Specific TaqMan Research Articles

Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

The proof is in the poo: Non-invasive method to detect endoparasitic infection.

Almost every animal trait is strongly associated with parasitic infection or the potential exposure to parasites. Despite this importance, one of the greatest challenges that researchers still face is to accurately determine the status and severity of the endoparasitic infection without killing and dissecting the host. Thus, the precise detection of infection with minimal handling of the individual will improve experimental designs in live animal research. Here, we quantified extracellular DNA from two species of endoparasitic worm that grow within the host body cavity, hairworms (phylum Nematomorpha) and mermithids (phylum Nematoda), from the frass of their insect host, a cave wētā (Orthoptera: Rhaphidophoridae) and an earwig (Dermaptera: Forficulidae), respectively. Frass collection was done at two successive time periods, to test if parasitic growth correlated with relative DNA quantity in the frass. We developed and optimized two highly specific TaqMan assays, one for each parasite-specific DNA amplification. We were able to detect infection prevalence with 100% accuracy in individuals identified as infected through post-study dissections. An additional infection in earwigs was detected with the TaqMan assay alone, probably because some worms were either too small or degraded to observe during dissection. No difference in DNA quantity was detected between sampling periods, although future protocols could be refined to support such a trend. This study demonstrates that a noninvasive and minimally stressful method can be used to detect endoparasitic infection with greater accuracy than dissection alone, helping improve protocols for live animal studies.

Establishment and preliminary application of quantitative real-time PCR assay for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA

Objective: To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples. Methods: The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells. Results: The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×102 copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect. Conclusion: A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.

Genetic markers of the risk of coronary heart disease and coronary artery thrombosis developing in the Kazakh population.

Surgical methods such as coronary artery bypass grafting and percutaneous coronary interventions (PCI) are widely used along with traditional conservative therapy in the treatment of coronary artery disease. The disease outcome directly depends on timely diagnosis and treatment. A significant role in predicting the effectiveness of treatment is given to personification of treatment and management of the patient. In this case, the determining component is its individual genetic status. The study groups included persons of Kazakh nationality which identify themselves, their biological parents, and biological grandparents on the maternal and paternal side as Kazakh. Research groups included 108 people at the age from 45 to 65 years of both sexes. Blood samples genotyping was carried out by PCR using highly specific TaqMan samples. Thermo Fisher cloud applicationwas used for genotypes determining on the base of an automatic algorithm. The article presents the results of the evaluation of gene polymorphisms associated with coronary artery restenosis in a population of Kazakh nationality. 3 SNPs were determined when searching for an association with stenting due to coronary artery thrombosis: rs7543130 (p=0.009324), rs6785930 (p=0.016858), rs7819412 (p=0.061325). Four polymorphisms associated with the risk of developing coronary heart disease were revealed during the study of polymorphisms among the people of the Kazakh population. Three SNPs were determined when searching for an association with stenting due to coronary artery thrombosis. It should be noted that the Bonferonni correction for multiple comparisons did not reveal significant polymorphisms associated with coronary artery disease, which requires further research with more quantity of samples.

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of four feline diarrhea-associated viruses.

Since their recent discovery, the prevalence of novel feline enteric viruses, including feline bocavirus 1 (FBoV-1), feline astrovirus (FeAstV), and feline kobuvirus (FeKoV), has been reported in China. Co-infections of these viruses with feline parvovirus (FPV) are common causes of diarrhea in cats. Viral co-infections are difficult to identify because of their non-specific clinical signs. To detect and identify these viruses, a quick and specific pathogen-testing approach is required. Here, we establish a real-time PCR (qPCR) based on multiple TaqMan probes for the simultaneous detection of FBoV-1, FeAstV, FeKoV, and FPV. Specific primers and TaqMan fluorescent probes were designed to ensure specificity. The results showed that the detection limit of single qPCR was up to 10 copies, and the detection limit of multiplex qPCR was up to 100 copies, with correlation coefficients >0.995 in all cases. Clinical sample detection revealed a 25.19% (34/135) total rate of co-infection among the viruses and a 1.48% (2/135) quadruple infection rate. Thus, this multiplex qPCR approach can serve as a quick, sensitive, and specific diagnostic tool for FBoV-1, FeAstV, FeKoV, and FPV identification, and it may be utilized for routine surveillance of these emerging and reemerging feline enteric viruses.

