Search Results (5)

Porcine reproductive and respiratory syndrome (PRRS) represents a critical infectious disease caused by the PRRS virus (PRRSV), posing a substantial threat to the global swine industry. In China, there is currently an epidemic trend characterized by the coexistence of multiple evolving genotypes. Effective prevention and control measures are contingent upon the availability of rapid, precise, and sensitive pathogen detection technologies. Addressing the need for swift differentiation of the predominant circulating strains, including the classical strains (PRRSV-C), the highly pathogenic strains (PRRSV-HP), and NADC30-like strains (PRRSV-NA), this study focuses on the NSP2 region of each lineage. It establishes a triple TaqMan-qPCR method capable of simultaneously genotyping these three lineages. The method demonstrated no cross-reactivity with other viruses, including porcine parvovirus (PPV), porcine transmissible gastroenteritis virus (TGEV), porcine pseudorabies virus (PRV), classical swine fever virus (CFSV), African swine fever virus (ASFV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (RV), and porcine circovirus (PCV2), thereby fully affirming its specificity. The sensitivity analysis demonstrated that the limit of detection (LOD) for the NSP2 gene in each lineage was 1 copy/μL based on the purified plasmids. Both inter-group and intra-group coefficients of variation (CV) were less than 4%, indicating high reproducibility. Comparative studies with commercial kits revealed that the developed TaqMan-qPCR method exhibited 100% relative sensitivity and a relative conformity rate exceeding 98%, suggesting its potential as a viable alternative to commercial kits. Furthermore, the analysis of 1049 clinical samples using the qPCR method indicated that the PRRSV-NADC30-like strains are currently the predominant circulating strain in clinical settings in Hebei Province. In conclusion, this study developed a triple TaqMan-qPCR method capable of simultaneously identifying PRRSV-C, PRRSV-HP and PRRSV-NA, enabling rapid and accurate identification of the PRRSV genotypes prevalent in pig populations. This provides a robust technical tool for the development of targeted immunization and prevention strategies. Full article

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: 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

Babesia microti is a protozoan that infects red blood cells, causing hemolytic anemia and flu-like symptoms in humans. Understanding co-infections is crucial for the better diagnosis, treatment, and management of tick-borne diseases. This study examined the prevalence of Babesia microti co-infection with other prevalent tick-borne pathogens in Pennsylvania. The dataset acquired from the Dr. Jane Huffman Wildlife Genetics Institute included passive surveillance data from Ixodes spp. from 2021 to 2023. Submitted ticks were screened for tick-borne pathogens using species-specific TaqMan qPCR. Of the 793 B. microti-positive ticks pulled for analysis, 65.0% were co-infected with other pathogens (n = 516). Notably, 60.9% of the B. microti-positive ticks were co-infected with Borrelia burgdorferi, 10.2% with Anaplasma phagocytophilum Ap-ha, and 7.5% carried a triple co-infection with B. burgdorferi and A. phagocytophilum Ap-ha. The rates of B. microti infection and its co-infections are on the rise, with patterns observed in Pennsylvania and other regions of the USA. While other studies have collected both nymphal and adult ticks to screen for co-infections in Pennsylvania, our study stood out as a unique contribution to the field by focusing exclusively on B. microti-positive ticks. The continued monitoring of tick-borne co-infections is vital to prevent misdiagnosis and ensure effective treatment regimens. Full article

Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens. Full article