Search Results (87)

Due to morphological similarities, adulterants are frequently substituted for Qingfengteng (QFT, Sinomenium acutum) in regional markets. This study developed a TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay targeting a 57-base pair (bp) fragment of the internal transcribed spacer 2 (ITS2) region for the specific detection of QFT. The method was validated using a diverse set of samples, including: (1) 19 batches of QFT and 8 batches of Beidougen (BDG, Menispermum dauricum), comprising both medicinal materials and decoction pieces; (2) 5 batches each of decoction pieces from Qingsheteng (QST, Periploca calophylla), Jishiteng (JST, Paederia foetida), Kuanjinteng (KJT, Tinospora sinensis), and Huibeiqingfengteng (HBQFT, Sabia discolor); (3) 6 batches of commercial QFT-containing tablets (with different batch numbers) and 6 batches of laboratory-prepared QFT aqueous decoctions (with different decocting time). Distinct cycle threshold (Ct) values and amplification curves unambiguously differentiated QFT from all adulterants. An external standard-based quantification approach was established to detect adulteration with BDG, the morphologically and genetically most similar adulterant. Recovery rates ranged from 81.79 to 102.38% in herbal mixed powders spiked with 1%, 5%, 50%, and 100% BDG. The method reliably detected QFT in processed tablets and freeze-dried decoctions, demonstrating high tolerance to deoxyribonucleic acid (DNA) degradation. This qPCR assay enables specific and quantitative detection of QFT in dried and processed samples using short amplicons (57 bp), thereby supporting quality control throughout the herbal production chain. Full article

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis and is primarily transmitted via contaminated water and food. Groundwater may also serve as a potential vector for HEV transmission. This study aimed to optimize real-time polymerase chain reaction (rtPCR) for the detection of HEV, employing both TaqMan probe- and SYBR Green-based methods. A total of 12 primer sets for the TaqMan probe-based method and 41 primer sets for the SYBR Green-based method were evaluated in order to identify the optimal combinations. Primer sets #4 (TaqMan probe-based) and #21 (SYBR Green-based) demonstrated the highest sensitivity and specificity, successfully detecting HEV in artificially spiked samples at 1 fg/μL. The TaqMan probe-based method facilitated rapid detection with minimized non-specific amplification, whereas the SYBR Green-based method allowed for broader primer exploration by leveraging melting curve analysis. Despite the absence of HEV detection in 123 groundwater samples, this study underscores the value of real-time PCR for environmental monitoring and paves the way for enhanced diagnostic tools for public health applications. Full article

Background/Objectives: The gene pool of the Apennine wolf is affected by admixture with domestic variants due to anthropogenic hybridisation with dogs. Genetic monitoring at the population level involves assessing the extent of admixture in single individuals, ranging from pure wolves to recent hybrids or wolf backcrosses, through the analysis of nuclear and mitochondrial DNA (mtDNA) markers. Although individually non-diagnostic, mtDNA is nevertheless essential for completing the final diagnosis of genetic admixture. Typically, the identification of wolf mtDNA haplotypes is carried out via sequencing of coding genes and non-coding DNA stretches. Our objective was to develop a fast real-time PCR assay to detect the mtDNA haplotypes that occur exclusively in the Apennine wolf population, as a valuable alternative to the demanding sequence-based typing. Methods: We validated a qualitative duplex real-time PCR that exploits the combined presence of diagnostic point mutations in two mtDNA segments, the NDH-4 gene and the control region, and is performed in a single-tube step through TaqMan-MGB chemistry. The aim was to detect mtDNA multi-fragment haplotypes that are exclusive to the Apennine wolf, bypassing sequencing. Results: Basic validation of 149 field samples, consisting of pure Apennine wolves, dogs, wolf × dog hybrids, and Dinaric wolves, showed that the assay is highly specific and sensitive, with genomic DNA amounts as low as 10⁻⁵ ng still producing positive results. It also proved high repeatability and reproducibility, thereby enabling reliable high-throughput testing. Conclusions: The results indicate that the assay presented here provides a valuable alternative method to the time- and cost-consuming sequencing procedure to reliably diagnose the maternal lineage of the still-threatened Apennine wolf, and it covers a wide range of applications, from scientific research to conservation, diagnostics, and forensics. Full article