Expression of the miR-9-5p, miR-125b-5p and its target gene NFKB1 and TRAF6 in childhood-onset systemic lupus erythematosus (cSLE)

Childhood- onset systemic lupus erythematosus (cSLE) is a multisystem inflammatory disease that can lead to severe clinical conditions resulting in early comorbidities. Several genetic, environmental, and immunological factors are known to influence the onset of the disease. MiRNAs have been already considered as potential actors involved in the development and activity of the SLE. Thus, understanding the behavior of these regulators can contribute to clarify the inflammatory process affecting SLE patients. Among miRNAs, miR-125b-5p and miR-9-5p targeting NFKB1 and TRAF6 genes can be involved in the etio-pathogenesis of the disease by modulating inflammation. In this study we evaluated miR-9-5p and miR-125b-5p expression and its target genes NFKB1 and TRAF6 in peripheral blood samples (PBMC) from the 35 cSLE patients and 35 healthy controls. MiRNAs and gene target expression have been evaluated by using RT-PCR with specific TaqMan® probes. Both miR-9-5p [Fold Change (FC) = −2.21; p = 0.002] and miR-125b-5p (FC= −3.30; p < 0.0001) and NFKB1 (FC = −1.84; p < 0.001) were downregulated in cSLE patients, while TRAF6 was upregulated (FC = 1.80; p = 0.006) in cSLE patients when compared to controls. A significant correlation was found between miR-125b-5p and its target gene NFKB1 [Spearman (r) = 0.47; p = 0.023]. Our results showed miR-125b-5p and miR-9-5p differential expression in cSLE patients, possibly contributing to better understanding the role of these regulators in cSLE development and disease pathogenesis.

Quantitative RT-PCR Assays for Quantification of Undesirable Mutants in the Novel Type 2 Oral Poliovirus Vaccine.

Emergence of mutations is an inherent property of RNA viruses with several implications for their replication, pathogenesis, and evolutionary adaptation. Oral poliovirus vaccine (OPV), developed by Albert Sabin, is composed of live attenuated polioviruses of three serotypes that can revert to neurovirulence during replication in cell culture and in vaccine recipients. Recently, a new modified variant of Sabin 2 virus was developed by introducing changes in its genome, making it more genetically stable to prevent the reversion. The new strain was used to manufacture novel OPV2 (nOPV2), which was approved by the World Health Organization for emergency use to stop outbreaks caused by circulating vaccine-derived poliovirus (cVDPV2). Manufacture of this improved vaccine requires close attention to the genetic heterogenicity to ensure that the levels of the undesirable mutations are limited. Preliminary studies using whole-genome Illumina sequencing (NGS) identified several genomic sites where mutations tend to occur with regularity. They include VP1-I143T amino acid change at the secondary attenuation site; VP1-N171D, a substitution that modestly increases neurovirulence in mice; and VP1-E295K, which may reduce the immunogenicity of the nOPV2. Therefore, to ensure the molecular consistency of vaccine batches, the content of these mutants must be quantified and kept within specifications. To do this, we have developed quantitative, multiplex, one-step reverse-transcriptase polymerase chain reactions (qmosRT-PCRs) as simple methods for quantification of these mutations. Each method uses specific short TaqMan probes with different dyes for the analysis of both mutants and non-mutants in the same sample. The quantification is done using calibration curves developed using validated reference materials. To evaluate the sensitivity and the linearity of the qmosRT-PCR method, the mutant viruses were spiked in non-mutant viruses, and nOPV2 batches were used to validate the method. The spiked samples and the nOPV2 batches were analyzed by qmosRT-PCR and NGS assays. The results showed that qmosRT-PCR is sensitive enough to detect around 1% of mutants. The percentages of mutants determined by qmosRT-PCR correlate well with the results of the NGS. Further, the analysis of the nOPV2 batches showed that the results of qmosRT-PCR correlated well with the results of NGS. In conclusion, the qmosRT-PCR is a specific, sensitive, and linear method. It could be used for quality control of the nOPV2 batches.

