比较单重、嵌套、半嵌套、多重和嵌套多重 PCR 方法在鉴定临床疑似感染性眼内炎的微生物病因中的应用。

Comparative evaluation of uniplex, nested, semi-nested, multiplex and nested multiplex PCR methods in the identification of microbial etiology of clinically suspected infectious endophthalmitis.

机构信息

Department of Microbiology & Molecular Biology, Aravind Eye Hospital & Postgraduate Institute of Ophthalmology, SN High Road, Tirunelveli 627001, Tamil Nadu, India.

出版信息

Curr Eye Res. 2013 May;38(5):550-62. doi: 10.3109/02713683.2013.772205. Epub 2013 Feb 28.

DOI:10.3109/02713683.2013.772205

PMID:23448514

Abstract

PURPOSE

This study is aimed to determine the utility of various polymerase chain reaction (PCR) methods in vitreous fluids (VFs) for detecting the infectious genomes in the diagnosis of infectious endophthalmitis in terms of sensitivity and specificity.

METHODS

This prospective and consecutive analysis included a total of 66 VFs that were submitted for the microbiological evaluation, which were obtained from 66 clinically diagnosed endophthalmitis patients presented between November 2010 and October 2011 at the tertiary eye care referral centre in South India. Part of the collected VFs were subjected to cultures and smears, and the remaining parts were utilized for five PCR methods: uniplex, nested, semi-nested, multiplex and nested multiplex after extracting DNA, using universal eubacterial and Propionibacterium acnes species-specific primer sets targeting 16S rRNA gene in all bacteria and P. acnes, and panfungal primers, targeting 28S rRNA gene in all fungi.

RESULTS

Of the 66 VFs, five (7.5%) showed positive results in smears, 16 (24%) in cultures and 43 (65%) showed positive results in PCRs. Among the 43 positively amplified VFs, 10 (15%) were positive for P. acnes genome, one for panfungal genome and 42 (62%) for eubacterial genome (including 10 P. acnes positives). Among 42 eubacterial-positive VFs, 36 were positive by both uniplex (first round) and multiplex (first round) PCRs, while nested (second round) and nested multiplex (second round) PCRs produced positive results in 42 and 41 VFs, respectively. Of the 43 PCR-positive specimens, 16 (37%) had positive growth (15 bacterial and one fungal) in culture. Of 50 culture-negative specimens, 27 (54%) were showed positive amplification, of which 10 were amplified for both P. acnes and eubacterial genomes and the remaining 17 were for eubacterial genome alone.

CONCLUSIONS

Nested PCRs are superior than uniplex and multiplex PCR. PCRs proved to be a powerful tool in the diagnosis of endophthalmitis, especially for detecting uncultured microbes.

摘要

目的

本研究旨在通过灵敏度和特异性，确定聚合酶链反应（PCR）方法在眼内液（VF）中检测感染性内源性眼内炎的传染性基因组的应用价值。

方法

本前瞻性连续分析共纳入 66 例 VF，这些 VF 来自 2010 年 11 月至 2011 年 10 月在印度南部的一家三级眼科转诊中心就诊的 66 例临床诊断为内源性眼内炎的患者。收集的部分 VF 进行培养和涂片，其余部分用于 5 种 PCR 方法：单重 PCR、嵌套 PCR、半嵌套 PCR、多重 PCR 和嵌套多重 PCR，方法是提取 DNA 后，使用针对所有细菌和痤疮丙酸杆菌 16S rRNA 基因的通用细菌和痤疮丙酸杆菌种特异性引物组，以及针对所有真菌 28S rRNA 基因的泛真菌引物组。

结果

66 例 VF 中，5 例（7.5%）涂片阳性，16 例（24%）培养阳性，43 例（65%）PCR 阳性。在 43 例扩增阳性的 VF 中，10 例（15%）痤疮丙酸杆菌基因组阳性，1 例泛真菌基因组阳性，42 例（62%）细菌基因组阳性（包括 10 例痤疮丙酸杆菌阳性）。在 42 例细菌阳性的 VF 中，36 例同时通过单重（第一轮）和多重（第一轮）PCR 阳性，而嵌套（第二轮）和嵌套多重（第二轮）PCR 分别在 42 例和 41 例 VF 中产生阳性结果。在 43 例 PCR 阳性标本中，16 例（37%）培养阳性（15 例细菌和 1 例真菌）。在 50 例培养阴性标本中，27 例（54%）显示阳性扩增，其中 10 例同时扩增痤疮丙酸杆菌和细菌基因组，其余 17 例仅扩增细菌基因组。