Quantitative real-time PCR with high -throughput automatable DNA preparation for molecular screening of Nosema spp. in Antheraea pernyi

文献类型： 外文期刊

第一作者： Li, Peipei

作者： Li, Peipei;Zhang, Yaozhou;Li, Peipei;Mi, Rui;Zhang, Bo;Yue, Dongmei;Ye, Bo;Zhao, Zhenjun;Wang, Linmei;Fan, Qi;Zhao, Rui;Li, Xiangcun;Jiang, Xiaobin;Zhu, Youmin;Bao, Chen

作者机构：

关键词： Nosema spp.; Diagnostic PCR; High-throughput; Automatable; Sensitive; Epidemiological studies

期刊名称：JOURNAL OF INVERTEBRATE PATHOLOGY （ 影响因子：2.841； 五年影响因子：3.368 ）

ISSN： 0022-2011

年卷期： 2019 年 164 卷

页码：

收录情况： SCI

摘要： Accurate diagnosis of pathogenic Nosema spp. in Antheraea pernyi samples is considered especially useful for reducing economic losses in sericulture and improving food safety by maintaining pathogen-free pupae. However, microscopy and immunologic methods have poor diagnostic sensitivity, while the more sensitive PCR methods remain costly and time-consuming for template preparation. To address this issue, we introduce a sensitive ALMS-qPCR method that combines fast, simple DNA extraction using Alkali Lysis followed by Magnetic bead Separation (ALMS) and quantitative real-time PCR (qPCR). This approach is especially fit for large-scale pathogen molecular screening, because the DNA preparation procedure is fast (< 0.94 min per sample) and is high-throughput (performs on a 96-well plate). It is cost-effective, since the most expensive materials can be made in the lab and can be recycled, while the automated procedure can help to minimize labor cost. Though the DNA preparation procedure was substantially simplified, common PCR inhibitory factors were not observed. The sensitivity of ALMS-qPCR is high and the limit of detection is 0.045 parasites/A. Large-scale screening of Nosema spp. in 3000 Antheraea pernyi samples confirmed the efficacy of the ALMS-qPCR method. Sensitivity is much higher than clinical microscopy, especially for host groups with low infection prevalence and levels. High throughput ALMS-qPCR, combining automated DNA preparation and sensitive qPCR, provides an enhanced approach for pebrine screening and epidemiological studies. The application of ALMS-qPCR in the sericulture industry will help to strengthen pebrine control and breed pathogen-free species, which means much safer food provision and better genetic resource conservation.

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