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

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods Pub Date : 2024-09-21 DOI:10.1016/j.jviromet.2024.115033

Zachary C. Ready , Laura Adamovicz , Maris Daleo , Amber Simmons , Matthew C. Allender

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引用次数: 0

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.

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开发和验证用于检测苏拉威西陆龟腺病毒的定量 PCR 检测方法。

2007年，100多只苏拉威西陆龟（Indotestudo forsteni）、两只印象陆龟（Manouria impress）和一只极度濒危的缅甸星龟（Geochelone platynota）死亡，原因是苏拉威西陆龟腺病毒（STADV；Siadenovirus属）。我们开发了一种针对 STADV DNA 聚合酶基因的 TaqMan 定量 PCR 检测方法，用于临床诊断和流行病学监测。该测定未能扩增出五种密切相关的螯腺病毒，这表明该测定具有高度的分析特异性。该测定的效率高（斜率 = -3.337；R2 = 0.999），测定间和测定内重复性高（变异系数为 7 至 1.00 × 101 目标拷贝/反应，检测限为 101 目标拷贝/反应，但间歇检测到 100 目标拷贝/反应）。这种 qPCR 检测方法为确定 STADV 流行病学特征提供了一种有价值的诊断工具，包括对北美贮藏宿主的潜在鉴定。

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来源期刊

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.