PCR-mtDNA技术鉴别检测不同动物肌肉组织和饲料中鸭源性成分

PCR-mtDNA for Detecting Components of Duck Origin in Foodstuff and Feedstuff

摘要:

以鸭肌肉组织DNA为模板, 利用PCR-mtDNA技术成功克隆出了鸭mtDNA COIII基因(GenBank Accession No. DQ655706)。对所克隆的序列分析表明, 其序列包括鸭细胞色素C氧化酶III(COIII)基因全序列784 bp, 通过同源性分析可知, 动物的线粒体DNA COIII基因是相对保守的, 利用此特性设计PCR-mtDNA方法鉴别检测鸭源性成分的特异性引物; 以各种动物肌肉组织及饲料DNA为模板进行PCR扩增、经反复验证筛选出只能扩增出鸭DNA的目的片段, 而不能扩增出其他动物DNA片段的特异性强、稳定性好的引物P3、P4; 利用此引物PCR扩增鸭DNA的特异性片段为226 bp, 对PCR产物进行测序分析可知与已克隆的鸭mtDNA COIII基因同源性达到100%, 证明了所筛选引物的准确性。通过对不同含量的DNA模板溶液进行PCR扩增的方法, 对筛选出的特异性引物P3、P4进行灵敏度试验, 结果分析表明灵敏度约为0.001%, 证明该PCR方法具有特异性强、灵敏度高的特点, 完全可作为鉴别不同动物肌肉组织和饲料中鸭源性成分的方法。

Abstract:

Mitochondrial cytochrome oxidase III(COIII) of duck was successfully amplified by PCR-mtDNA with duck muscle DNA as the template (GenBank Accession No. DQ655706). Cloning sequence analysis shows that the 784 bp nucleotides of COIII gene were contained. Through homology analysis, we confirmed that the cytochrome oxidase III(COIII) was relatively conservative. The method of PCR-mtDNA can be designed to detect the components of duck origin. And then, the method of PCR can be applied to amplify with the muscle DNA of various animal and feedstuff as the template, repeated verification, the primer ( P3, P4 ) with strong specificity and good stability is screened, which can only amplify the sequence of duck. The special sequence contains 226bp, the amplified product of 226 bp was sequenced and analyzed, it showed 100% homology with duck mtDNA COIII gene, which proved the accuracy of the special primer. The test that used different concentration of DNA with P3 and P4 is the sensitive experiment by PCR. The result showed that the primer has much specialty and rather sensitivity. So it is a way to detect the duck origin in the muscle of various animal and feedstuff.