A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle

A multiplex polymerase chain reaction (PCR) test was developed for identify ing gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discrim inating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and ex ternal (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as wel l as the 3'-end of the small subunit rDNA and 5'-end of the large subunit r DNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern ch aracterized by a single DNA fragment for Ostertagia ostertagi (257 bp), Hae monchus placei (176 bp), Oesophagostomum radiatum (329 bp), Trichostrongylu s colubriformis (243 bp) and Cooperia oncophora (151 bp). In a similar mann er, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemo nchus contortus, Trichostrongylus axel, Cooperia surnabada and Cooperia pun ctata. With respect to H. contortus, a closely migrating doublet was genera ted suggesting size heterogeneity in the ETS which is consistent with multi ple rDNA repeat units within this species. PCR analyses using mixtures of m onospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitati ve technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs. Published by Els evier Science B.V.