一种直接对牙龈炎龈沟液进行PCR扩增的方法

A method for direct PCR amplification of bacteria in gingival cervial fluid in gingivitis

目的利用加入突变TaqDNA聚合酶等的优化方法实现人类牙齿龈沟液中的细菌及人类基因组片段的直接扩增。方法随机选取10例牙龈炎患者。用无菌注射器分别吸取10μL的龈沟液。然后各吸取2μL,标记为第一组,分别加入突变TaqDNA聚合酶及DMSO,通过改变PCR反应体系,直接实现龈沟液中细菌的16sRNA及MAOA-VNTR基因的扩增;再分别吸取2μL,标记为第二组,此组先采用微量DNA提取试剂盒来实现DNA的提取,然后扩增龈沟液中细菌的16sRNA及MAOA-VNTR基因。结果直接扩增法所获得的扩增产物比传统提取DNA法多2个,两种方法的转化子阳性率无差别;MAOA-VNTR可扩增出单倍体,串联体等。结论直接PCR法可以用于龈沟液中细菌的初步检测以及人类基因组小片段的扩增,也可应用于牙龈炎或牙周炎患者的口腔龈沟液研究及临床诊断。

Objective To achieve the direct amplification of bacteria in gingival cervical fluid（ GCF） and fragments of the human genome by using the optimization method of joining mutant TaqDNA polymerase. Methods Ten cases of patients with gingivitis were selected randomly,and 10 μL of GCF from them were absorbed by a sterile syringe respectively. The samples were divided into two groups,the first group was added with mutant TaqDNA polymerase and DMSO in 2 μL GCF respectively,the 16 sRNA and MAOA-VNTR gene of bacteria were amplifed directly by changing the reaction system of PCR. The second group,DNA was extracted using DNA extraction kit firstly and then amplified the 16 sRNA and MAOA-VNTR gene of bacteria in2 μL GCF. Results Two more amplification products were obtained by direct amplification rather than traditional method. There was no significant difference between the two methods on transformants positive rate; Haploidy and concatemer were amplified by MAOA-VNTR. Conclusion The direct PCR method can not only be used to detect bacteria in GCF and small fragments of human being＇s genome,but also be applied to the study of GCF and clinical diagnosis in gingivitis and periodontitis.