Acute coronary syndrome (ACS) remains the leading cause of mortality in developed countries. Although recent advances have improved our understanding of the pathophysiology of ACS and its primary consequence, myocardial infarction, many questions remain regarding the molecular and cellular changes occurring during and after an infarction. This study aimed to evaluate the expression levels of the zyxin (ZYX) gene in patients with ACS, stable coronary artery disease (stable CAD), and healthy controls. RNA was extracted from PBMCs and analyzed by quantitative real-time PCR (qRT-PCR). Gene expression was measured using TaqMan Gene Expression Assays and the number of ZYX mRNA molecules was quantified based on qRT-PCR kinetics. Kruskal–Wallis was used to compare gene expression levels among the three groups. A significantly higher number of ZYX gene copies was observed in both the ACS and stable CAD groups than in healthy controls (p < 0.0001 and p < 0.001, respectively). A statistically significant difference was also observed between the ACS and stable CAD groups (p = 0.004). The increased expression of zyxin observed in patients with ACS and stable CAD may reflect cellular repair mechanisms activated in response to myocardial injury. Full article

Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV P72 gene, CSFV 5’UTR region, PRRSV ORF6, PCV2 cap gene, and PRV gB gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control. Full article

Microbial detection in milk is crucial for food safety and quality, as beneficial and harmful microorganisms can affect consumer health and dairy product integrity. Identifying and quantifying these microorganisms helps prevent contamination and spoilage. The study employs advanced molecular techniques to detect and quantify the genomic DNA for the target hydrolytic enzyme coding genes lipA and aprX based on the multi-align sequence conserved region, specific primer pair, and hydrolysis probes designed using the singleplex qPCR and multiplex qPCR. Cultured isolates and artificially contaminated sterilized ultra-high-temperature (UHT) milk were analyzed for their specificity, cross-reactivity, and sensitivity. The finding indicated that strains with lipA and aprX genes were amplified while the other strains were not amplified. This indicated that the designed primer pairs/probes were very specific to the target gene of interest. The specificity of each design primer pair was checked using SYBR Green qPCR using 16 different isolate strains from the milk sample. The quantification specificity of each strain target gene was deemed to be with a mean Ct value for positive pseudomonas strain > 16.98 ± 1.76 (p < 0.0001), non-pseudomonas positive strain ≥ 27.47 ± 1.25 (p < 0.0001), no Ct for the negative control and molecular grade water. The sensitivity limit of detection (LOD) analyzed based on culture broth and milk sample was >10⁵ and >10⁴ in PCR amplification while it was >10⁴ and >10³ in real-time qPCR, respectively. At the same time, the correlation regression coefficient of the standard curve based on the pure culture cell DNA as the DNA concentration serially diluted (20 ng/µL to 0.0002 ng/µL) was obtained in multiplex without interference and cross-reactivity, yielding R² ≥ 0.9908 slope (−3.2591) and intercepting with a value of 37, where the efficiency reached the level of 95–102% sensitivity reached up to 0.0002 ng/µL concentration of DNA, and sensitivity of microbial load was up to 1.2 × 10² CFU/mL. Therefore, multiplex TaqMan qPCR simultaneous amplification was considered the best method developed for the detection of the lipA and aprX genes in a single tube. This will result in developing future simultaneous (three- to four-gene) detection of spoilage psychrotrophic bacteria in raw milk. Full article