New real‐time PCR assay for detecting the blueberry and cranberry twig blight pathogen

AbstractDiaporthe vaccinii is a fungal pathogen of concern to Vaccinium species, responsible for twig blight disease. This fungus may survive in woody tissues without displaying symptoms, which makes the diagnostics more challenging. To provide a rapid and efficient diagnostic tool for D. vaccinii, a new and specific TaqMan real‐time PCR assay was developed using translation elongation factor 1 gene region. The assay was specific to D. vaccinii and did not cross‐react with any other tested Vaccinium pathogens. The sensitivity of the assay was found to be 1 pg of the pathogen DNA in water or in host DNA, which is equivalent to less than 20 hyphal cells. The assay was also used to test infected plant tissues, which determined that the assay can detect the pathogen from symptomatic as well as non‐symptomatic material. Furthermore, D. vaccinii was detected after mixing infected asymptomatic tissue with healthy stem tissues at a proportion of 1:10 by weight. To co‐amplify the D. vaccinii and the plant cytochrome oxidase gene as internal control in a single reaction, a duplex assay was successfully developed without any reduction in sensitivity. Inter‐laboratory testing of the assay showed consistent results, demonstrating that the developed assay is robust and transferrable to other laboratories.

The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus.

Simple SummaryAfrican swine fever (ASF) was first reported in August 2018 in China, and the naturally gene-deleted ASFV strain was first identified in 2020 in this country. The vaccine candidates that deleted some virulent genes from the virulent parental strains have also been reported in many countries. To differentiate the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. After optimizing the reaction conditions, a multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed. The assay was further used to test 4239 clinical samples, and 534 samples were positive for ASFV, of which 30 samples lacked B646L, I177L, MGF505-2R and/or EP402R genes. The assay showed high specificity, sensitivity and repeatability, and it provided a reliable method for evaluating ASFV in clinical samples.African swine fever virus (ASFV) causes African swine fever (ASF), a devastating hemorrhagic disease of domestic pigs and wild boars. Currently, the MGF505R, EP402R (CD2v) and I177L gene-deleted ASFV strains were confirmed to be the ideal vaccine candidate strains. To develop an assay for differentiating the wild-type and gene-deleted ASFV strains, four pairs of specific primers and TaqMan probes targeting the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes were designed. A multiplex real-time qPCR assay for the differential detection of the wild-type and gene-deleted ASFV strains was developed after optimizing the reaction conditions, including the annealing temperature, primer concentration and probe concentration. The results showed that the multiplex real-time qPCR assay can specifically test the ASFV B646L (p72), I177L, MGF505-2R and EP402R (CD2v) genes with a limit of detection (LOD) of 32.1 copies/μL for the B646L (p72) gene, and 3.21 copies/μL for the I177L, MGF505-2R and EP402R (CD2v) genes. However, the assay cannot test for the classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine circovirus type 2 (PCV2), PCV3 and pseudorabies virus (PRV). The assay demonstrated good repeatability and reproducibility with coefficients of variation (CV) less than 1.56% for both the intra- and inter-assay. The assay was used to test 4239 clinical samples, and the results showed that 12.60% (534/4239) samples were positive for ASFV, of which 10 samples lacked the EP402R gene, 6 samples lacked the MGF505-2R gene and 14 samples lacked the EP402R and MGF505-2R genes. The results indicated that the multiplex real-time qPCR developed in this study can provide a rapid, sensitive and specific diagnostic tool for the differential detection of the ASFV B646L, I177L, MGF505-2R and EP402R genes.

A New TaqMan Real-Time PCR Assay for Detecting the Blueberry Pathogen Monilinia vaccinii-corymbosi.

Monilinia vaccinii-corymbosi (Mvc) is an important fungal pathogen of blueberry, causing mummy berry disease. While the symptoms of the advanced stages of the disease can be obvious, diagnosing its early stages can be challenging. To facilitate fast and sensitive screening of asymptomatic or latently infected plant material for Mvc, we developed a specific TaqMan real-time PCR assay targeting the internal transcribed spacer (ITS) region. The assay was shown to be specific to Mvc and did not cross react with any of the other tested Monilinia species or other blueberry pathogens. Using the multicopy ITS region ensured high analytical sensitivity, enabling very low concentrations of Mvc DNA (0.1 pg) to be detected both in water and host DNA matrix. Comparable results were obtained in interlaboratory testing, showing that the assay is robust, and can be effectively used in other laboratories. Assay sensitivity was also confirmed on infected plant tissue, showing that it is effective in detecting the pathogen in infected asymptomatic stem tissue, as well as infected tissue that was mixed with healthy tissue at a ratio of 1:10 by weight. The assay was duplexed with a plant internal control (cytochrome oxidase gene) for simultaneous amplification of the pathogen and plant internal control in a single reaction. This new diagnostic tool can be used for sensitive and rapid screening of blueberry plants for the presence of Mvc in many different settings, e.g., for breeding programs, research, or biosecurity diagnostics.

SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR.

Background: Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (CTL2, also known as SLC44A2). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare SLC44A2*2 allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of SLC44A2 in mixed populations with different proportions. Methods: DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in SLC44A2. The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. SLC44A2 frequency was determined by two-channel ddPCR. Results: The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of SLC44A2*1 and SLC44A2*2 in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%–70.8%) and 35.1% (95% CI, 29.2%–41%), respectively. The ratio of SLC44A2*2 alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the SLC44A2 frequency in the population to be accurately inferred. Conclusion: This new method of detecting SLC44A2 alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the SLC44A2 frequency by ddPCR using the probes that we designed.

Aberrant hydroxymethylation in promoter CpG regions of genes related to the cell cycle and apoptosis characterizes advanced chronic myeloid leukemia disease, poor imatinib respondents and poor survival

BackgroundThere is strong evidence that disease progression, drug response and overall clinical outcomes of CML disease are not only decided by BCR/ABL1 oncoprotein but depend on accumulation of additional genetic and epigenetic aberrations. DNA hydroxymethylation is implicated in the development of variety of diseases. DNA hydroxymethylation in gene promoters plays important roles in disease progression, drug response and clinical outcome of various diseases. Therefore in this study, we aimed to explore the role of aberrant hydroxymethylation in promoter regions of different tumor suppressor genes in relation to CML disease progression, response to imatinib therapy and clinical outcome.MethodsWe recruited 150 CML patients at different clinical stages of the disease. Patients were followed up for 48 months and haematological/molecular responses were analysed. Haematological response was analysed by peripheral blood smear. BCR/ABL1 specific TaqMan probe based qRT-PCR was used for assessing the molecular response of CML patients on imatinib therapy. Promoter hydroxymethylation of the genes was characterized using MS-PCR.ResultsWe observed that promoter hydroxymethylation of DAPK1, RIZ1, P16INK4A, RASSF1A and p14ARFARF genes characterize advanced CML disease and poor imatinib respondents. Although, cytokine signalling (SOCS1) gene was hypermethylated in advanced stages of CML and accumulated in patients with poor imatinib response, but the differences were not statistically significant. Moreover, we found hypermethylation of p14ARF, RASSF1 and p16INK4A genes and cytokine signalling gene (SOCS1) significantly associated with poor overall survival of CML patients on imatinib therapy. The results of this study are in agreement of the role of aberrant DNA methylation of different tumor suppressor genes as potential biomarkers of CML disease progression, poor imatinib response and overall clinical outcome.ConclusionIn this study, we report that promoter hydroxymethylation of DAPK1, RIZ1, P16INK4A, RASSF1A and p14ARFARF genes is a characteristic feature of CML disease progressions, defines poor imatinib respondents and poor overall survival of CML patients to imatinib therapy.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri.

The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. A comparative genomics approach was used to identify O-acetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10fg genomic DNA and 1CFUml-1 bacterial culture. No change in the detection threshold (1CFUml-1 ) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

Molecular Diagnosis and Management of Leptospiral Mastitis

Background: Bovine Leptospirosis is one of the leading causes of abortion, infertility, stillbirths and sudden drop in milk production to complete agalactia in cattle and buffaloes. An outbreak in water buffaloes with the history of sudden appearance of bloody/pinkish milk from all four quarters and decrease in milk production to complete agalactia was investigated in village Baghot, Mahendergarh, Haryana, India. Methods: A total of fifty eight (n=58) biological samples (milk=38, blood=11 and urine =9) samples) from 17 buffaloes were collected from Baghot and its neighbouring villages of district Mahendergarh, Haryana, India. All collected samples were processed for molecular diagnosis using pathogenic Leptospira specific TaqMan probe based real-time PCR and nucleotide sequencing. Result: The pathogenic Leptospira spp. was detected in real time PCR from milk samples of 6 animals (35.2%) out of 17 buffaloes. None of the blood and urine sample was found positive for pathogenic leptosipra spp. PCR amplified product were further sequenced and confirmed pathogenic Leptospira spp. The affected animals in the village were treated with parenteral administration of Streptopenicillin, Tranexamic acid and Ascorbic acid for 3-5 days, in recommended doses. Treated animals responded positively to the therapeutic regimen. Furthermore, animal owners and veterinary staff were instructed to take protective measures to ensure no further spread to animals and humans.

