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引用次数: 0
摘要
所有基于 PCR 和 DNA 的血粉分析,都要求从采集标本到提取 DNA 的整个过程中,有效保存蚊子血粉中的宿主 DNA。一旦蚊子吸食了宿主动物的血液,蚊子中肠中的酶就会开始消化血餐中的宿主细胞和细胞成分,从而降解聚合酶链反应(PCR)扩增目标的宿主 DNA 模板。如果没有有效的保存方法,宿主 DNA 通常在进食 48 小时后就无法通过聚合酶链式反应检测到。蚊子血餐的保存方法效果各异,野外工作的后勤工作也会限制保存血餐和保持宿主 DNA 完整性的选择。本方案介绍了一种有效、方便的血粉保存方法,适合在偏远地区进行野外工作,因为在这些地区可能无法将血粉低温保存在 -20°C 或 -80°C 的温度下。该方法使用弗林德斯技术协会(FTA)卡,这是一种经过化学处理的卡,可以裂解细胞并保存核酸。预计这种方法还能保存充血蚊子腹部内病原体(包括 RNA 病毒)的 DNA 或 RNA。
Preservation of Field-Collected Mosquito Blood Meals.
All PCR- and DNA-based blood meal analyses require host DNA from a mosquito blood meal to be effectively preserved between the time when the specimen is collected and the extraction of DNA. As soon as a mosquito ingests blood from a host animal, digestion of host cells and cellular components within the blood meal by enzymes in the mosquito midgut begins to degrade the host DNA templates that are the targets of polymerase chain reaction (PCR) amplification. Without effective preservation, host DNA is typically undetectable by PCR 48 h after feeding, because of digestion. Preservation methods for mosquito blood meals vary in their efficacy, and the logistics of fieldwork can limit the options for preservation of blood meals and maintenance of the integrity of host DNA. This protocol describes a method of blood meal preservation that is effective, convenient, and amenable to fieldwork in remote locations where cryopreservation at -20°C or -80°C may not be feasible. It uses a Flinders Technology Associates (FTA) card, which is a chemically treated card that lyses cells and allows nucleic acids to be preserved. This method is also expected to preserve the DNA or RNA of pathogens present within the engorged mosquito abdomen, including RNA viruses.
Cold Spring Harbor protocolsBiochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
3.00
自引率
0.00%
发文量
163
期刊介绍:
Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online. Cold Spring Harbor Protocols is an interdisciplinary journal providing a definitive source of research methods in cell, developmental and molecular biology, genetics, bioinformatics, protein science, computational biology, immunology, neuroscience and imaging. Each monthly issue details multiple essential methods—a mix of cutting-edge and well-established techniques.