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结合聚合酶链反应(polymerase chain reaction,PCR)和酶切方法,对鹰嘴豆象Callosobruchus analis(Fabricius)和四纹豆象Callosobruchus maculatus(Fabricius)的分子检测技术进行了研究。根据两种豆象m tDNACOⅠ基因序列的比对结果设计了2对特异性PCR引物,并筛选到2个特异性内切酶SnaⅠ和MboⅠ,通过PCR反应(最适退火温度为55℃)和PCR产物的酶切反应检测2个近缘种。可靠性检测表明,2对引物针对不同地理种群和个体以及不同DNA浓度的鹰嘴豆象和四纹豆象均能扩增出相应的目的条带。限制性内切酶验证结果表明,PCR技术具有较高的准确性和灵敏度,弥补了形态鉴定的不足。
The molecular detection techniques of Callosobruchus analis (Fabricius) and Callosobruchus maculatus (Fabricius) were studied by polymerase chain reaction (PCR) and restriction enzyme digestion. Two pairs of specific PCR primers were designed according to the results of the sequence comparison of the two cDNA sequences of m tDNACOⅠand two specific endonucleases SnaⅠ and MboⅠ were screened. The PCR reaction (optimum annealing temperature was 55 ℃) and PCR The enzyme digestion reaction detected two closely related species. The reliability test showed that two pairs of primers could amplify the corresponding bands according to different geographic populations and individuals as well as the chickpea and four-legumes with different DNA concentrations. The results of restriction endonuclease test showed that PCR technology has higher accuracy and sensitivity, which make up for the lack of morphological identification.