Mazoteras Paloma, Bispo Paulo José Martins, Höfling-Lima Ana Luisa, Casaroli-Marano Ricardo P
Department of Surgery, School of Medicine, University of Barcelona (UB) , Barcelona , Spain .
Curr Eye Res. 2015 Jul;40(7):697-706. doi: 10.3109/02713683.2014.957327. Epub 2014 Oct 6.
Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification.
Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out.
The column-based extraction method was the most time-effective, achieving DNA detection limits ≥10(2) and 10(3 )CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4).
The column-based extraction method for very small inocula in small volume samples (50-100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.
比较三种从眼内液中提取DNA的不同方法,随后通过通用PCR扩增检测细菌和真菌DNA。
评估了三种从房水和玻璃体液中提取DNA的方法,以比较它们的相对效率。本研究使用了细菌(革兰氏阳性和阴性)和真菌菌株:大肠杆菌、表皮葡萄球菌和白色念珠菌。分析了DNA提取和PCR扩增的质量、定量及检测限。对20例临床疑似眼内炎患者的13份房水样本和14份玻璃体样本进行了验证程序。
基于柱的提取方法最省时,细菌和真菌的DNA检测限分别≥10(2)和10(3)CFU/100µL。PCR扩增分别检测到100fg、1pg和10pg的大肠杆菌、表皮葡萄球菌和白色念珠菌基因组DNA。PCR从20例临床疑似眼内炎患者采集的27份眼内样本中检测出90.0%的病原体,而标准微生物技术仅能检测出60.0%。最常见的微生物是链球菌属,在38.9%(n = 7)的患者中发现,葡萄球菌属在22.2%(n = 4)的患者中发现。
对于小体积样本(50 - 100µL)的房水和/或玻璃体液中非常少量的接种物,基于柱的提取方法能使所有样本进行PCR扩增,且质量足以进行后续测序并在大多数样本中鉴定微生物。