title: A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle.
abstract: A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discriminating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as well as the 3'-end of the small subunit rDNA and 5'-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by a single DNA fragment for Ostertagia ostertagi (257bp), Haemonchus placei (176bp), Oesophagostomum radiatum (329bp), Trichostrongylus colubriformis (243bp) and Cooperia oncophora (151bp). In a similar manner, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogeneity in the ETS which is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitative technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs.
authors.affiliation: US Department of Agriculture, ARS, Immunology and Disease Resistance Laboratory, Bldg. 1180, BARC-East, Beltsville, MD 20705, USA. zarlenga@anri.barc.usda.gov
authors.forename: D S
authors.initials: DS
authors.lastname: Zarlenga
authors.forename: M
authors.initials: M
authors.lastname: Barry Chute
authors.forename: L C
authors.initials: LC
authors.lastname: Gasbarre
authors.forename: P C
authors.initials: PC
authors.lastname: Boyd
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000469:parasitology*; D018509:DNA, Helminth / Q000032:analysis; D005243:Feces / Q000469:parasitology; D006189:Haemonchus / Q000302:isolation & purification; D010028:Ostertagia / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D017183:Strongylida / Q000302:isolation & purification*; D017206:Strongylida Infections / Q000469:parasitology / Q000662:veterinary*; D013320:Strongyloidea / Q000302:isolation & purification; D014251:Trichostrongyloidea / Q000302:isolation & purification
chemical_list: D018509:DNA, Helminth