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

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

魏霜;孟茹;乾义柯;孙凯;刘中勇;鄞杰平;周广彪;吴希阳

汕头出入境检验检疫局,广东汕头,515041;伊犁出入境检验检疫局,新疆伊宁,835000;暨南大学理工学院食品科学与工程系,广州,510632

收稿日期:2017-01-25 修回日期:2017-01-25 出版日期:2017-01-25 发布日期:2017-01-25

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

WEI Shuang;MENG Ru;QIAN Yi-ke;SUN Kai;LIU Zhong-yong;YIN Jie-ping;ZHOU Guang-biao;WU Xi-yang

Received:2017-01-25 Revised:2017-01-25 Online:2017-01-25 Published:2017-01-25

摘要/Abstract

摘要： [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(Lactoba-cillus rhamnosus)的gyrB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 Green Ⅰ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.

Abstract: [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 sequences 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.rhamnosus 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 established in this study can detect L.rharanosus accurately and rapidly in the microbial fertilizers and other probiotic products.

魏霜;孟茹;乾义柯;孙凯;刘中勇;鄞杰平;周广彪;吴希阳. 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[J]. .

WEI Shuang;MENG Ru;QIAN Yi-ke;SUN Kai;LIU Zhong-yong;YIN Jie-ping;ZHOU Guang-biao;WU Xi-yang. Real-time PCR for Detection of Lactobacillus rhamnosus Based on Dual Priming Oligonucleotide System[J]. .