Efficient polymerase chain reaction detection of the second internal transcribed spacer of mucosa-derived larvae is dependent on the larval extraction method.
Methods for estimating abundance of arrested gastrointestinal larvae in large mammal hosts by digestion of the gastrointestinal mucosa are well established. The effects of digestion on the success of species identification using the polymerase chain reaction (PCR) are, however, unknown. In this study, the relationship between numerical recovery of arrested larvae and the success of PCR-typing for the second internal transcribed spacer of ribosomal genes was characterized. Fresh and prefrozen mucosa of 4 sheep yielded very similar rates of recovery and PCR detection. When sheep mucosa were digested with neutral N-acetyl cysteine, recovery increased, whereas PCR detection remained constant (60-80%) with digest duration (1-16 hr). In contrast, when sheep and Svalbard reindeer mucosa were digested with acid-pepsin, recovery increased, whereas PCR detection declined to 0 with digest duration. Thus, to optimize recovery and PCR analysis of arrested gastrointestinal nematode larvae, acid-pepsin digestion of 1-2 hr for PCR detection and 16 hr for recovery, or neutral N-acetyl cysteine digestion of 8-16 hr for both assays, should be used.
Irvine
R J
RJ
Centre for Ecology and Hydrology Banchory, Hill of Brathens, Banchory, Aberdeenshire AB31 4BW, U.K. ji@ceh.ac.uk
Dallas
J F
JF
D000111:Acetylcysteine; D000818:Animals; D018509:DNA, Helminth / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D003670:Deer / Q000469:parasitology*; D006851:Hydrochloric Acid; D007814:Larva; D009348:Nematoda / Q000235:genetics / Q000302:isolation & purification*; D010434:Pepsin A; D016133:Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep / Q000469:parasitology*; D013997:Time Factors
D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer; D010434:Pepsin A; D006851:Hydrochloric Acid; D000111:Acetylcysteine