Acute gastroenteritis (AGE) has one of the highest rates of morbidity and mortality among children under five years old worldwide. It is estimated that 1.7 billion cases of childhood diarrheal diseases occur annually, leading to up to 443,832 deaths. Approximately 90% of these cases are viral, with human norovirus being the main cause in countries that have implemented rotavirus vaccines. The objective of this study was to describe the prevalence and genetic diversity of norovirus in child outpatients and inpatients under five years old at the Regional Hospital of Antofagasta. From 1 January to 31 October 2019, a total of 121 stool samples were collected to detect the presence of norovirus GI and GII using Taqman™-based real-time RT-PCR. Norovirus RNA was detected in 50 (41.3%) samples, of which 96% were typed as GII.4 Sydney (42% GII.4 Sydney[P16] and 54% GII.4 Sydney[P4 New Orleans]). Furthermore, most (92%) of the positive specimens were from children under two years of age and a majority were detected in April (38%) and May (20%). Our findings highlight the high burden of norovirus in hospitalized children with AGE and the importance of molecular strain surveillance to support vaccine development. Full article

Background: Porcine circovirus disease (PCVD), caused by porcine circovirus (PCV), is a significant swine disease characterized by porcine dermatitis, nephrotic syndrome, and reproductive disorders in sows. Given the overlapping clinical presentations of PCV2, PCV3, and PCV4, a rapid and accurate method for their differential detection is essential. Methods: In this study, specific primers and probes were designed based on the conserved regions of the ORF1 genes of PCV2 and PCV4, as well as the ORF2 gene of PCV3. Results: A TaqMan triple real-time PCR method was developed, demonstrating excellent specificity, sensitivity, and repeatability, with limits of detection (LODs) of 53.3 copies/µL, 12.0 copies/µL, and 13.8 copies/µL for PCV2, PCV3, and PCV4, respectively. Using this method, 500 clinical porcine tissue samples collected from 23 provinces across China between 2023 and 2024 were analyzed. The results showed detection rates of 75.20% (376/500) for PCV2, 17.60% (88/500) for PCV3, and 4.40% (22/500) for PCV4. The detection rate of triple coinfections involving PCV2, PCV3, and PCV4 was 0.80% (4/500). PCV2 consistently presented significantly higher positive detection rates across all growth stages, and its viral copy number was significantly greater than those of PCV3 and PCV4 (* p < 0.05). Forty PCV2 ORF2 genes, fourteen PCV3 ORF2 genes, and three PCV4 ORF2 genes were identified. These included four PCV2a genotypes, thirty-five PCV2d genotypes, and one PCV2e genotypes; two PCV3a genotypes and six each of PCV3b and PCV3c genotypes; and two PCV4a genotypes and one of PCV4b genotype. Conclusions: The triple qPCR method established in this study provides a rapid, specific, and accurate approach for the detection and differentiation of PCV2, PCV3, and PCV4 genotypes. Full article

The Vibrio genus represents a critical group of bacterial pathogens in the marine environment globally, leading to massive mortality in the aquaculture industry. Diagnosing vibriosis, an infection caused by Vibrio species, in clinical samples poses challenges due to its non-specific clinical manifestations. In this study, we developed a TaqMan probe-based multiplex real-time PCR method for the simultaneous detection and quantification of four Vibrio pathogens: Vibrio anguillarum (Va), Vibrio alginolyticus (Val), Vibrio harveyi (Vh), and Vibrio scophthalmi (Vsc). The assay targets conserved intra-species regions and specific inter-species regions using specific primers and TaqMan probes to ensure specificity. Sensitivity analysis demonstrated that the multiplex real-time PCR assay could simultaneously detect the four different bacteria, with detection limits of 26–60 copies per reaction, making it 100 times more sensitive than conventional PCR assays. Additionally, the assay exhibited high reproducibility, with intra- and inter-group coefficients of variation below 1.4%. A total of 63 clinical samples was analyzed using this established assay, which successfully detected both single and mixed infections. These results demonstrate that the multiplex quantitative PCR assay is a rapid, specific, and sensitive diagnostic tool for the detection of Va, Val, Vh, and Vsc, making it suitable for monitoring these bacteria in both single- and co-infected clinical samples. Full article

