New micro-amount of virion enrichment technique (MiVET) to detect influenza A virus in the duck feces
New micro-amount of virion enrichment technique (MiVET) to detect influenza A virus in the duck feces
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新型微量病毒粒子富集技术（MiVET）检测鸭粪便中的甲型流感病毒
DOI：
10.1111/tbed.13027
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
K.
中科院分区：
文献类型：
--
作者：
Yamazaki;W.;Makino;R.;Nagao;K.;Mekata;H.;Tsukamoto;K.
包括高致病性禽流感在内的跨界动物疾病在全世界造成巨大的经济损失。虽然查明传播的来源和传播途径很重要，但流行病学证据不完整往往阻碍了这类战略的实施。由于污染水平非常低，从环境样品中分离/检测微量病原体很少成功。本文描述了微量病毒粒子富集技术（MiVET）的发展，这是一种简单且高度灵敏的方法，结合使用包含多克隆抗体和蛋白G包被的磁珠的复合物进行病毒粒子捕获，以及简单的十二烷基苯磺酸钠（SDBS）洗脱用于低体积样品。通过真实的实时逆转录聚合酶链反应（rRT-PCR），使用人工加标样本中的甲型禽流感病毒（AIV）评价了MiVET的性能。使用4种AIV（H3 N2、H4 N2、H5 N2和H7 N7）人工加标50 ml磷酸盐缓冲液（PBS）和1 ml 10%-25%鸭粪便上清液。MiVET系统成功地浓缩了PBS和粪便样品中的AIV，比传统的RNA提取方法分别具有至少2和1个对数的功效。从样品制备开始到最终RNA提取，MiVET可在<30 min内完成。MiVET有效地防止了粪便样品中抑制剂的影响，并且不需要特殊设备。这是这种新型系统的首次报道，预计可用于检测微量的各种兽医和人类病毒，以阐明其在环境中的循环动力学，并以更高的检测能力进行快速灵敏的诊断。
Transboundary animal diseases, including highly pathogenic avian influenza, cause vast economic losses throughout the world. While it is important to identify the sources and propagation routes of the spread, such strategies are often hindered by incomplete epidemiological evidence. Isolation/detection of micro‐amounts of pathogens from environmental samples is rarely successful due to the very low contamination level. This paper describes the development of the micro‐amount of virion enrichment technique (MiVET), a simple and highly sensitive method that combines the use of a complex comprising a polyclonal antibody and protein G‐coated magnetic beads for virion capture, and simple sodium dodecyl benzenesulfonate (SDBS) elution for low volume samples. The performance of the MiVET was evaluated using avian influenza A viruses (AIVs) in artificially spiked samples by real‐time reverse transcription polymerase chain reaction (rRT‐PCR). Four AIVs, H3N2, H4N2, H5N2 and H7N7, were used to artificially spike 50 ml of phosphate‐buffered saline (PBS) and 1 ml of 10%–25% duck faecal supernatants. The MiVET system successfully concentrated AIVs in both PBS and faecal samples with at least 2 and 1 log greater efficacy, respectively, than conventional RNA extraction methods. The MiVET could be completed in <30 min from the beginning of sample preparation to final RNA extraction. The MiVET effectively prevented the effects of inhibitors in faecal samples, and did not require special equipment. This is the first report of this novel type of system, which is expected to be useful for the detection of micro‐amounts of various veterinary and human viruses to elucidate their circulation dynamics in the environment, and for rapid and sensitive diagnosis with greater detection power.