实时荧光定量PCR检测感病蝗虫体内绿僵菌rDNA基因

Detection of Metarhizium anisopliae rDNA in Haemalymph of Infected Adult Locust by Real-time Fluorescence Quantitative PCR

根据绿僵菌23SrDNA的转录间隔区(ITS)和5.8S的基因序列,设计检测感病蝗虫体内绿僵菌菌体DNA分子的特异引物,通过优化感病蝗虫菌体DNA快速制备方法,建立了基于荧光定量PCR的定量检测体系。该检测方法专一、灵敏,检测下限为10pg/μl绿僵菌,并且能从刚侵染48h的东亚飞蝗血淋巴中,早期定量检测到在虫体血淋巴中增殖的绿僵菌的rDNA。

According to the internal transcription spacer （ITS） sequence of 23S rDNA and the gene sequence of 5.8S rDNA of Metarhizium anisopliae var. acridum CQMa102 , a specific primer pair of fluorescence quantitative PCR was designed, and a modified extraction method of DNA from the infected haemolymph of male adult locust was optimized. By combination of the specific primer pair and the DNA extraction method, a premycoses quantitative deaection approach for rDNA of insect pathogen in the haemolymph of infected locust was established using real-time fluorescent PCR, which is rapid, sensitive and specific. With this method, rDNA of Metarhizium anisopliae in the haemolymph of locust could be detected 36h post-inoculation.