优化并应用逆转录聚合酶链反应来确定传染性眼内炎中的细菌活力。

Optimization and application of a reverse transcriptase polymerase chain reaction to determine the bacterial viability in infectious endophthalmitis.

机构信息

Larsen and Toubro Microbiology Research Centre, Kamal Nayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

出版信息

Curr Eye Res. 2012 Dec;37(12):1114-20. doi: 10.3109/02713683.2012.704476. Epub 2012 Jul 3.

DOI:10.3109/02713683.2012.704476

PMID:22757687

Abstract

PURPOSE

To develop RNA based assay - reverse transcriptase polymerase chain reaction (RT-PCR) to detect viable bacteria in intraocular specimens obtained from patients with infectious endophthalmitis.

MATERIALS AND METHODS

Thirty-five intraocular specimens (19 vitreous fluid and 16 aqueous humor) collected from patients with typical infectious endophthalmitis were subjected to conventional and molecular microbiological investigations. Culture negative, eubacterial genome PCR positive intraocular specimens were subjected to denaturing high performance liquid chromatography (dHPLC) for separation of mixed genomes and subsequently identified by PCR based DNA sequencing. In parallel, RT-PCR was performed to detect the presence of viable bacteria in intraocular specimens.

RESULTS

Among 35 intraocular specimens, single bacterial genome was detected in 9 (25.7%) and two or more genomes in 26 (74.28%) intraocular specimens. Eubacterial genome was detected by RT-PCR in 29 (82.85%) specimens. PCR based dHPLC followed by PCR based DNA sequencing revealed the presence of 65 bacterial genomes in 35 intraocular specimens. Five novel genera namely Terrabacter species, Facklamia species, Xylella fastidiosa, Duganella species and Synechococcus species were detected.

CONCLUSION

RT-PCR serves as a rapid and reliable tool to detect viable bacteria causing endophthalmitis.

摘要

目的

开发基于 RNA 的检测方法——逆转录聚合酶链反应（RT-PCR），以检测患有感染性眼内炎患者的眼内标本中的存活细菌。

材料和方法

对 35 份来自患有典型感染性眼内炎患者的眼内标本（19 份玻璃体和 16 份房水）进行了常规和分子微生物学研究。对培养阴性、细菌基因组 PCR 阳性的眼内标本进行变性高效液相色谱（dHPLC）分离混合基因组，然后通过基于 PCR 的 DNA 测序进行鉴定。同时，进行 RT-PCR 以检测眼内标本中存活细菌的存在。

结果

在 35 份眼内标本中，9 份（25.7%）标本中检测到单个细菌基因组，26 份（74.28%）标本中检测到两个或更多基因组。RT-PCR 检测到 29 份（82.85%）标本中的细菌基因组。基于 PCR 的 dHPLC 随后进行基于 PCR 的 DNA 测序，在 35 份眼内标本中发现了 65 个细菌基因组。检测到了 5 个新属，分别是 Terrabacter 种、Facklamia 种、Xylella fastidiosa、Duganella 种和 Synechococcus 种。