Kinchington P R, Turse S E, Kowalski R P, Gordon Y J
Charles T. Campbell Laboratory for Infectious Diseases, Department of Ophthalmology, University of Pittsburgh, Pennsylvania 15213.
Invest Ophthalmol Vis Sci. 1994 Nov;35(12):4126-34.
To evaluate the application of polymerase chain reaction (PCR) methodology as a potential diagnostic tool for the detection of adenovirus DNA in ocular swab samples.
Oligonucleotides derived from the adenovirus hexon gene were used to amplify a 306-base pair (bp) product by PCR. Radiolabeled oligonucleotides derived from sequences within the amplified product were used as specific probes. Specificity was determined against DNA of 13 adenovirus serotypes (types 1 to 11, inclusive, and types 19 and 37) and from nonadenoviral DNAs. Limits of detection were determined by PCR amplification of known amounts of purified adenovirus serotype 2 DNA. The assay was tested on 107 ocular swab samples and correlated to results obtained from tissue culture and a commercial immunoassay (Adenoclone).
The 306-bp PCR product was amplified from all adenovirus serotypes tested, but not from negative control DNAs. As little as 15 fg of adenovirus type 2 DNA could be detected by PCR and ethidium bromide stain. Using a simplified sample preparation procedure, 46 of 58 adenovirus culture-positive but Adenoclone-negative swabs were positive by PCR (79% sensitivity). All (11 of 11) Adenoclone-positive clinical eye swabs tested were positive by PCR (100% sensitivity). Only 1 of 38 nonadenoviral ocular swab samples was positive by PCR (97% specificity).
PCR appeared to be highly suitable for the diagnosis of adenovirus in ocular swabs, offering important improvements in speed over tissue culture isolation and in sensitivity over immunoassay.
评估聚合酶链反应（PCR）方法作为检测眼拭子样本中腺病毒DNA的潜在诊断工具的应用。
使用源自腺病毒六邻体基因的寡核苷酸通过PCR扩增出306个碱基对（bp）的产物。将源自扩增产物序列的放射性标记寡核苷酸用作特异性探针。针对13种腺病毒血清型（包括1至11型以及19和37型）的DNA和非腺病毒DNA确定特异性。通过对已知量的纯化腺病毒2型DNA进行PCR扩增来确定检测限。该检测方法在107份眼拭子样本上进行测试，并与组织培养和商业免疫测定（腺病毒克隆检测法）的结果相关联。
从所有测试的腺病毒血清型中均扩增出306 bp的PCR产物，但从阴性对照DNA中未扩增出。通过PCR和溴化乙锭染色，可检测到低至15 fg的腺病毒2型DNA。使用简化的样本制备程序，58份腺病毒培养阳性但腺病毒克隆检测法阴性的拭子中有46份通过PCR检测为阳性（灵敏度79%）。所有测试的11份腺病毒克隆检测法阳性的临床眼拭子均通过PCR检测为阳性（灵敏度100%）。38份非腺病毒眼拭子样本中只有1份通过PCR检测为阳性（特异性97%）。
PCR似乎非常适合用于眼拭子中腺病毒的诊断，与组织培养分离相比，在速度上有重要改进，与免疫测定相比，在灵敏度上有提高。