Establishment of a duplex TaqMan RT-PCR for the differential detection of RHDV GI.1 and GI.2

BackgroundRabbit haemorrhagic disease (RHD) is a highly contagious and acute fatal hepatitis of the European rabbit (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). Up to 2010, all RHD viruses (RHDV) isolated belonged to one genotype. In 2010, a new genotype of RHDV (RHDV2/b, currently designated GI.2 based on phylogenetic analysis) emerged in France. The aim of this study was to develop a rapid, simple, specific and sensitive TaqMan real-time PCR assay for the classic strain of RHDV and RHDV2 detection. Specific primers and probes were designed for the VP60 gene of RHDV and RHDV2 within the conserved region of viral genome. ResultsThis study was demonstrated to be highly specific for RHDV and RHDV2, without cross-reactions with other non-targeted viruses. The detection limit of this work was 102 copies of RHDV and RHDV2, respectively. The coefficient of variation of the assay was less than 5% for both intra-assay and inter-assay. The reproducibility of method was assessed using plasmids and the coefficient of variation obtained was 0.2–3.70. Of 79 clinical samples, 68 were positive samples (86.08%), of which 60 were classic RHDV variants (75.9%), 4 were co-infected (5.06%) and 8 were RHDV2 (10.12%), those results are more sensitivity compare with conventional RT-PCR RT-PCR. ConclusionsIn conclusion, this duplex TaqMan RT-qPCR based on VP60 gene of RHDV and RHDV2 could be a valuable tool in diagnose and molecular epidemiological study of the RHDV and RHDV2.

Vitamin D Receptor and Binding Protein Gene Variants in Patients with Essential Tremor.

Several studies have shown an association between some variants in the vitamin D receptor (VDR) and the GC vitamin D binding protein (GC) genes with the risk for Parkinson's disease or other neurological disorders. VDR rs2228570 has shown an association with essential tremor (ET) in a previous study. The aim of this study is to look for the association between several common variants in these genes and the risk for ET. We genotyped 272 patients diagnosed with familial ET and 272 age-matched controls using specific TaqMan assays for VDR rs2228570, VDR rs731236, VDR rs7975232, VDR rs739837, VDR rs78783628, GC rs7041, and GC rs4588 single nucleotide variants (SNVs). We found an association between GC rs7041 SNV and ET using recessive, codominant, and allelic models. Despite our results did not find an association between VDR rs2228570 and ET, the pooled data with those by a previous report suggest this association under recessive, codominant, and allelic models. None of the SNVs studied was related to the age at onset of tremor in ET patients. Data from the current study suggest an association between GC rs7041 and VDR rs2228570 SNVs and ET risk.

Circulating Serum MiRNA-8074 as a Novel Prognostic Biomarker for Multiple Myeloma.

MiRNA-8074 is a molecule with the potential to regulate the expression of key genes related to the pathogenesis of multiple myeloma (MM), i.e., TP53, MYC, MAPK1, and KIAA. We analyzed the predictive and prognostic value of miRNA-8074 expression in MM patients. In total, 105 newly diagnosed MM patients treated with thalidomide (n = 27), bortezomib (n = 41) and bortezomib with thalidomide (n = 37) were studied. For miRNA analysis, the column method and the Real-Time PCR technique with specific TaqMan Fast Advanced Master Mix and TaqMan probes were used. Factors that were associated with a significant reduction in progression-free survival (PFS) included: ECOG > 1, ISS stage III, low hemoglobin, thrombocytopenia, hypoalbuminemia, abnormal renal function, elevated creatinine, GFR < 60 mL/min/1.73 m2, elevated LDH, del(17p), t(11;14), the use of a single drug regimen (thalidomide or bortezomib) and high miRNA-8074 expression (HR = 2.01, 95% CI: 1.16–3.49; p = 0.0233). In addition to the known prognostic factors, such as ECOG > 1, Durie–Salmon stage III, diagnosis of light chain disease or non-secreting MM, renal failure, hypoalbuminemia, hypercalcemia, high β2-microglobulin, elevated LDH, and t(14;16), a high expression of miRNA-8074 was significantly associated with a higher risk of death (HR = 4.12, 95% CI: 2.20–7.70; p = 0.0009). In summary, miRNA-8074 may be a useful diagnostic tool to assess the prognosis in MM patients.

