高通量DNA快速提取方法及其在柞蚕微孢子虫分子诊断中的应用

High-Throughput DNA Extraction Technology and Its Applications in the Molecular Diagnosis of Nosema pernyi

针对柞蚕微孢子虫分子检测受限于繁琐的DNA提取而局限于实验室使用的瓶颈问题,以成本较低的碱裂解法结合磁珠抽提,建立了一种全程在96孔板上使用排枪操作的高通量DNA提取方法(HT提取),对比HT-冻融法和HT-SDS法2种裂解方法获得的DNA在常规PCR与荧光定量PCR(qPCR)中的检测下限,并进行微粒子病筛查的应用。结果表明,HT提取法与传统方法相比,无繁琐预处理,操作快速、通量高,污染低、抗干扰、提取DNA品质好,主要耗材能重复使用,可自动化,人力物力成本较低;HT-SDS法具有更稳定的裂解效果,获得的DNA用于常规PCR、qPCR检测下限可达45和0045个/μL;HT-冻融法在常规PCR与qPCR中的检测下限为45和045个/μL,而传统抽提方法的常规PCR检测下限仅为110个/μL。结合HT-SDS法和qPCR,对3 000个样品进行微粒子病筛查,结果显示,其检测灵敏度显著高于现行镜检技术,最高高出近11倍,且对低带毒品种优势尤为显著,有望实现在柞蚕种茧微粒子病检疫及柞蚕种质资源保护中的应用,促进柞蚕业发展。

Since Nosema pernyi detection is confined to laboratory due to time-consuming DNA preparation,a highthroughput method performed on a 96-well plate was introduced using low-cost alkaline lysis followed by magnetic bead separation,which was named as HT-extraction. Meanwhile,limit of detection(LOD)in conventional PCR and qPCR using DNA as template were compared by 2 alkaline lysis approaches including HT-freeze-thaw method and HTSDS method,and then optimized method was applied for pebrine screening. Results showed that in contrast to conventional method,HT-extraction is quicker in preprocessing and performance,better in throughput and extracted DNA,and less in pollution and interference. Besides,its major consumables can be recycled and automatized,and cost of human and material resources can be lowered down. HT-SDS method had higher stability,by which LOD of conventional PCR and qPCR using DNA as template were 4 5 spores/μL and 0 045 spores/μL respectively. LOD of conventional PCR and qPCR using DNA as template by HT-freeze-thaw method were 45 spores/μL and 0 45 spores/μL. However,LOD of conventional PCR using DNA as template by conventional method was 110 spores/μL. In conclusion,based on 3 000 samples for pebrine screening using HT-SDS method and qPCR,HT extraction and qPCR detection method is 11 times more sensitive in pebrine screening than current microscopy detection,especially for detecting hosts with low infection. This work will help to strengthen pebrine control and protection of pathogen-free eggs for the development of tussah industry.