高特异性聚合酶链反应法鉴别大、小黄鱼鱼鳔及其混淆品

Identification of Swimming Bladder of Pseudosciaena crocea and Pseudosciaena polyactis and Their Adulterants by Highly Specific PCR

目的:建立可鉴别大、小黄鱼鱼鳔及其混淆品的高特异性聚合酶链式反应(PCR)方法。方法:根据大、小黄鱼及6种常见混淆品线粒体12S r RNA基因序列,设计两对专用于大、小黄鱼鱼鳔的鉴别引物Fish-F1、Fish D-R1与Fish-F1、Fish X-R2;采用PCR法在不同的复性温度扩增,确立特异性反应条件。结果:在94℃预变性5 min;94℃变性30 s,60.4℃复性30 s,72℃延伸30 s,30个循环;72℃延伸5 min扩增条件下,大黄鱼鱼鳔得到约670 bp的扩增带,而小黄鱼和混淆品鱼鳔在同样的条件下无扩增带。在94℃预变性5 min;94℃变性30 s,64.8℃复性30 s,72℃延伸30 s,30个循环;72℃延伸5 min扩增条件下,小黄鱼鱼鳔得到约670 bp的扩增带,而大黄鱼和混淆品鱼鳔在同样的条件下无扩增带。结论:设计的鉴别引物对大、小黄鱼鱼鳔有高度的特异性。本方法简单、准确、快速、重现性好、稳定性高,能有效鉴别大、小黄鱼鱼鳔及其混淆品。

OBJECTIVE： To develop PCR method for the identification of swimming bladder of Pseudosciaena crocea and Pseudosciaena polyactis and their adulterants. METHODS ： Based on the sequences of mitochondrial 12S rRNA gene of P. crocea and P. polyactis and 6 kinds of common adulterants, two pairs of highly specific primers （Fish-F1 and FishD-R1, Fish-F1 and FishX-R2） were designed for distinguishing P. crocea and R polyactis respectively. The primers were amplified by PCR at different annealing temperatures to confirm specific reaction condition. RESULTS： 12S rRNA gene was pre-denatured for 5 min at 94 ℃; 12S rRNA gene was denatured for 30 s at 94 ℃, and then renatured for 30 s at 60.4 ℃, finally extended for 30 s at 72 ℃, the tri- al repeated for 30 cycles; a fragment about 670 bp was amplified only from swimming bladder ofP. crocea but not from P. polyac- tis or adulterants when 12S rRNA gene was amplified for 5 min at 72 ℃. 12S rRNA gene was pre-denatured for 5 min at 94 ℃ ; 12S rRNA gene was denatured for 30 s at 94 ℃, and then renatured for 30 s at 64.8 %, finally extended for 30 s at 72 ℃, the tri- al repeated for 30 cycles; a fragment about 670 bp was amplified only from swimming bladder ofP. polyactis but not from P. poly- actis or adulterants when amplified for 5 min at 72 ℃. CONCLUSIONS： Designed specific primers are highly specific to swim- ming bladder of P. crocea and P. polyactis. Established method is stable, simple, accurate, rapid and reproducible, and can be used for the identification of P. crocea, P. polyactis and their adulterants.