An easily-automated assay for the physiological state quantification of Pseudomonas sp. M18.
In order to foreknow poorly performing cultures before wasting energy to scale them to large cultures, industrial microbial fermentation can greatly benefit from knowledge of the physiological state of cells. The method currently proposed is an easily automated physiological state determination method. We have designed one universal rRNA-specific probe for bacteria and developed novel signal probe hybridization (SPH) assay featuring no RNA extraction and no PCR amplification steps necessary to quantify the physiological state of microbial cells. The microbial cell was lysed with sonication and SDS. Signal probes were applied to hybridize and protect the rRNA target. S1 nuclease was then applied to remove the excessive signal probes, the single-stranded RNA and the mismatch RNA/DNA hybrids. The remaining signal probe was captured with a corresponding capture probe immobilized on a microplate and quantified with a horseradish peroxidase-conjugated color reaction. We then systemically optimized our assay. Results showed that the cell limit of detection (LOD) and the cell limit of quantification (LOQ) were 2.64 x 10(4) cells and 9.86 x 10(4) cells per well of microplate, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of signal probe were 49.0 fM and 344.0 fM respectively. Using this technique, we quantified the 16S rRNA levels during the fermentation process of Pseudomonas sp. M18. Our results indicate that the 16S rRNA levels can directly inform us about the physiological state of microbial cells. This technique has great potential for application to the microbial fermentation industry.
Dong
Dexian
D
MOE Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China.
Zhou
Kejun
K
Zhou
Quan
Q
Huang
Xianqing
X
Xu
Yuquan
Y
Li
Rongxiu
R
D001331:Automation; D005285:Fermentation; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics / Q000502:physiology*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S