School of Applied Sciences, Ellison Building, University of Northumbria, Newcastle upon Tyne, UK.
Lett Appl Microbiol. 2010 Sep;51(3):272-7. doi: 10.1111/j.1472-765X.2010.02891.x. Epub 2010 Jun 20.
The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF).
Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT-PCR-DGGE and qPCR analysis of the bacterial and fungal communities was carried out.
Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae.
The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4 degrees C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.
本研究旨在定量和定性评估样本储存对囊性纤维化 (CF) 患者痰液样本中代谢活跃的微生物群落的影响。
收集等量的两份痰液样本，一份立即浸入 RNAlater 并冷藏，另一份在室温下储存 24 小时，随后加入 RNAlater。提取 mRNA，并进行细菌和真菌群落的 RT-PCR-DGGE 和 qPCR 分析。
两种方案之间观察到细菌群落存在显著差异，但真菌群落分析未见显著差异。qPCR 分析表明，室温储存会导致大肠杆菌和铜绿假单胞菌的统计学显著增加，流感嗜血杆菌的统计学显著减少。
使用分子技术分析 CF 痰液中的代谢活跃微生物群落表明，加入 RNAlater 后应将样本储存在 4°C 以准确描述 CF 肺部微生物组。此外，室温储存呼吸道样本可能会导致铜绿假单胞菌过度表达，并掩盖其他具有临床意义的生物体的存在。
