Search Results (259)

Chicken coccidiosis is a major parasitic disease in poultry that causes substantial economic losses worldwide. The accurate and simultaneous diagnosis of mixed infections with multiple Eimeria species remains challenging using conventional methods. In this study, we developed a multiplex TaqMan-MGB qPCR assay targeting the ITS1 region for simultaneous detection and quantification of four major pathogenic species: E. acervulina, E. necatrix, E. maxima, and E. tenella. The assay exhibited high specificity without cross-reactivity and achieved a sensitivity 100-fold greater than conventional PCR, with detection limits ranging from 10¹ to 10² copies/μL. Standard curves demonstrated strong linearity (R² ≥ 0.95) and amplification efficiency (95–113%). Repeatability was robust, with intra- and inter-assay CVs below 2%. Applied to 165 clinical samples collected from poultry farms in Fujian Province, China (2022–2024), the assay detected an overall Eimeria infection rate of 93.3%, higher than conventional PCR (89.7%). Epidemiological analysis revealed E. tenella (78.8%, [130/165]) and E. necatrix (78.8%, [130/165]) were dominant, while mixed infections were frequent (83.3%, [136/154]), including dual (45.5%, [70/154]), triple (31.2%, [48/154]), and quadruple (11.7%, [18/154]) infections. This multiplex TaqMan-MGB qPCR provides a rapid, sensitive, and quantitative tool for detecting multiple Eimeria species, greatly improving diagnostic efficiency and supporting clinical diagnosis, surveillance, and control strategies. Full article

Background: Legg–Calvé–Perthes disease (LCPD) is a rare avascular osteonecrosis of the proximal femoral epiphysis and typically occurs during the childhood growth phase. LCPD is a complex illness of unknown origin, which is considered the main difficulty in the study of this disease. Various theories on LCPD etiology have been proposed; however, no consensus has been reached about its origin. Our research objective was to evaluate the polymorphisms FVL rs6025, FVIII rs5987061, FIX Malmö rs6048, PAI-1 rs1799889, eNOS rs17899983/rs2070744, IL-23R rs1569922/rs154655686/7539625, and TNF-α rs180062, and their relationship with LCPD. Methods: A blood sample was taken from each study participant. Complete blood count, coagulation times and factors, antithrombotic proteins, and homocysteine (Hcy) were determined using a coagulometric method. DNA was obtained and genotyped using real-time PCR with TaqMan probes. Genotypic and allelic distributions were analyzed using comparative analysis, the Hardy–Weinberg equilibrium, and OR. Results: This study included 46 children: 23 with LCPD (cases) and 23 without (controls). Statistically significant differences were found in Prothrombin Time, Factor V, and Factor IX activity, as well as Hcy concentration; these values suggest the presence of hypercoagulable states in patients, which can cause thrombotic events. On the other hand, significant differences were also found in the neutrophil–lymphocyte ratio and systemic immune-inflammation index, showing major inflammation states in the patient group. Moreover, statistically significant differences were found in the IL-23R rs1569922 polymorphism; it was found that carriers of the T/T and C/T genotypes have an increased risk of developing LCPD. Conclusions: Our results show greater hemostatic activity and inflammation in the group of patients included in this study, supporting various theories previously proposed. Therefore, we believe that LCPD is a multifactorial condition in which hemostatic, inflammatory, and genetic factors play a central and triggering role in the disease. Full article

Background/Objectives: Emerging evidence implicates impaired DNA repair mechanisms in the pathogenesis of Parkinson’s disease (PD), particularly in the context of oxidative stress and environmental exposures. This study investigated the association between five polymorphisms in nucleotide excision repair (NER) pathway genes and PD susceptibility in a northern Mexican mestizo population. Methods: We conducted a case–control study including 137 patients with clinically diagnosed PD and 137 age- and sex-matched controls. Genomic DNA was isolated from peripheral blood, and genotyping of ERCC1 (rs11615), ERCC2 (rs13181), XPA (rs1800975), XPC (rs2228001), and XPF (rs1799801) was performed using TaqMan real-time PCR assays. Associations between genotype frequencies and PD were evaluated using logistic regression models adjusted for age, sex, and pesticide exposure. Results: A significantly higher prevalence of pesticide exposure was observed in PD patients than in controls (OR 2.08, 95% CI 1.18–3.68; p = 0.01). The XPC rs2228001 C/C genotype was independently associated with increased PD risk in males (OR 3.25, 95% CI 1.07–9.85; p = 0.042), even after adjusting for uric acid, pesticide exposure, and cognitive status (MMSE score). No significant associations were found for other NER-related polymorphisms. Male PD patients also exhibited significantly lower serum uric acid levels than controls (p = 0.046), supporting a link between oxidative stress and disease vulnerability. Conclusions: Our findings suggest a sex-specific genetic contribution to PD susceptibility involving the XPC rs2228001 variant, particularly in the context of pesticide exposure. These results underscore the relevance of DNA repair pathways in PD pathogenesis and highlight the importance of integrated models incorporating genetic and environmental risk factors. Full article

