Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan.
Microbiol Immunol. 2012 Jan;56(1):10-20. doi: 10.1111/j.1348-0421.2011.00405.x.
A sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was developed for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli by using specific primers. Counts of the enteric pathogens spiked in human stools were quantified at the lower detection limit of 10(3) cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 10(5) to 10(6) cells/g stool. The bacterial counts determined by RT-qPCR were almost equivalent to those determined by the culture method and fluorescence in situ hybridization (FISH) during the course of in vitro culture. Bacterial rRNA in the stools was stable for at least 4 weeks when the stools were kept as the suspensions in RNA-stabilizing agent, RNAlater®, even at 37(o) C. These data suggested that the rapid and high sensitive rRNA-targeted RT-qPCR was applicable for the accurate quantification of viable enteric pathogens, such as V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli.
建立了一种灵敏的 rRNA 靶向逆转录定量聚合酶链反应（RT-qPCR）方法，用于检测霍乱弧菌/拟态弧菌、副溶血性弧菌/algino 溶血性弧菌和空肠弯曲菌/结肠弯曲菌，使用特异性引物。通过 RT-qPCR 在 10(3)细胞/g 粪便的较低检测限下定量检测人粪便中添加的肠道病原体，与常规定量聚合酶链反应（qPCR）在 10(5)至 10(6)细胞/g 粪便的检测限形成鲜明对比。在体外培养过程中，通过 RT-qPCR 确定的细菌计数与培养方法和荧光原位杂交（FISH）确定的细菌计数几乎相当。当粪便保持在 RNA 稳定剂 RNAlater®中作为悬浮液时，粪便中的细菌 rRNA 至少稳定 4 周，即使在 37°C 时也是如此。这些数据表明，快速和高灵敏的 rRNA 靶向 RT-qPCR 适用于准确定量检测活的肠道病原体，如霍乱弧菌/拟态弧菌、副溶血性弧菌/algino 溶血性弧菌和空肠弯曲菌/结肠弯曲菌。
