酸奶中长双歧杆菌PCR计数方法的建立

Study on a new method for counting Bifidobacterium longum in yogurt by PCR

根据细菌保守序列设计一对通用引物,扩增细菌16S rDNA序列,作为阳性标记;根据长双歧杆菌菌株的16S rRNA和23S rRNA之间的间区基因序列设计定性PCR引物。用BBL琼脂平板对长双歧杆菌混合发酵的酸奶进行培养计数,随机挑选部分长出的菌落,提取DNA做PCR鉴定。将PCR技术与传统平板计数技术结合起来,初步建立酸奶中长双歧杆活菌计数方法。抽提方法操作简便、高效、灵敏,决定着PCR计数方法的准确高效,本研究着重研究了菌体DNA抽提方法的影响因素,以及抽提方法通用性,同时也研究了PCR引物的特异性。将PCR技术与传统平板计数技术结合起来,初步建立酸奶中长双歧杆活菌计数方法。

This study tried to initially establish a method for counting Bifidobacterium Iongum in yogurt by using polymerase chain reaction（PCR） technique and traditional plate count method technique.Based on the conserved sequence of 16S rDNA sequences of bacteria,a pair of universal primer were designed as a positive mark. And a pair of special primers were also designed and synthesized according to 16SN23S rRNA gene of the Bifidobacteriurn Iongum. The randomly selected colonies of the mixed fermented yogurt culturing on BBL agar plate were abstracted and identified by PCR. PCR technology combines with the traditional plate count method technology,established the method for counting the living Bifidobacterium Iongum in the mixed fermented yogurt . Operation of extraction method must be simple,efficient,responsive and accurate,and it determined the efficient of PCR counting method.This study focused on the factors that influencing the bacterial DNA extraction,as well as the versatility of the bacterial DNA extraction,but also the specificity of the PCR primers for Bifidobacterium Iongum. A method for counting Bifidobacterium Iongum in yogurt was initially established by using polymerase chain reaction（PCR） technique and traditional plate count method technique.