Detection of pathogenic Leptospira spp. in herpetofauna in Central Appalachia.

Leptospirosis is a water borne zoonotic disease of global significance that is caused by pathogenic species of the genus Leptospira. Pathogenic leptospires live in the kidneys of reservoir or infected animals and are shed in their urine contaminating water, soil, etc. Rodents are considered the primary reservoir of leptospirosis, but little is known about the role of herpetofauna (non-avian reptiles and amphibians) in the epidemiology of the disease. To address this, various species of amphibians and reptiles in the Cumberland Gap Region of the Central Appalachia were screened for the presence of Leptospira spp. Kidneys harvested from of a total of 116 amphibians and reptiles belonging to seven species of snakes, seven species of salamanders, seven species of frogs/toads, seven species of turtles and one species of lizards were tested using a highly specific TaqMan based qPCR that targets lipl32 gene of pathogenic Leptospira spp. Overall, 15 of the tested 116 amphibians and reptiles were positive (12.9%; 95% CI: 7.4%-20.4%). Of the 101 amphibians, 11 were positive (10.9%; 95% CI: 5.6%-18.7%), and 4 of the 15 reptiles tested positive (26.7%; 95% CI: 7.8%-55.1%). The amplified gene fragments of lipl32 from qPCR positive kidneys were sequenced and found to be identical with known pathogenic Leptospira spp. These results suggest that although the proportion of reptiles and amphibians transmitting pathogenic Leptospira spp. within the environment may be low as compared to rodents, they pose a risk to other susceptible hosts that share their habitats and may have role in maintaining a baseline infection in the environment.

Honey polyphenolic fraction inhibits cyclooxygenase-2 expression via upregulation of microRNA-26a-5p expression in pancreatic islets

Objectives Honey total polyphenolic fraction (HTPF) is reported to have anti-disease potential, however the role of HTPF in the regulation of microRNAs (miRNAs) has never been investigated. This study was undertaken to investigate the potential of HTPF against inflammation via regulation of miRNAs in pancreatic islets of Langerhans. Methods Pancreatic islets were isolated from C57BL/6 mice and HTPF was purified from honey. Bioinformatics algorithms were used to determine miRNA target genes. Expression of miRNA and mRNA was determined using their specific taqman assays. Pairing between miRNA and 3′ untranslated region (3′UTR) of mRNA was confirmed using luciferase reporter clone containing the 3′UTR of mRNA sequences and results were verified by transfection of mouse pancreatic β-cell line Min6 with miRNA inhibitors. Results The data showed that mmu-miR-26a-5p is a direct regulator of cyclooxygenase-2 (COX-2) expression and HTPF inhibits COX-2 expression or prostaglandin E2 (PGE2) production via up-regulating mmu-miR-26a-5p expression. Transfection of islets with anti-miR-26a-5p significantly enhanced COX-2 expression and PGE2 production ( p &lt; .01), while HTPF treatment significantly inhibited anti-miR-26a-5p transfection-induced COX-2 expression or PGE2 production ( p &lt; .05). These findings were further verified in pancreatic β-cells Min6. Moreover, the data also determined that HTPF also inhibits glucose-induced nuclear transcription factor (NF)-κB activity. Conclusion HTPF suppresses glucose-induced PGE2 production and activation of NF-κB via negative regulation of COX-2 and mmu-miR26a-5p. These novel pharmacological actions of HTPF on glucose-stimulated pancreatic islets provide new suggestions that HTPF or HTPF-derived compounds inhibit glucose induced inflammation in pancreas by up-regulating the expression of microRNAs.