应用种特异性PCR技术快速鉴定辣椒实蝇

【目的】辣椒实蝇Bactrocera latifrons(Hendel)为我国重要的检疫性有害生物,其寄主范围广泛,危害严重。由于传统鉴定方法受到饲养周期、饲养条件、虫态等因素的限制,使得果蔬进出口贸易通关速度、疫情快速鉴定受到较大的影响,因此迫切需要开发关于实蝇的快速鉴定识别的技术。【方法】本研究基于mt DNA COI序列设计了一对能够准确鉴定辣椒实蝇的种特异性引物FL680和RL1057,选用辣椒实蝇作为阳性对照,选用番石榴实蝇B.correcta(Bezzi)、桔小实蝇B.dorsalis(Hendel)和颜带实蝇B.cilifer(Hendel)等20种实蝇作为阴性对照,进行PCR扩增并将PCR产物进行电泳检测。【结果】仅目标种辣椒实蝇能够扩增出清晰且单一的约378 bp的条带,其余实蝇种类均未出现条带。将本实验建立的种特异性PCR(SS-PCR)鉴定方法应用于实际检疫工作中并得到了验证,表明该方法具有强的种特异性。【结论】本文提出辣椒实蝇快速鉴定识别技术可应用于实蝇的疫情监测和口岸的检疫检测工作。

Rapid identification of Bactrocera latifrons (Diptera: Tephritidae) by using species-specific PCR technique

【Aim】 Bactrocera latifrons (Hendel) is one of the most important plant quarantine pests in plant production and trade, and it has wide host range and causes serious damage. Traditional identification methods are limited by such factors as raising period, feeding conditions and developmental stage, thus limiting the clearance rate of customs and quick identification of infestation situation of fruits. For this reason, a rapid identification technique for fruit fly species is desperately needed. 【Methods】 A pair of primers, FL680 and RL1057, was designed and synthesized based on the mtDNA COI sequence. For the PCR amplification and gel electrophoresis, B. latifrons (Hendel) was chosen as the positive control and other 20 species of fruit flies including B. correcta (Bezzi), B. dorsalis (Hendel) and B. cilifer (Hendel) were used as the negative controls. 【Results】 A clear and single 378 bp band was amplified only from B. latifrons, but not from the other 20 fruit flies. The SS-PCR identification method established in this experiment was applied and verified in inspection and quarantine, indicating that this method has high species specificity. 【Conclusion】 The rapid identification method developed here for B. latifrons can be applied in infestation monitoring and quarantine surveillance of fruit flies in ports.