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

Background/Objectives: The location and distribution of excess fat, rather than overall adiposity, are stronger predictors of cardiometabolic risk and are commonly assessed using the waist-to-hip ratio (WHR). Fat distribution in women has a heritable component, yet the genetic factors that influence it remain poorly understood. We aim to assess the association between obesity-related polymorphisms with WHR and cardiometabolic risk in overweight and obese women. Methods: A cohort study was conducted in 512 women (56.1 ± 6.4 years; body mass index (BMI) ≥ 25 kg/m²). WHR was calculated, and participants were classified into android (WHR > 0.85) or gynoid (WHR ≤ 0.85) obesity groups. We genotyped 15 SNPs previously associated with obesity using TaqMan real-time PCR. Different genetic models (dominant, recessive, and allelic) were analysed, and bivariate and multivariate analyses were performed to compare the fat distribution groups. Results: Of the 15 SNPs studied, only 3 presented a significant association with WHR > 0.85. PSRC1 rs599839 in a dominant model (AA + GA vs. GG) with OR = 3.18 (p = 0.041), SLC30A8 rs1326634 in a recessive model (CC vs. TC + TT) (OR = 2.38; p = 0.004), both showing increased susceptibility to central obesity. KIF6 rs20455 offers protection in a recessive model (CC vs. TC + TT) with an OR of 0.47 (p = 0.043). After adjusted multivariate analysis, only SLC30A8 and diabetes remained independently associated with an increased risk of android obesity (OR = 2.50; p = 0.003 and OR = 3.63; p = 0.004, respectively). Conclusions: The SLC30A8 variant was significantly associated with android fat distribution and high cardiometabolic risk in overweight/obese women. Identifying genetic factors that influence fat distribution may help specify targeted lifestyle changes or pharmacological interventions to reduce risk. 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

Peste des petits ruminants virus (PPRV) has been classified into four lineages based on the nucleocapsid and fusion genes, with lineage IV strains being the most widely distributed. In Africa, recent epidemiological data revealed that PPRV lineage IV is increasingly displacing other lineages in prevalence, suggesting a competitive advantage in viral transmission and adaptability. Moreover, a lineage IV strain was the only confirmed strain in Europe and Asia. In this study, a one-step Taqman quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay for lineage IV PPRV was established by targeting the hemagglutinin (H) gene. The results indicated that this method could detect approximately six copies of PPRV RNA, indicating high sensitivity. No cross-reactions with related viruses or other lineages of PPRV were observed. The results of a repeatability test indicated that the coefficient of variation values were low in both the inter-assay and intra-assay experimental groups. Detection of field samples indicated that all positive samples could be detected successfully using the developed method. This RT-qPCR assay provides a valuable tool to facilitate targeted surveillance and rapid differential diagnosis in regions with active circulation of PPRV lineage IV, enabling timely epidemiological investigations and strain-specific identification. Full article

Background/Objectives: The influence of single-nucleotide polymorphisms (SNPs) on hepatocellular carcinoma (HCC) in terms of etiological factors remains to be explored. This study evaluated the distribution of PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs6834314 and overall survival of HCC patients with metabolic dysfunction-associated steatotic liver disease (MASLD-HCC) and viral-related HCC (VIRAL-HCC). Methods: This study included 564 patients with HCC: 254 with MASLD-HCC and 310 with VIRAL-HCC. The SNPs were determined by real-time PCR using TaqMan assays. Results: The mean ages of patients with MASLD-HCC and VIRAL-HCC were 68.4 vs. 60.9 years (p < 0.001), with a significant difference between groups. The prevalence of PNPLA3 GG genotype in MASLD-HCC was significantly higher in MASLD-HCC than in VIRAL-HCC (24.0% vs. 15.5%, OR = 1.86, 95% CI = 1.14–3.05, p = 0.009). Similarly, the prevalence of TM6SF2 TT genotype in MASLD-HCC and VIRAL-HCC was 7.1% vs. 2.6% (OR = 3.39, 95% CI = 1.36–9.21, p = 0.003), while HSD17B13 GG genotype in the corresponding groups was 7.1% vs. 12.6% (OR = 0.53, 95% CI = 0.27–1.01, p = 0.039). The overall median survival of MASLD-HCC was significantly shorter than that of the VIRAL-HCC group (42 vs. 66 months, p = 0.035). In Cox regression hazard analysis, HSD17B13 GG genotype was significantly associated with a lower mortality rate in MASLD-HCC (HR = 0.38, 95% CI = 0.18–0.81, p = 0.011). In contrast, PNPLA3 and TM6SF2 were not associated with overall survival in patients with MASLD-HCC or VIRAL-HCC. Conclusions: Our data demonstrated that the prevalence of the SNPs significantly differed between MASLD-HCC and VIRAL-HCC. The HSD176B13 GG genotype was also associated with a survival benefit in Thai patients with MASLD-HCC. Thus, assessing the HSD176B13 genotype might be beneficial in risk stratification and potential therapeutic inhibition of HSD17B13 among patients with MASLD-HCC. 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

Background: Osteoporosis is a chronic metabolic condition characterized by progressive loss of bone mass and disruption of the bone spatial architecture. Pathological changes are influenced by multiple factors, including genetic predispositions. Identifying risk factors for osteoporosis is crucial for recognizing at-risk populations, implementing preventive strategies, and supporting diagnostics. Type I collagen, composed of two chains—α1(I) and α2(I), encoded by the COL1A1 and COL1A2 genes, respectively—plays a key role in the mechanical strength of tissues, including bones. The aim of this study was to assess the effect of the rs17166249 and rs412777 polymorphisms in the COL1A2 gene on bone mineral density (BMD) in postmenopausal women. Methods: The study included 570 unrelated women: 119 diagnosed with osteoporosis, 96 with osteopenia, and 355 healthy controls. Polymorphisms in the COL1A2 gene were analyzed using real-time PCR with specific primers and TaqMan probes. Results: The results showed no significant differences in the distribution of genotypes and alleles of rs412777 between the groups. However, the rs17166249 T allele was found to be more prevalent in the osteoporosis group, although the association was not statistically significant after adjusting for confounders. Furthermore, no significant correlations were observed between the genotypes of either SNP and BMD parameters such as T-score, Z-score, and BMD measurements. Conclusion: These findings suggest that while the COL1A2 gene may have a modest influence on bone health, its role in osteoporosis risk remains inconclusive, highlighting the need for further studies to explore additional genetic and environmental factors. Full article