A conventional PCR (cPCR) remains an effective molecular technique for the diagnosis of canine monocytic ehrlichiosis. However, agarose gel electrophoresis requires additional time after thermal cycling. In the present study, we developed a PCR-based lateral flow biosensor (PCR-LFB) to detect Ehrlichia canis (E. canis). Lateral flow strips allow for the simple and rapid detection of PCR products and provide an alternative to gel electrophoresis. The sensitivity, specificity, and detection limit of PCR-LFB were compared to those of TaqMan probe-based real-time PCRs (qPCRs). The PCR-LFB was performed with 5′ 6-FITC and biotin-labeled primers specific to E. canis, targeting the dsb gene. The detection limit of the PCR-LFB assay was 10⁻⁶ for the target DNA sequence in a 10-fold dilution of the recombinant plasmid, which is 10 times lower than that of qPCR. Among the confirmed qPCR results in the 30 dog samples, false-positive results were not detected by the PCR-LFB. Compared to qPCR, the sensitivity and specificity of PCR-LFB were 63.6% (95% CI; 42.9–80.2%) and 100% (95% CI; 67.5–100%), respectively. The Kappa value of the PCR-LFB is in moderate agreement with the qPCR (κ = 0.483). Perfect agreement (κ = 1) was observed between cPCR and PCR-LFB. Lower cost and shorter time consumption were demonstrated using PCR-LFB. Full article

Objectives: Vulvar squamous cell carcinoma (VSCC) is a rare gynecologic malignancy, with most cases arising from differentiated vulvar intraepithelial neoplasia (dVIN). Approximately one-third of VSCC cases originate from high-grade squamous intraepithelial lesions (HSILs), which are associated with persistent infection by varieties of high-risk human papillomavirus (hrHPV). This study aimed to quantify the circulating microRNAs (miRNAs) in the plasma of patients with premalignant conditions (dVIN and HSILs) and VSCC using TaqMan Low-Density Arrays. Methods: Plasma samples were collected from 40 patients, including those treated for HSILs, dVIN, and VSCC. Quantitative real-time PCR (qRT-PCR) identified the circulating miRNAs differentially expressed in the plasma of VSCC patients compared to patients with precancerous lesions. Results: A total of 31 differentially expressed miRNAs (DEMs) were found to be significantly upregulated in plasma from VSCC patients compared to precancerous cases. None of the analyzed miRNAs were able to distinguish VSCC cases based on hrHPV tumor status. Conclusions: This study provides strong evidence that a distinct set of miRNAs can differentiate between plasma samples from VSCC patients and those with precancerous lesions. Thus, these DEMs have potential diagnostic and prognostic value. “Predisposing” DEMs could be developed as biomarkers to aid in the assessment of vulvar lesions, helping to exclude or confirm progression toward cancer. Full article

This study investigated the diagnostic efficiencies of two assays for the detection of Mycobacterium tuberculosis complex: (1) the reciprocal-flow real-time polymerase chain reaction (PCR)-based GeneSoC assay and (2) the real-time PCR based GENECUBE MTB assay with quenching probe. These assays were performed for stored clinical samples and results were compared with the confirmed results based on culture and COBAS TaqMan MTB assay. A total of 53 samples (20 confirmed positives and 33 confirmed negatives) were included in the performance analysis. The GeneSoC assay showed concordance in all 53 samples, regardless of specimen type, while the GENECUBE MTB assay showed concordance in 19 of the 20 confirmed positive samples and all 33 confirmed negative samples. The overall agreement was 100.0% for the GeneSoC assay and 98.1% for the GENECUBE MTB assay. Positive and negative percent agreements were 100.0% each for the GeneSoC assay and 95.0% and 100.0%, respectively, for the GENECUBE MTB assay. Both the GeneSoC and GENECUBE MTB assays exhibited excellent performance in detecting M. tuberculosis complex. The GeneSoC assay is useful for independent assays of individual samples, whereas the GENECUBE MTB assay is suitable for batch assays of multiple samples. Full article

