Deng Wenxin, Feng Shiqian, Stejskal Vaclav, Opit George, Li Zhihong
Department of Plant Biosecurity, College of Plant Protection, China Agricultural University, Beijing 100193, China.
Sanya Institute of China Agricultural University, Yazhou Bay Science and Technology City, Yazhou District, Sanya 572025, Hainan, China.
J Econ Entomol. 2023 Oct 10;116(5):1911-1921. doi: 10.1093/jee/toad139.
Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) is a booklouse pest that is a threat to commodity storage security worldwide. Accurate and sensitive methods of L. bostrychophila on-site identification are essential prerequisites for its effective management. Evidence suggests that L. bostrychophila contains 3 intraspecific biotypes that are morphologically indistinguishable but can be discriminated at the level of mitochondrial genome organization and sequences. The traditional molecular identification methods, such as DNA barcoding and PCR-RFLP, are instrumentally demanding and time-consuming, limiting the application of the identification in the field. Therefore, this study developed a new CRISPR/Cas12a-based visual nucleic acid system based on the mitochondrial gene coding for NADH dehydrogenase subunit 2 (nad2), combined with recombinase polymerase amplification (RPA) to accurately identify L. bostrychophila from 4 other common stored-product booklice, and also differentiate 3 biotypes of this species at the same time. The entire identification process could be completed at 37 °C within 20 min with high sensitivity. The system could stably detect at least 1 ng/μl of DNA template. The green fluorescence signal produced by the trans-cleaving of the single-stranded DNA reporter could be observed by the naked eye under blue light. Additionally, the suggested system combined with the crude DNA extraction method to extract DNA rapidly, enabled identification of all developmental stages of L. bostrychophila. With crude DNA, this novel diagnostic system successfully identified an unknown booklouse by holding the reaction tubes in the hand, thus can be considered as an accurate, rapid, highly sensitive, and instrument-flexible method for on-site visual identification of L. bostrychophila.
嗜卷书虱（Liposcelis bostrychophila Badonnel，啮目：书虱科）是一种书虱害虫，对全球商品储存安全构成威胁。准确且灵敏的嗜卷书虱现场鉴定方法是对其进行有效管理的必要前提。有证据表明，嗜卷书虱包含3个种内生物型，它们在形态上无法区分，但可在线粒体基因组组织和序列水平上加以鉴别。传统的分子鉴定方法，如DNA条形码技术和PCR-RFLP，对仪器要求高且耗时，限制了其在现场鉴定中的应用。因此，本研究基于编码烟酰胺腺嘌呤二核苷酸脱氢酶亚基2（nad2）的线粒体基因，开发了一种新的基于CRISPR/Cas12a的可视化核酸系统，并结合重组酶聚合酶扩增技术（RPA），以从其他4种常见的储藏物书虱中准确鉴定嗜卷书虱，同时区分该物种的3个生物型。整个鉴定过程可在37℃下20分钟内完成，灵敏度高。该系统可稳定检测至少1 ng/μl的DNA模板。单链DNA报告基因经反式切割产生的绿色荧光信号在蓝光下可用肉眼观察到。此外，所建议的系统与粗DNA提取方法相结合，能够快速提取DNA，实现对嗜卷书虱所有发育阶段的鉴定。利用粗DNA，这种新型诊断系统通过手持反应管成功鉴定了一种未知书虱，因此可被视为一种用于嗜卷书虱现场可视化鉴定的准确、快速、高灵敏且仪器灵活的方法。
