Dalapathy S, Lily T K, Roy S, Madhavan H N
Microbiology Research Centre, Vision Research Foundation, Chennai, India.
J Clin Virol. 1998 Jul 24;11(1):77-84. doi: 10.1016/s0928-0197(98)00021-x.
The standard virus isolation method for detecting adenovirus is time consuming and direct detection of viral antigens in smears has its limitations. Therefore a rapid and a reliable method to identify virus in clinical specimens is desirable.
To develop and evaluate nested PCR as a tool for detecting adenovirus from conjunctival swabs of patients with acute conjunctivitis during an epidemic.
A total of 201 patients with acute conjunctivitis were seen between August and November 1996. Conjunctival swabs from the most recently affected eyes were collected from 20 random patients and processed for antigen detection in direct smears, for adenovirus, enterovirus (EV70) and coxsackievirus A24 variant and adenovirus isolation by culture. Nested PCR was performed using oligonucleotides to amplify 1004 basepair (bp) and 956 bp fragments of DNA coding for adenovirus hexon protein. The neutralisation test, to type the adenovirus, was done on four isolates selected at random.
The PCR could detect 0.0032 fg of adenovirus DNA (corresponding to 8.3 x 10(-3) adenovirus particles). The EV70 and coxsackievirus A24 antigens were not detected. The specimens were positive for adenovirus by all three techniques in seven patients: (a) by direct smear and PCR in 2; (b) by virus isolation and PCR in 2; and (c) by PCR alone in five patients. In one patient the direct smear alone was positive. The PCR required 3 days to detect the virus, antigen detection provided diagnosis the same day and virus isolation required 8-27 days. A total of four isolates selected at random were identified as serotype 7a.
The nested PCR is a reliable and rapid technique for detection of adenovirus from conjunctival swabs. The adenovirus serotype 7a was the likely causative agent of this epidemic conjunctivitis.
检测腺病毒的标准病毒分离方法耗时较长，而直接检测涂片上的病毒抗原有其局限性。因此，需要一种快速且可靠的方法来鉴定临床标本中的病毒。
开发并评估巢式聚合酶链反应（nested PCR）作为在疫情期间从急性结膜炎患者结膜拭子中检测腺病毒的工具。
1996年8月至11月期间共诊治了201例急性结膜炎患者。从20例随机选取的患者中采集最近受影响眼睛的结膜拭子，进行直接涂片抗原检测、检测腺病毒、肠道病毒（EV70）和柯萨奇病毒A24变种，并通过培养进行腺病毒分离。使用寡核苷酸进行巢式PCR，以扩增编码腺病毒六邻体蛋白的1004碱基对（bp）和956 bp的DNA片段。对随机选取的4株分离株进行腺病毒分型的中和试验。
PCR可检测到0.0032 fg的腺病毒DNA（相当于8.3×10⁻³个腺病毒颗粒）。未检测到EV70和柯萨奇病毒A24抗原。7例患者的标本通过所有三种技术检测均为腺病毒阳性：（a）2例通过直接涂片和PCR检测为阳性；（b）2例通过病毒分离和PCR检测为阳性；（c）5例仅通过PCR检测为阳性。1例患者仅直接涂片呈阳性。PCR检测病毒需要3天，抗原检测可在当天提供诊断，病毒分离需要8至27天。随机选取的4株分离株均被鉴定为7a型。
巢式PCR是一种从结膜拭子中检测腺病毒的可靠且快速的技术。腺病毒7a型可能是此次流行性结膜炎的病原体。
