A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax |
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| Institution: | 1. Graduate School Khon Kaen University, 123 Mittraphap Highway, Khon Kaen 40002, Thailand;2. Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand;3. Department of Pathology, Faculty of Medicine, Khon Kaen University, 123 Mittraphap Highway, Khon Kaen 40002, Thailand;4. Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, 123 Mittraphap Highway, Khon Kaen 40002, Thailand;5. Department of Parasitology and Entomology, Faculty of Public Health, Mahidol University, 420/1 Ratchawithi Road, Ratchathewi District, Bangkok 10400, Thailand;6. Department of Epidemiology, Faculty of Public Health, Khon Kaen University, 123 Mittraphap Highway, Khon Kaen 40002, Thailand;7. Department of Clinical Microscopy, Faculty of Associated Medical Sciences, Khon Kaen University, 123 Mittraphap Highway, Khon Kaen 40002, Thailand;1. Pediatric Infectious Disease Unit, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel;2. Parasitology Laboratory, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel;1. Internal Medicine Department, Groupement Hospitalier Sud, Hospices Civils de Lyon, France;2. Thoracic Surgery Department, Groupement Hospitalier Sud, Hospices Civils de Lyon, France;3. Visceral Surgery Department, Groupement Hospitalier Sud, Hospices Civils de Lyon, France;4. Laboratory of Parasitology, Groupement Hospitalier Nord, Hospices Civils de Lyon, France;5. Université Claude Bernard, Lyon 1, France;1. Laboratory of Helminthology, Institute of Veterinary Medicine, Zaisan, Ulaanbaatar, Mongolia;2. Department of Parasitology, Asahikawa Medical University, Asahikawa, Japan;3. Laboratory of Protozoology, Institute of Veterinary Medicine, Zaisan, Ulaanbaatar, Mongolia;4. Department of Medical Biology and Histology, School of Biomedicine, Health Science University of Mongolia, Ulaanbaatar, Mongolia;5. Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, TX, USA;1. Department of Veterinary Parasitology, United Graduate School of Veterinary Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan;2. Department of Veterinary Parasitology, Faculty of Applied Biological Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan;3. Division of Experimental Animals, Center for Promotion of Medical Research and Education, Graduate School of Medicine, Nagoya University, Tsurumai 65 Showa-ku, Nagoya 466-8550, Japan |
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| Abstract: | This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380 bp for An. minimus, 180 bp for An. harrisoni, 150 bp for An. aconitus, 310 bp for An. varuna, 276 bp for P. falciparum, and 300 bp for P. vivax. The sensitivities were 0.5 pg/μl (25 sporozoites/μl) for P. falciparum DNA and between 0.5 and 5 pg/μl (25–250 sporozoites/μl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes. |
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