Wilkes Rebecca P, Anis Eman, Dunbar Dawn, Lee Pei-Yu A, Tsai Yun-Long, Lee Fu-Chun, Chang Hsiao-Fen G, Wang Hwa-Tang T, Graham Elizabeth M
1 Clinical Virology Laboratory, University of Tennessee Veterinary Medical Center, Knoxville, TN, USA.
*Current address: Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia, Tifton, GA, USA.
J Feline Med Surg. 2018 Apr;20(4):362-369. doi: 10.1177/1098612X17712847. Epub 2017 Jun 7.
Objectives Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection.
目的 猫白血病病毒（FeLV）是一种γ逆转录病毒，可引发猫造血系统疾病，这些疾病通常是致命的。在需求点（PON）进行快速准确的检测有助于预防病毒传播和临床疾病管理。本研究评估了用于检测前病毒DNA的绝缘等温PCR（iiPCR）以及用于检测病毒RNA和前病毒DNA的逆转录（RT）-iiPCR在PON处检测FeLV的性能。方法 使用溶血支原体、猫冠状病毒、猫疱疹病毒、猫杯状病毒和猫免疫缺陷病毒检测分析特异性。体外转录RNA、人工质粒、FeLV毒株美国典型培养物保藏中心VR-719和一株临床FeLV分离株用于分析灵敏度测定。开展一项回顾性研究，纳入116份已通过病毒分离、实时PCR和ELISA检测的临床血浆和血清样本，以及一项前瞻性研究，纳入150份临床血浆和血清样本，以评估基于iiPCR的方法检测FeLV的临床性能。结果 计算得出RT-iiPCR的95%检测限为16个RNA拷贝和5个DNA拷贝，iiPCR为6个DNA拷贝。两种反应的分析灵敏度与参考实时PCR（qPCR）相当，且未检测到5种非目标猫病原体。RT-iiPCR和iiPCR的临床性能与qPCR（n = 8）分别有98.82%的一致性（kappa[κ]=0.97）和100%的一致性（κ = 1.0）。在回顾性研究（n = 1）和前瞻性研究（n = 150）中，自动核酸提取/RT-iiPCR系统与病毒分离检测血浆或血清中FeLV的一致性分别为95.69%（κ = 0.95）和98.67%（κ = 0.85）。结论及意义 这些结果表明，RT-iiPCR和iiPCR检测均可作为PON处检测FeLV的可靠工具。
