梭子蟹肌孢虫SYBR Green I实时定量PCR检测方法的建立与应用  

Establishment and application of a SYBR Green I real-time q PCR detection of Ameson portunus

作　　者：陈甜甜[1,2] 房文红[1] 王元[1] 李新苍[1] 赵姝[1] 周俊芳[1] 

机构地区：[1]农业部东海渔业资源开发利用重点实验室,中国水产科学研究院东海水产研究所,上海200090 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《南方水产科学》2017年第6期22-29,共8页South China Fisheries Science

基　　金：公益性行业(农业)科研专项经费项目(201303047);中央级公益性科研院所基本科研业务费专项资金(2014T03)

摘　　要：梭子蟹肌孢虫(Ameson portunus)是感染三疣梭子蟹(Portunus trituberculatus)的常见病原之一,该研究以其SSU rRNA基因为靶标建立了SYBR Green I实时定量PCR检测方法。结果显示:该方法扩增效率为101.45%,检测灵敏度下限可达2.0×10~1拷贝·μL^(-1),特异性、组内重复性和组间重复性均好。利用该荧光定量PCR方法对一批来自"牙膏病"流行区的养殖三疣梭子蟹样品进行检测,梭子蟹肌孢虫检出率为82.35%。检测结果显示,活蟹的每毫克肌肉平均带虫量大幅低于死蟹,表明该地区梭子蟹的死亡与肌孢虫的感染密切相关,而且,当梭子蟹肌孢虫载量达到1.0×10~9拷贝·mg^(-1)时三疣梭子蟹存在暴发疾病甚至死亡的风险。另外,通过对比检测发现,梭子蟹肌孢虫套式PCR方法对这批样品的检出率为64.71%,表明该研究建立的荧光定量PCR方法具有更高的灵敏度。Ameson portunus is one of the common pathogens of the swimming crab （Portunus trituberculatus）. In this study, a SYBR Green I real time qPCR method whose amplification efficiency reached up to 101.45% with a detection limit as low as 2.0×10^1 copies·μL^-1 was established by targeting A.portunus SSU rRNA gene. The method was A. portunus-specific with good repeatability within and between groups. Then the qPCR method was used to detect farmed swimming crabs froma “toothpaste disease” epidemic area, and the detection rate of A.portunus was 82.35%. The results show that the average muscle load per milligram of the live crabs was lower than that of the dead crabs significantly, which indicates that the death of crabs in the region is related with infection of A.portunus closely. When the load of A.portunus was 1.0×10^9 copies·mg^-1 in the muscle tissue, P.trituberculatus had a risk of illness or even death. Besides, the detection rate for the same samples by a previously established nested-PCR method was only 64.71%, showing that the qPCR method in the present study had higher sensitivity and can provide good technical support for the early diagnosis and prevention of ＂toothpaste disease＂.

关 键 词：梭子蟹肌孢虫 三疣梭子蟹 实时定量PCR方法 SYBR Green I 

分 类 号：S945[农业科学—水产养殖]