Quantitative Detection ofNeospora caninumin Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR

Abstract

We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10 ؊1 tachyzoite per assay was observed with excellent linearity (R 2 ‫؍‬ 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques ( ‫؍‬ 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection.

Absolute Quantification of Plasmid DNA by Real-time PCR with Genomic DNA as External Standard and Its Application to a Biodistribution Study of an HIV DNA Vaccine

“…Secondly, the inhibitory effect of gDNA on real-time PCR must be addressed, and a universal reference must be established for absolute quantification across tissue types. An inhibitory effect of the gDNA matrix on amplification efficiency in realtime PCR had been mentioned in previous studies 6,7,13 but was finally fully validated in the present study.…”

“…Incorporation of fluorescencebased detection systems into real-time PCR instruments not only allows kinetic detection of the accumulation of PCR products over the cycling period, but also provides greater sensitivity for amplicon detection as compared to conventional gel-based detection. Major uses of real-time PCR include analysis of differential mRNA expression, 1,2 SNP detection, 3 cancer marker quantification, 4,5 biodistribution of pathogen, [6][7][8] and plasmid DNA vaccine. 9,10 Absolute quantification to measure the copy number of a particular target present in cells or tissue remains technically difficult due to the lack of methodology that can comprehensively account for all the variation that can occur during sample preparation.…”

Experimental neosporosis in bulls: Parasite detection in semen and blood and specific antibody and interferon-gamma responses

“…caninum DNA from nested-PCR positive semen samples was quantified by a real-time PCR test based on the Nc-5 sequence using the double-stranded DNAbinding (dsDNA) dye SYBR Green I. Amplification, data acquisition, and data analysis of reactions for the Neospora Nc5 sequence were carried out in the ABI 7700 Prism Sequence Detector (Applied Biosystems), as described by Collantes-Fernández et al [10]. N. caninum organism number was quantified by interpolation of the corresponding Ct values (cycle threshold: the fractional cycle number reflecting a positive PCR result) using a DNA standard curve equivalent to 10 À1 -10 4 tachyzoites.…”

Effects of purine nucleotide administration on purine nucleotide metabolism in brains of heroin-dependent rats

“…The cycle threshold values (Ct) were calculated and exported to Microsoft Excel for analysis. Quantification was performed by experimental determination of Ct, defined as the cycle at which the fluorescence exceeds 10 times the standard deviation of the mean baseline emission for the early cycles (Collantes-Fernandez et al, 2002). The Pfaffl mathematical model was used for qRT-PCR data analyses (Pfaffl, 2001).…”