A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations.
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http://dx.doi.org/10.1016/j.mrfmmm.2014.05.013 | DOI Listing |
J Vet Diagn Invest
January 2025
Department of Obstetrics, Gynecology and Reproductive Biology, Massachusetts General Hospital, Boston, MA, USA.
Equid alphaherpesvirus 4 (EqAHV4; , ; equine rhinopneumonitis virus) has seldom been associated with complications such as abortion and myeloencephalopathy, given the low tendency of this virus to induce viremia. We investigated the frequency of EqAHV4 viremia in horses with fever and respiratory signs. Case selection included all equids with EqAHV4 quantitative real-time PCR (qPCR)-positive nasal secretions (defined as EqAHV4 qPCR-positive cases) submitted to a diagnostic laboratory.
View Article and Find Full Text PDFJ Vet Diagn Invest
January 2025
Brookfield Zoo Chicago, Brookfield, IL, USA.
Bacteria in the complex and nontuberculous mycobacteria may affect a variety of animal species under human care and pose public health risks as zoonotic pathogens. A case of sudden onset of lethargy and increased respiratory effort in a 5-y-old, intact female reindeer () under managed care had progressed to severe dyspnea despite aggressive treatment. The animal was euthanized due to poor prognosis.
View Article and Find Full Text PDFSci Rep
January 2025
Institute of Emerging Infectious Diseases, Korea University, Seoul, Republic of Korea.
This study aimed to evaluate the usefulness of amplicon-based real-time metagenomic sequencing applied to cerebrospinal fluid (CSF) for identifying the causative agents of bacterial meningitis. We conducted a 16S rRNA amplicon sequencing using a nanopore-based platform, alongside routine polymerase chain reaction (PCR) testing or bacterial culture, to compare its clinical performance in pathogen detection on CSF samples. Among 17 patients, nanopore-based sequencing, multiplex PCR, and bacterial culture detected potential bacterial pathogens in 47.
View Article and Find Full Text PDFTrop Anim Health Prod
January 2025
Department of Biology, College of Science, Sultan Qaboos University, PC. 123, Muscat, Sultanate of Oman.
Bluetongue virus (BTV) has emerged as a significant concern in Oman, affecting various animal species, including camels. This cross-sectional study aimed to assess the seroprevalence of BTV in camels and explore the associated risk factors within the northern region of Oman. Between October 2016 and March 2017, 439 serum samples and 100 blood samples were collected from camels in five governorates.
View Article and Find Full Text PDFReprod Sci
January 2025
Department of Physiology, College of Basic Medicine, Chongqing Medical University, Chongqing, 400016, China.
Polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder affecting women of reproductive age. Oxidative stress (OS) is suggested to play a significant role in the development of PCOS. Using antioxidants to reduce OS and maintain a healthy balance in the body could be a novel treatment approach for PCOS.
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