Open AccessConventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes
Author(s) -
Ronald J. Sayler,
Courtney J. Walker,
Fiona L. Goggin,
Paula Agudelo,
T. L. Kirkpatrick
Publication year - 2012
Publication title -
plant disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.663
H-Index - 108
eISSN - 1943-7692
pISSN - 0191-2917
DOI - 10.1094/pdis-12-11-1033-re
Subject(s) - biology , taqman , rotylenchulus reniformis , nematode , meloidogyne incognita , heterodera , dna extraction , real time polymerase chain reaction , polymerase chain reaction , internal transcribed spacer , veterinary medicine , gene , genetics , ribosomal rna , ecology , medicine
Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.