Development and evaluation of a single step duplex PCR for simultaneous

Development and evaluation of a single step duplex PCR for simultaneous 1 detection of Fasciola hepatica and Fasciola gigantica 2 (Fasciolidae; Trematoda; Platyhelminthes) 3 Thanh Hoa Le, Khue Thi Nguyen, Nga Thi Bich Nguyen, Huong Thi Thanh Doan, 4 Xuyen Thi Kim Le, Chau Thi Minh Hoang, and Nguyen Van De 5 Department of Immunology, Institute of Biotechnology, Vietnam Academy of Science and 6 Technology, 18. Hoang Quoc Viet Rd, Cau Giay, Hanoi, Vietnam. 7 Department of Parasitology, Hanoi Medical University, 1. Ton That Tung st, Dong Da, Hanoi, 8 Vietnam 9 ABSTRACT 10 A single-step multiplex PCR (hereafter, duplex PCR) has been developed for simultaneous 11 detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in 12 distribution in many countries of North and East Africa, Central and Southeast Asia and are 13 similar in egg morphology, difficult for identification from fecal samples. Based on a 14 comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, 15 two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. 16 gigantica), and a single reverse FHGR (common for both species). Conventional PCR followed 17 by sequencing was applied using species-specific primer pairs to verify specificity of primers 18 and identity of Fasciola DNA templates. Duplex PCR (using 3 primers) was assayed to test with 19 the DNA extracted from adult worms, miracidia and eggs, producing an amplicon of 1031 bp for 20 F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other 21 common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an 22 echinostomid, another fasciolid and a taeniid cestode. The sensitivity assay showed that the 23 duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng 24 DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults; 4 eggs), and 25 25 field-collected stools of ruminants and humans, revealed specific bands of the correct size and 26 Fasciola species present. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate 27 identification of Fasciola species in areas of distributional and zonal overlap. 28