Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection |
| |
| Institution: | 1. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Longzihu Campus of Henan Agricultural University, No. 15 Longzihu University Area, Zhengdong New District, Zhengzhou, 450046, PR China;2. School of Biotechnology and Food, Shangqiu Normal University, Wenhua Road 298, Shangqiu, 476000, PR China;1. State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China;2. Mudanjiang Forestry Central Hospital, Mudanjiang, Heilongjiang Province, China;3. Shanghai Institute of Medical Genetics, Shanghai Jiaotong University, Shanghai, China;4. Departments of Pathology and Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA;1. Department of Medical Microbiology and Hygiene, University of Mainz, Obere Zahlbacherstrasse 67, D-55131 Mainz, Germany;2. IDEXX Diavet, Schlyffistrasse 10, CH-8806 Bäch, Switzerland;3. IDEXX Laboratories, Mörikestrasse 28/3, D-71636 Ludwigsburg, Germany;4. Clinic for Swine and Small Ruminants, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany;5. Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald - Insel Riems, Germany;6. Department of Biology, Montclair State University, 1 Normal Ave., Montclair, NJ, 07043, USA;7. Sackler Institute for Comparative Genomics, The American Museum of Natural History, Central Park West and 79th Street, New York, NY, 10024, USA;1. Laboratoire de Microbiologie, Ecole Nationale de Médecine Vétérinaire, Institution de la Recherche et de l’Enseignement Supérieur Agricoles, Université de La Manouba, Sidi Thabet, Tunisia;2. Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy;3. Laboratoire d’Entomologie, Institut Pasteur de Tunis, Tunis, Tunisia |
| |
| Abstract: | Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species.We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described.The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 102 copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections. |
| |
| Keywords: | Duplex PCR qPCR Real-time PCR |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
