Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA.
A real-time fluorogenic polymerase chain reaction (PCR) assay based on SYBR green that allows for sensitive, reproducible, and accurate quantification of Babesia gibsoni (Asian genotype). DNA from peripheral blood of infected dogs was developed. Standard curves were created by plotting the input amount of a standard template, constructed with plasmid DNA containing 182 base pairs (bp) of the p18 gene, against threshold cycle numbers. The curves showed a wide dynamic range (1,000,000-fold input) and high correlation values (>0.99). The PCR amplification efficacy of the standard template was similar to that of intact genomic DNA obtained from peripheral blood with B. gibsoni infection. The detection limit of the assay was 9 parasites/microl of blood with B. gibsoni infection. The intra-assay and interassay coefficients of variation of the threshold cycles ranged from 0.70% to 1.89% and from 1.18% to 1.92%, respectively. This assay system was found to be reproducible and accurate for the quantification of parasite DNA in experimentally infected dogs and far more sensitive than traditional microscopic examination.
Department of Small Animal Medicine, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori, Japan.
Aya
A
Matsuu
Satomi
S
Ono
Hiromi
H
Ikadai
Tsuyoshi
T
Uchide
Saiki
S
Imamura
Misao
M
Onuma
Shozo
S
Okano
Seiichi
S
Higuchi
D000818:Animals; D001403:Babesia / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics*; D004283:Dog Diseases / Q000175:diagnosis / Q000469:parasitology; D004285:Dogs; D005260:Female; D005456:Fluorescent Dyes; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary*; D012680:Sensitivity and Specificity
D016054:DNA, Protozoan; D005456:Fluorescent Dyes