title: Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification.
abstract: Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70min at an isothermal temperature of 63°C. The sensitivity of LAMP with the detection limit of 362fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.
authors.affiliation: Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou, PR China.
authors.forename: Xuelian
authors.initials: X
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Dayang
authors.initials: D
authors.lastname: Zou
authors.forename: Xuesong
authors.initials: X
authors.lastname: Wang
authors.forename: Zhan
authors.initials: Z
authors.lastname: Yang
authors.forename: Zhitao
authors.initials: Z
authors.lastname: Yin
authors.forename: Qian
authors.initials: Q
authors.lastname: Cui
authors.forename: Wei
authors.initials: W
authors.lastname: Shang
authors.forename: Huan
authors.initials: H
authors.lastname: Li
authors.forename: Xiao
authors.initials: X
authors.lastname: Wei
authors.forename: Jing
authors.initials: J
authors.lastname: Cui
authors.forename: Zhongquan
authors.initials: Z
authors.lastname: Wang
authors.forename: Liuyu
authors.initials: L
authors.lastname: Huang
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000235:genetics; D007814:Larva / Q000235:genetics; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D017160:Trichinella spiralis / Q000302:isolation & purification*; D014235:Trichinellosis / Q000469:parasitology*
chemical_list: D018509:DNA, Helminth