PCR-RFLP法三种凝胶电泳快速鉴定分枝杆菌的比较研究

Identification of mycobacterimn species by 3 types of gel electrophoresiss of PCR- RFLP

目的:比较2%一般琼脂糖凝胶、2%Metaphor琼脂糖凝胶和10%非变性聚丙烯酰胺凝胶分析分枝杆菌标准株酶切片段的准确性。方法:采用通用引物对分枝杆菌hsp65基因序列进行PCR扩增,限制性内切酶BstE Ⅱ和Hae Ⅲ对扩增产物进行消化,分别采用2%一般琼脂糖凝胶、2%Metaphor琼脂糖凝胶和10%非变性聚丙烯酰胺凝胶比较分枝杆菌标准株酶切片段的准确性。结果:与标准酶切图谱相比,所有10%非变性聚丙烯酰胺凝胶酶切片段差异均在15 bp以内,而2%Metaphor琼脂糖凝胶和2%一般琼脂糖凝胶则超过15 bp,甚至少数条带不能显示。结论:三种方法中以PCR-RFLP非变性聚丙烯酰胺凝胶法的准确性较高。

Objective： To compare the accuracy of the fi＇agment sizes of PCR- RFLP using 2% agarose gel, 2% Metaphor agarose gel and 10% polyacrylamide gel. Methods： PCR was performed targeting the common hsp65 gene in mycobacteria. Two restriction enzymes, BstE Ⅱ and Hae Ⅲ, were used to digest the PCR products. The abovementioned three gels were used to compare the accuracy of the fragment sizes. Results： Compared with standard restriction patterns, all the fragments generated by digestion on 10% polyacrylamide gel had a size difference by less than ± 15 bp, 2% Metaphor agarose gel and 2% agarose gel by more than ± 15 bp, and few even no bands. Conclusion： 10% polyaerylamJde gel yields more precise estimates of fragment sizes.