Use of chimeric DNA-RNA primers in quantitative PCR for detection of Ehrlichia canis and Babesia canis.
To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine beta-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.
Peleg
Ofer
O
Genaphora Ltd, Tel Aviv 69710, Israel. ofer.peleg@gmail.com
Baneth
Gad
G
Eyal
Osnat
O
Inbar
Jacob
J
Harrus
Shimon
S
D000199:Actins / Q000235:genetics; D000818:Animals; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics*; D004285:Dogs; D041061:Ehrlichia canis / Q000235:genetics / Q000302:isolation & purification*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
D000199:Actins; D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; C034477:RNA primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012313:RNA