双歧杆菌属特异性定量引物的设计及验证

Design and evaluation of Bifidobacterium genus-specific primer for quantification

摘要:

[目的] 旨在设计一对双歧杆菌属特异性引物以检测不同样品中低丰度双歧杆菌的含量。[方法] 在NCBI中下载57株双歧杆菌全基因组序列，以其共有单拷贝核心基因为目的片段设计双歧杆菌属特异性引物；并对引物进行PCR初筛和特异性复筛；之后借助ddPCR（Droplet Digital PCR，微滴式数字PCR）依次对筛选出的引物进行特异性、灵敏度和实用性验证。[结果] 引物Bif-D-9特异性最好，可扩增出4株双歧杆菌而不能扩增20株非双歧杆菌中的任何一株菌；同时通过ddPCR仪定量稀释后的DNA，其扩增结果呈线性下降趋势，证明其灵敏度较好；另外，Bif-D-9结合ddPCR定量出婴儿粪便中双歧杆菌的拷贝数为71 copies/μL，母亲粪便中双歧杆菌的拷贝数为2.7 copies/μL，证明了该方法的实用性。[结论] 引物Bif-D-9具有双歧杆菌属特异性，且灵敏度较高、实用性较好，适用于复杂样品中双歧杆菌属定量。

关键词:双歧杆菌属;特异性引物;定量;验证;微滴式数字PCR

Abstract:

[Objective] The aim of this study was to design a pair of absolute quantitative specific primers for detecting the number of the low content Bifidobacterium in different samples.[Methods] The complete genome sequence of 57 strains of Bifidobacterium was downloaded from NCBI, and the specific primers of Bifidobacterium were designed based on their common single-copy core genes. Primers were screened by PCR and rescreened by specificity. Then ddPCR (Droplet Digital PCR) was used to verify the specificity, sensitivity and practicability of the selected primers.[Results] The specificity of Bif-D-9 primer was best, and 4 strains Bifidobacterium were amplified but 20 strains of non-Bifidobacterium could not be amplified. The diluted DNA was quantitatively by ddPCR, and the amplification results show a linear decreasing trend, which proves that the sensitivity is better. At the same time, Bif-D-9 combined with ddPCR can quantitatively determine the copy number of Bifidobacterium in infant feces is 71 copies/μL, and the maternal feces contained 2.7 copies/μL, which proves the practicality is good.[Conclusion] The primer Bif-D-9 was high specificity, high sensitivity and accuracy of Bifidobacterium, and is suitable for the quantitative study of Bifidobacterium in complex environmental samples.

Key words:Bifidobacterium;genus-specific primer;quantitative;evaluation;droplet digital PCR