title: Detection of Babesia canis vogeli and Hepatozoon canis in canine blood by a single-tube real-time fluorescence resonance energy transfer polymerase chain reaction assay and melting curve analysis.
abstract: A real-time fluorescence resonance energy transfer polymerase chain reaction (qFRET PCR) coupled with melting curve analysis was developed for detection of Babesia canis vogeli and Hepatozoon canis infections in canine blood samples in a single tube assay. The target of the assay was a region within the 18S ribosomal RNA gene amplified in either species by a single pair of primers. Following amplification from the DNA of infected dog blood, a fluorescence melting curve analysis was done. The 2 species, B. canis vogeli and H. canis, could be detected and differentiated in infected dog blood samples (n = 37) with high sensitivity (100%). The detection limit for B. canis vogeli was 15 copies of a positive control plasmid, and for H. canis, it was 150 copies of a positive control plasmid. The assay could simultaneously distinguish the DNA of both parasites from the DNA of controls. Blood samples from 5 noninfected dogs were negative, indicating high specificity. Several samples can be run at the same time. The assay can reduce misdiagnosis and the time associated with microscopic examination, and is not prone to the carryover contamination associated with the agarose gel electrophoresis step of conventional PCR. In addition, this qFRET PCR method would be useful to accurately determine the range of endemic areas or to discover those areas where the 2 parasites co-circulate.
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Amornmas
authors.initials: A
authors.lastname: Kongklieng
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Pewpan M
authors.initials: PM
authors.lastname: Intapan
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Thidarut
authors.initials: T
authors.lastname: Boonmars
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Tongjit
authors.initials: T
authors.lastname: Thanchomnang
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Penchom
authors.initials: P
authors.lastname: Janwan
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Oranuch
authors.initials: O
authors.lastname: Sanpool
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Viraphong
authors.initials: V
authors.lastname: Lulitanond
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Piyanan
authors.initials: P
authors.lastname: Taweethavonsawat
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat).
authors.forename: Sudchit
authors.initials: S
authors.lastname: Chungpivat
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases (Kongklieng, Intapan, Boonmars, Thanchomnang, Janwan, Sanpool, Lulitanond, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandDepartments of Parasitology (Kongklieng, Intapan, Boonmars, Maleewong), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandMicrobiology (Lulitanond), Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandFaculty of Medicine, Mahasarakham University, Mahasarakham, Thailand (Thanchomnang, Sanpool)Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand (Janwan)Parasitology Unit, Department of Pathology, Faculty of Veterinary Science Chulalongkorn University, Bangkok, Thailand (Taweethavonsawat, Chungpivat) wanch_ma@kku.ac.th.
authors.forename: Wanchai
authors.initials: W
authors.lastname: Maleewong
mesh_terms: D000818:Animals; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis*; D003044:Coccidia / Q000235:genetics / Q000302:isolation & purification*; D003048:Coccidiosis / Q000175:diagnosis / Q000662:veterinary*; D017931:DNA Primers; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000662:veterinary; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D012337:RNA, Ribosomal, 18S
keywords: Babesia; Hepatozoon; real-time fluorescence resonance energy transfer polymerase chain reaction