Search Results (9)

Ujimqin sheep, known for its distinctive multi-vertebrae phenotypes (T13L7, T14L6, and T14L7) and economic value, has garnered significant attention. However, conventional phenotypic detection methods suffer from low efficiency and high costs. In this study, based on a key SNP locus (ABCD4 gene, Chr7:89393414, C > T) identified through a genome-wide association study (GWAS), a TaqMan-MGB (minor groove binder) genotyping system was developed. the objective was to establish a high-throughput and efficient molecular marker-assisted selection (MAS) tool. Specific primers and dual fluorescent probes were designed to optimize the reaction system. Standard plasmids were adopted to validate genotyping accuracy. A total of 152 Ujimqin sheep were subjected to TaqMan-MGB genotyping, digital radiography (DR) imaging, and Sanger sequencing. the results showed complete concordance between TaqMan-MGB and Sanger sequencing, with an overall agreement rate of 83.6% with DR imaging. For individuals with T/T genotypes (127/139), the detection accuracy reached 91.4%. This method demonstrated high specificity, simplicity, and cost-efficiency, significantly reducing the time and financial burden associated with traditional imaging-based approaches. the findings indicate that the TaqMan-MGB technique can accurately identify the T/T genotype at the SNP site and its strong association with the multi-vertebrae phenotypes, offering an effective and reliable tool for molecular breeding of Ujimqin sheep. Full article

To establish a rapid real-time RT-PCR method for differentiating wild-type classical swine fever virus (CSFV) strains from vaccine strains (HCLV), we designed a universal primer targeting the NS3 gene to detect wild-type CSFV strains and vaccine strains simultaneously, and two TaqMan-MGB probes were designed to differentiate between wild-type and vaccine strains. After optimizing the RT-qPCR conditions, a rapid dual TaqMan-MGB RT-qPCR method for the detection and identification of CSFV and HCLV was developed. The results showed that method could specifically detect CSFV and HCLV with no cross-reactivity with other swine pathogens. The analytic sensitivity for the NS3 gene of CSFV and HCLV were 1.67 × 10¹ copies/μL, respectively. For precision testing, the repeatability and reproducibility of the test was less than 2%. This method was successfully used for the rapid detection of 193 biological samples collected from CSFV-vaccinated pigs. This fast and accurate detection technology can be used for the detection of CSFV and is suitable for differentiating between wild-type CSFV strains and vaccine strains. Full article

Equine piroplasmosis (EP) is a parasitic disease caused by Theileria equi (T. equi), Babesia caballi (B. caballi) and Theileria haneyi (T. haneyi). This disease is considered to be reportable by the World Organization for Animal Health (WOAH). Real-time quantitative PCR (qPCR) is regarded as a straightforward, rapid and sensitive diagnostic method to detect pathogens. However, qPCR has not been employed in the various epidemiological investigations of T. haneyi. In this study, we developed a new qPCR method to detect T. haneyi based on the chr1sco (chromosome 1 single-copy open reading frame (ORF)) gene, which has no detectable orthologs in T. equi or B. caballi. A TaqMan MGB probe was used in the development of the qPCR assay. A plasmid containing the chr1sco gene was constructed and used to establish the standard curves. The novel qPCR technique demonstrated great specificity for detecting additional frequent equine infectious pathogens and sensitivity for detecting diluted standard plasmids. This qPCR was further validated by comparison with an optimized nested PCR (nPCR) assay in the analysis of 96 clinical samples. The agreement between the nPCR assay and the established qPCR assay was 85.42%. The newly established method could contribute to the accurate diagnosis of T. haneyi infections in horses. Full article

We report the optimization of a high-throughput, compliant DNA extraction method that uses standard format 96-well plates and a commercial automated DNA purification system (ABI PRISM^® 6100 Nucleic Acid PrepStation). The procedure was set up for maize and soybean, the most common GMO crops and the main ingredients of several foodstuffs, and compared with an EU-validated CTAB-based method. Optimization of the DNA extraction was achieved by applying self-prepared buffers (for DNA extraction, binding, and washing) on the PrepStation loaded with proprietary glass-fiber-coated purification plates. Quantification of extracted DNA was performed by real-time PCR using previously reported endogenous soybean lectin and maize starch synthase genes and a novel plant-specific universal TaqMan MGB probe that targets the 18S rRNA multiple copy gene. Using serial dilutions of both maize and soybean genomic DNAs, we show low PCR sensitivity and efficiency for the official TransPrep DNA extraction protocol compared to the CTAB-based one. On the other hand, using serial dilutions of a standard reference plasmid containing a 137 bp sequence cloned from the 18S rRNA plant-specific ribosomal gene, we demonstrate the high PCR sensitivity and efficiency of the optimized DNA extraction protocol setup with self-prepared buffers. The limits of detection and quantification of the 18S rDNA reiteration were consistent with the calculated values, supporting the suitability of the DNA extraction procedure for high-throughput analyses of large populations and small amounts of tissue. Full article

