Development and validation of a quantitative PCR assay for detection of Sulawesi tortoise adenovirus

Abstract

In 2007, a mortality event involving over 100 Sulawesi tortoises (Indotestudo forsteni), two Impressed tortoises (Manouria impress) and a critically endangered Burmese star tortoise (Geochelone platynota) was attributed to Sulawesi tortoise adenovirus (STADV; genus Siadenovirus). We developed a TaqMan quantitative PCR assay targeting the DNA polymerase gene of STADV for use in clinical diagnosis and epidemiologic surveillance. This assay failed to amplify five closely-related chelonian adenoviruses, indicating high analytical specificity. The assay performed with high efficiency (slope = −3.337; R² = 0.999) and high inter- and intra-assay repeatability (coefficient of variation <1.36 % at all standard curve dilutions). Dynamic range included 1.00 × 10⁷ to 1.00 × 10¹ target copies per reaction and limit of detection was 10¹ target copies per reaction, though 10⁰ target copies per reaction were intermittently detected. This qPCR assay provides a valuable diagnostic tool for characterization of STADV epidemiology, including potential identification of the North American reservoir host.