Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA.
Palo Alto Medical Foundation Toxoplasma Serology Laboratory, National Reference Center for the Study and Diagnosis of Toxoplasmosis, Palo Alto, California, USA.
Br J Ophthalmol. 2019 Apr;103(4):569-572. doi: 10.1136/bjophthalmol-2018-313064. Epub 2019 Jan 12.
is the most common infectious cause of posterior uveitis worldwide. Two multicopy targets (B1 and Rep529) are commonly used in PCR assays, but studies evaluating these targets in ocular fluid samples are limited. Herein, we determine the analytical characteristics of a single-reaction, internally controlled, dual-target, real-time PCR and evaluate the clinical performance of this assay in intraocular fluid samples obtained at a reference ophthalmologic centre in the USA.
Lower limits of detection for the B1 and Rep529 components of the dual-target assay were determined using serial dilutions of cultured strain Z185. The dual-target assay was then used to test 148 archived intraocular samples (132 vitreous,16 aqueous humour) collected at the Francis I. Proctor Foundation between January 2010 and December 2015 for testing by a nested, conventional PCR targeting the B1 gene.
The 95% lower limits of detection for the dual-target assay was determined to be 1.05 tachyzoites/mL for B1 and 0.83 tachyzoites/mL for Rep529. Using archived clinical intraocular specimens, the dual-target assay demonstrated 97.2% positive per cent agreement (n=35/36; 95% CI 83.7% to 99.9%) and 99.1% negative per cent agreement (n=111/112; 95% CI 94.4% to 100%) compared with the nested, conventional B1 PCR.
This single-reaction, internally controlled, dual-target (B1, Rep529) real-time PCR for the detection of DNA in intraocular specimens demonstrated excellent agreement with nested, conventional, B1 PCR. The dual-target design may ensure detection when variation is present in one of two target regions.
是全球最常见的引起后葡萄膜炎的感染性原因。两种多拷贝靶标（B1 和 Rep529）常用于 PCR 检测，但关于这些靶标在眼内液样本中的研究有限。本文旨在确定单反应、内部对照、双靶标实时 PCR 的分析特性，并评估该检测方法在位于美国的一家参考眼科中心获得的眼内液样本中的临床应用效能。
采用连续稀释培养株 Z185 的方法，确定双靶标检测中 B1 和 Rep529 两个组分的检测下限。然后，采用双靶标检测方法对 Francis I. Proctor 基金会于 2010 年 1 月至 2015 年 12 月期间收集的 148 份存档眼内样本（132 份玻璃体、16 份房水）进行检测，这些样本由巢式常规 B1 基因 PCR 检测进行过检测。
双靶标检测的 95%检测下限确定为 B1 为 1.05 速殖子/ml，Rep529 为 0.83 速殖子/ml。使用存档的临床眼内标本，双靶标检测与巢式常规 B1 PCR 相比，阳性百分符合率为 97.2%（n=35/36；95%CI 83.7%至 99.9%），阴性百分符合率为 99.1%（n=111/112；95%CI 94.4%至 100%）。
该用于检测眼内标本中 DNA 的单反应、内部对照、双靶标（B1、Rep529）实时 PCR 与巢式常规 B1 PCR 具有极好的一致性。双靶标设计可确保在两个靶标区域之一存在变异时进行检测。
