基于DPO引物的实时荧光PCR检测微生物肥料中的鼠李糖乳杆菌

Real- time PCR for Detection of Lactobacillus rhamnosus Based on Dual Priming Oligonucleotide System

【目的】建立一种快速、准确的鼠李糖乳杆菌检测方法。【方法】根据已报道的鼠李糖乳杆菌(Lactobacillus rhamnosus)的gyr B基因保守序列,设计用于检测鼠李糖乳杆菌的特异性DPO引物,结合SYBR Green I实时荧光PCR技术,建立鼠李糖乳杆菌的实时荧光PCR检测方法,评价其特异性和灵敏度,并将建立的检测方法用于实际样品的检测中。【结果】该方法对2株鼠李糖乳杆菌能得到阳性扩增,而其余10种乳酸菌及阴性对照没有扩增曲线,而且在退火温度为50~60℃不影响其特异性;该实时荧光PCR检测灵敏度比农业部标准中普通PCR检测高10倍;用10种微生物肥料及其它益生菌产品进行了验证,检出情况与产品标识一致。【结论】研究建立的SYBR GreenⅠ实时荧光PCR能够准确、高效的检测微生物肥料及其他益生菌产品中的鼠李糖乳杆菌。

[ Objective ] To develop a method for the detection of Lactobacillus rhamnosus. [ Method ] The species- specific DPO （dual priming oligonucleotide） primers were designed based on thegyrB gene se- quences of L. rhamnosus. A SYBR Green I real -time PCR assay based on DPO primers was developed for the detection of L. rhamnosus. The specificity and sensitivity of the assay have been estimated. [ Result ] The results showed that this method was of high specificity, and only two L. rhamnosuz strains can be amplified. The other 10 L. spp. strains gave negative results. The detection sensitivity of this real -time PCR was more than 10 times higher than the conventional PCR. [ Conclusion] SYBR Green I real -time PCR method estab- lished in this study can detect L. rhamnosus accurately and rapidly in the microbial fertilizers and other probi- otic products.