To establish a rapid and sensitive detection method for the porcine reproductive and respiratory syndrome virus (PRRSV), gene-specific primers and a TaqMan probe were designed based on the M gene of PRRSV, and a new stable fully pre-mixed reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) reaction mixture was developed. A simple and rapid RT-qPCR detection method for PRRSV was developed by optimizing nucleic acid amplification conditions. The results showed that the method was able to specifically detect PRRSV without cross-reactivity with the other 11 porcine susceptible viruses. The sensitivities of the assay were 3.12 × 10⁰ copies/μL and 10⁰ TCID₅₀/μL for M gene and virus, respectively, and the repeatability and reproducibility (relative standard deviation, CV) of the assay were less than 2.5%. Based on the new fullly pre-mixed RT-qPCR reaction mixture, the RT-qPCR detection method may provide a new, simple, and rapid method for accurately detecting PRRSV. Full article

Dyserythropoietic anemia and myopathy syndrome (DAMS) with neonatal losses was recently characterized as an autosomal recessive disorder caused by an EHBP1L1 frameshift variant in English Springer Spaniels (ESSPs). The frequency and dissemination of the mutation remained unknown. The EHBP1L1 protein is essential for muscle function, and the Rab8/10-EHBP1L1-Bin1-dynamin axis participates in nuclear polarization during the enucleation of erythroblasts. Lack of EHBP1L1 function decreases enucleation, leading to increased numbers of nucleated erythrocytes, which are characteristic of DAMS. A genotyping survey for the EHBP1L1 variant was conducted based upon submitted samples of ESSPs from Europe. DNA was extracted, and a real-time PCR assay, with allele-specific TaqMan probes for EHBP1L1 wild-type and frameshift deletion, was applied. Between September 2022 and August 2024, 803 samples were received from 18 European countries. The EHBP1L1 mutant allele frequency was 9.7%, including 4 homozygous dogs and 148 heterozygotes. The mutant EHBP1L1 allele was found in 13 countries. A total of 6 homozygous and 73 heterozygous ESSPs reported on an open database could be tracked to an original common ancestor. Although the survey is biased, it indicates that the mutant EHBP1L1 variant is disseminated in the breed and across Europe. The genotyping of ESSPs is recommended to diagnose DAMS and guide breeders. Full article

Roe deer meat is a prized game product in many European countries. However, concerns exist regarding the accuracy of the amount of declared roe deer in processed game meat foods. This study aimed to develop a reliable method for the detection and quantification of roe deer in commercialized game meat products. A TaqMan probe-based quantitative real-time PCR (qPCR) assay was designed, targeting a single-copy 120-bp region of the roe deer agouti signaling protein (ASIP) encoding gene. The method employed the normalized ∆Cq approach to establish a calibration curve for roe deer detection and quantification within 0.05–50% (w/w) in complex raw and processed matrices. The method proved to be specific for roe deer identification, achieving limits of detection and quantification of 0.04 ng of roe deer DNA and 0.05% (w/w) of roe deer in simulated pâté. Following validation with blind samples, highlighting the precision and trueness of the approach, the assay was applied to 46 market samples from four European origins (Poland, Portugal, France, and Spain). The analysis revealed significant discrepancies between declared roe deer content and actual levels in all roe deer labeled products. The global analysis of results, combining the previous survey on red deer species with present roe deer data, identified 61% of mislabeled/adulterated samples due to the absence of deer species, substitution of roe deer with red deer, substitution of fallow deer with other deer species and red deer with pork, and undeclared addition of roe deer. This study demonstrates the effectiveness of the developed qPCR method for accurate roe deer meat authentication in foods, showing its usefulness as a tool for routine food inspection to ensure labeling compliance. Full article