title: Development and application of loop-mediated isothermal amplification assays based on ITS-1 for rapid detection of Toxoplasma gondii in pork.
abstract: The loop-mediated isothermal amplification (LAMP) assay is a novel method that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. In this study, we established a LAMP assay with six primers targeting a highly conserved region of Toxoplasma gondii ITS-1 sequence. The amplification protocol completes within 30 min under isothermal condition in a 65°C water bath while specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Eimeria tenella, Cryptosporidium parvum, Listeria monocytogenes and Streptococcus suis. The detection limit of the LAMP assay was 0.9 fg T. gondii genomic DNA, a sensitivity that was 10-fold higher than that of a conventional PCR assay. Both LAMP assay and conventional PCR were applied to detect T. gondii genomic DNA in 118 diaphragm samples obtained from pig farms in Zhejiang Province, China. Our results showed that the LAMP assay is more sensitive than conventional PCR (13.56% and 9.32%). The LAMP assay established in this study provides a simple, specific, sensitive and rapid method of T. gondii genomic DNA detection, hence is expected to plays an important role in the monitoring of T. gondii contamination in various food products.
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Xunhui
authors.initials: X
authors.lastname: Zhuo
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Huang
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Jiaqing
authors.initials: J
authors.lastname: Luo
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Haijie
authors.initials: H
authors.lastname: Yu
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Baolong
authors.initials: B
authors.lastname: Yan
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China. Electronic address: afdu@zju.edu.cn.
authors.forename: Aifang
authors.initials: A
authors.lastname: Du
mesh_terms: D000818:Animals; D021901:DNA, Intergenic / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics*; D016266:Food Parasitology / Q000379:methods*; D008460:Meat / Q000469:parasitology*; D021141:Nucleic Acid Amplification Techniques; D011091:Polyesters; D012680:Sensitivity and Specificity; D013552:Swine; D014122:Toxoplasma / Q000302:isolation & purification*
chemical_list: D021901:DNA, Intergenic; D016054:DNA, Protozoan; D011091:Polyesters; C016240:polycaprolactone
keywords: ITS-1; Loop-mediated isothermal amplification (LAMP); Toxoplasma gondii