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis. Full article

Retinoic acid inducible gene G (RIG-G) is an inducible gene produced during the treatment of acute promyelocytic leukemia with all-trans retinoic acid (ATRA). However, it is unclear the expression level of RIG-G gene in the peripheral blood of healthy subjects and patients with acute promyelocytic leukemia (APL or AML-M3). In the present study, we established the TaqMan-MGB fluorescent probe qPCR (real-time polymerase chain reaction) method for the first time to detect the expression of RIG-G gene in APL. Twenty APL patients were selected, and their RIG-G expression levels were quantified to assess the correlation between the expression of peripheral blood and bone marrow samples. U test was used to analyze the expression level of RIG-G in the peripheral blood of 40 normal specimens and 20 APL patients to observe the prognostic monitoring effect of RIG-G gene in the ATRA treatment process. ROC (receiver operating characteristic curve) was used to analyze and test the diagnostic efficiency of RIG-G gene for APL patients. There is a strong positive correlation between the expression of RIG-G in peripheral blood and bone marrow of APL patients. The expression level of RIG-G in peripheral blood of APL patients is significantly lower than that in healthy controls (p < 0.001). The changes in the expression level of RIG-G in peripheral blood changed indicates the remission and recurrence of APL patients after ATRA treatment, and the ROC curve shows that it has a better diagnostic power for APL. In summary, the TaqMan-MGB real-time PCR method we have established has successfully run. The detection of RIG-G gene expression in peripheral blood can effectively monitor the disease changes of APL patients and avoid harmful bone marrow puncture injury. Full article

Sacbrood virus (SBV) is a common honey bee virus disease. SBV variants and strains identified in Asian honey bees, Apis cerana, have created confusion in identifications. Although the regional names indicated the expansions of the virus in new regions, pathogenesis, and genomes of these variants are not distinct enough to be a separate virus species. However, current SBV qPCR methods may not detect newly identified A. cerana SBV variants (Ac SBV) according to the genome sequences. Since these Ac SBV can naturally infect A. mellifera and possibly other hymenopterans, ignorance of Ac SBV variants in detection methods is simply unwise. In this report, we updated the qPCR method based on Blanchard’s design that used conserved regions of VP1 to design a TaqMan method with an MGB (minor groove binder) probe. We tested the method in bees and hornets, including A. mellifera, A. cerana, and Vespa velutina. The updated primers and the probe can match published SBV and Ac SBV genomes in databases, and this updated method has reasonable sensitivity and flexibility to be applied as a detection and quantification method before the discovery of variants with more mutated VP1 gene. Full article

Background: Activation of Toll-like-receptor 4 (TLR4) causes chronic inflammation that can result in obesity and metabolic syndrome (MeS). Aim: This study aimed to investigate the role of TLR4 polymorphisms of TLR4D299G/T399I, and its impact on protein expression of TLR4 in obese female subjects. Methodology: A prospective cross-sectional association study was performed on Arab female subjects from Qatar University. The subjects were categorized according to BMI classifications into two groups: “obese; n = 69” and “non-obese; n = 136”. Anthropometric measurements, weight (kg), height (m) and waist circumference (WC) were evaluated, and the body mass index (BMI) was calculated. Fasting blood samples were collected, and assessment of glucose, lipid profile, C-reactive protein (CRP), leptin, IL-6 and insulin was performed. Insulin resistance was computed using HOMA-IR. Genotyping of the TLR4 polymorphisms of TLR4D299G (rs4986790) and TLR4T399I (rs4986791) was performed by the 5′ nuclease assay by TaqMan MGB probe. Flow cytometry was used to evaluate the monocyte cell surface expression of TLR4. Results: The frequency distribution of the genotype revealed that homozygous AA is the most frequent among obese subjects (86.4%) for (TLR4D299G, A > G) and the homozygous CC genotype is the most frequent (92.4%) for (TLR4T399I, C > T). Haplotype analysis of TLR4 D299G/T399I showed that GT carriers had a significant association with increased probability of insulin resistance (odds ratio = 4.73; 95% CI 1.19–18.90; p-value = 0.016). The monocyte cell surface of TLR4 was significantly higher by 1.3 folds in obese compared to non-obese subjects. Conclusions: TLR4 D299G/T399I haplotype polymorphism is associated with an increased risk of insulin resistance with the upregulation of TLR4 protein expression in obese subjects. Full article

Fusarium spp. and Magnaporthiopsis maydis are soil-inhabiting fungi and respectively the causal agents of fusarium ear rot and late wilt, two important diseases that can affect maize, one of the most important cereal crops worldwide. Here, we present two sensitive real-time PCR TaqMan MGB (Minor Groove Binder) assays that detect and discriminate several Fusarium spp. (F. oxysporum, F. verticillioides, and F. graminearum) from M. maydis. The method is based on selective real-time qPCR amplification of the internal transcribed spacer (ITS) region and allows the quantification of the fungi. The applicability of this newly developed TaqMan methodology was demonstrated in a field experiment through the screening of potentially infected maize roots, revealing a high specificity and proving to be a suitable tool to ascertain Fusarium spp. and M. maydis infection in maize. Its high sensitivity makes it very efficient for the early diagnosis of the diseases and also for certification purposes. Thus, qPCR through the use of TaqMan probes is here proposed as a promising tool for specific identification and quantification of these soil-borne fungal pathogens known to cause disease on a large number of crops. Full article