香港瘰螈eDNA引物和Taq Man探针的设计与确认  

Design and Verification of Primers and TaqMan Probe Specific for Paramesotriton hongkongensis eDNA

作　　者：杨泰昌 李嘉华 张颖 刘彦芹 袁智勇[3] YEUNG Tai-Cheong;LEE Ka-Wa;ZHANG Ying;LAU Anthony;YUAN Zhi-Yong(Environmental Laboratory,Ocean Park Corporation,Hong Kong;Department of Biology,Hong Kong Baptist University,Hong Kong;College of Forestry,Southwest Forestry University,Kunming 650224,China)

机构地区：[1]香港海洋公园环境实验室,中国香港 [2]香港浸会大学生物系,中国香港 [3]西南林业大学生物多样性保护学院,昆明650224

出　　处：《动物学杂志》2020年第5期624-636,共13页Chinese Journal of Zoology

基　　金：香港海洋公园环境实验室营运基金项目。

摘　　要：环境DNA(e DNA)技术是近年来新兴的一种野外水生生物调查方法。本研究旨在设计一组香港瘰螈(Paramesotriton hongkongensis)特异的引物和TaqMan探针并确立此eDNA方法在水样中的实用性,以期进一步调查其野外种群分布。测定11种瘰螈物种的线粒体细胞色素b(Cyt b)基因序列,然后使用Primer Express 3.0软件设计引物和TaqMan探针。通过比对NCBI基因库及进行退火温度梯度测试,验证引物和TaqMan探针的特异性。通过测试不同浓度的引物和TaqMan探针,优化q PCR扩增效率。然后检测养殖不同数量香港瘰螈的水缸水体中eDNA,以评估已建立的q PCR方法灵敏度。同时,测定香港瘰螈e DNA降解速度,以估计其在水中的持续时间。实验结果显示,本研究所设计的引物和探针只对香港瘰螈呈阳性扩增,而对同属的其他10个物种均呈阴性。优化的q PCR效率为93.9%、最低检测浓度为10DNA拷贝数。已建立的qPCR方法能灵敏地检测出在养殖1只香港瘰螈24h实验水缸水体的DNA拷贝数为(13.56±3.35)/ml水样。另外,降解实验发现,采用0.45μm孔径滤膜已有效检测香港瘰螈e DNA并能监测15 d。本研究成功设计并建立了一种能在水环境中检测香港瘰螈是否存在的e DNA技术,以期应用于野外考察。Environmental DNA(eDNA)detection method is considered as a new survey tool for the wildlife aquatic animals in recent years.To further investigate the Hong Kong newt(Paramesotriton hongkongensis)population distribution in the wild,we thus aimed to design a set of specific primers and TaqMan probe and verify the feasibility of this eDNA method for water samples.The mitochondrial cytochrome b genes of 11 Paramesotriton species were sequenced,and then the primers and TaqMan probe were designed using Primer Express Software 3.0.The primers and TaqMan probe specificity were verified by comparing to the NCBI GenBank and testing with the annealing temperature gradient.The qPCR amplification efficiency was optimized by testing different concentrations of primers and TaqMan probe.Then,eDNA was detected in the water samples in aquarium tanks with different numbers of Hong Kong newt to assess the sensitivity of the established qPCR method.Also,the decay rate of Hong Kong newt eDNA was determined to estimate its lasting time in water.The results showed that the designed primers and TaqMan probe only amplified eDNA of Hong Kong newt but not the other 10 closely related newts(Table 3 and 4).The optimized qPCR achieved the efficiency of 93.9%and the limit of detection down to 10 DNA copies(Fig.2 and Table 5).The established qPCR method was sensitive enough to detect 13.56±3.35 DNA copies/ml in the aquarium tank with 1 Hong Kong newt after housing for 24 h(Fig.3).In addition,the decay rate experiment demonstrated that the use of 0.45μm pore size filter membrane was effective to detect and monitor the Hong Kong newt eDNA in 15 d(Fig.6).In this study,an eDNA detection method was successfully designed and established to detect the presence or absence of Hong Kong newt in water environments,which can be potentially utilized in the field inspection.

关 键 词：香港瘰螈 环境DNA 线粒体细胞色素B基因 TaqMan qPCR 

分 类 号：Q75[生物学—分子生物学] Q958