University of Florida, Institute of Food and Agricultural Sciences, Department of Entomology and Nematology, Florida Medical Entomology Laboratory, Vero Beach, Florida 32962, USA
University of Florida, Institute of Food and Agricultural Sciences, Department of Entomology and Nematology, Florida Medical Entomology Laboratory, Vero Beach, Florida 32962, USA.
Cold Spring Harb Protoc. 2024 Oct 1;2024(10):pdb.prot108290. doi: 10.1101/pdb.prot108290.
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.
所有基于 PCR 和 DNA 的血食分析都需要在标本采集和提取 DNA 之间有效地保存蚊子血食中的宿主 DNA。一旦蚊子从宿主动物中吸食血液，蚊子中肠内的酶就开始消化血食中的宿主细胞和细胞成分，从而开始降解聚合酶链反应 (PCR) 扩增的宿主 DNA 模板。如果没有有效的保存，由于消化作用，宿主 DNA 在进食后 48 小时通常无法通过 PCR 检测到。蚊子血食的保存方法在效果上有所不同，野外工作的后勤工作可能会限制血食保存和宿主 DNA 完整性维护的选择。本方案描述了一种有效的、方便的血食保存方法，适用于在偏远地区进行野外工作，在这些地区，-20°C 或-80°C 的冷冻保存可能不可行。它使用 Flinders Technology Associates (FTA) 卡，这是一种经过化学处理的卡片，可以裂解细胞并保存核酸。这种方法还预计可以保存存在于饱食蚊子腹部内的病原体的 DNA 或 RNA，包括 RNA 病毒。
