall survival.

Expression Pattern of Trace Amine-Associated Receptors during Differentiation of Human Pluripotent Stem Cells to Dopaminergic Neurons.

Trace amine-associated receptors (TAARs), which were discovered only in 2001, are known to be involved in the regulation of a spectrum of neuronal processes and may play a role in the pathogenesis of a number of neuropsychiatric diseases, such as schizophrenia and others. We have previously shown that TAARs also have interconnections with the regulation of neurogenesis and, in particular, with the neurogenesis of dopamine neurons, but the exact mechanisms of this are still unknown. In our work we analyzed the expression of TAARs (TAAR1, TAAR2, TAAR5, TAAR6, TAAR8 and TAAR9) in cells from the human substantia nigra and ventral tegmental areas and in human pluripotent stem cells at consecutive stages of their differentiation to dopaminergic neurons, using RNA sequencing data from open databases, and TaqMan PCR data from the differentiation of human induced pluripotent stem cells in vitro. Detectable levels of TAARs expression were found in cells at the pluripotent stages, and the dynamic of their expression had a trend of increasing with the differentiation and maturation of dopamine neurons. The expression of several TAAR types (particularly TAAR5) was also found in human dopaminergic neuron-enriched zones in the midbrain. This is the first evidence of TAARs expression during neuronal differentiation, which can help to approach an understanding of the role of TAARs in neurogenesis.

THU235 Usefulness Of Digital Droplet Polymerase Chain Reaction (ddPCR) For Molecular Diagnosis Of McCune-Albright Syndrome In DNA From Peripheral Blood Leukocytes

Abstract Disclosure: A.G. de Faria: None. L.R. Montenegro: None. A.A. Jorge: None. R.S. Jallad: None. R.M. Martin: None. M.C. Fragoso: None. A.P. Canton: None. C.E. Seraphim: None. F.R. Tinano: None. N.C. Pinto: None. B.B. Mendonca: None. A. Latronico: None. V.N. Brito: None. Background: McCune-Albright Syndrome (MAS) is a rare congenital disorder caused by post-zygotic activating mutations in GNAS gene. Due to the mosaic pattern of this disease, mutation abundance is frequently low in several tissues, including blood cells. The emergence of digital droplet Polymerase Chain Reaction (ddPCR) is a breakthrough technology of quantitative PCR useful for a targeted detection and quantification of rare events. Objective: This study aims to identify the GNAS mutation in peripheral blood leukocytes of patients with MAS using ddPCR. Patients and Methods: Thirty-four patients (25 female and 9 male) with typical (classic triad) or atypical (one or two features) MAS were included. DNA was extracted from peripheral blood and was analyzed through ddPCR method. Two ddPCR mutation detection assays were used, each one targeting the p.R201C or p.R201H mutations. For each assay, the Taqman PCR system was used with two fluorescence probes: the first labeled with HEX, targeting de (WT allele), and the second labeled 6-FAM, targeting the (mutated allele). The mixture (reagents + DNA) was divided into 20,000 nanodrops, which was placed on a PCR plate and amplified by PCR. After amplification by PCR, the fluorescence of each droplet was read on the 200 Droplet Digital PCR system (Bio-Rad), being classified as positive (drop containing the target sequence) or negative. Results were analyzed using Quantasoft software (Bio-Rad) to determine the total amount of positive droplets in the original sample. The results were reported in fractional abundance (FA), corresponding to the percentage of mutated alleles within the total alleles. Results: Among all patients, the most common MAS feature was osteofibrous dysplasia (OFD) (73.5%), followed by peripheral precocious puberty (PPP) (64.7%), and café-au-lait skin spots (52.9%). Acromegaly or hyperthyroidism was diagnosed in 6 and 4 patients, respectively. Classical MAS triad, 2 clinical features, or 1 clinical feature (PPP or OFD) were present in 15, 9, and 10 patients, respectively. GNAS mutations were identified in 12/34 (35.3%) MAS patients: the p.R201C in 5 and the p.R201H in 7. Among them, 8 patients presented typical MAS and 4 patients with two MAS clinical features, respectively. Considering only typical MAS group, the positive molecular test rate was 53.3% (8/15). GNAS mutations were not identified in patients with only one MAS feature. There was a positive correlation between a positive molecular test and the number of MAS clinical features (p = 0.01). Median FA was 1.45%, ranged from 0.25% to 8.4%. There was no association between FA rate and the number of MAS clinical features (p=1.0). Conclusion: ddPCR representeds a more sensitive tool for identifying GNAS mutations in peripheral blood leukocytes, even with low abundance, in patients with classical and atypical MAS. Presentation: Thursday, June 15, 2023