根据Gen Bank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei)(EHP)SSU r DNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green I实时荧光定量PCR(q PCR)检测方法。结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线...根据Gen Bank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei)(EHP)SSU r DNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green I实时荧光定量PCR(q PCR)检测方法。结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线为1个单峰,构建的方法对8.3×101–8.3×108 copies/μl的EHP SSU r DNA片段的检测响应具有良好的线性关系,扩增产物阈值循环数(Ct)与模板起始量的对数[log(Sq)]的关系为Ct=–3.369 log(Sq)+39.364(R2=0.992),扩增效率为98.1%,检测灵敏度下限为8.3×101 copies/μl,在线性范围内具有良好的组内和组间重复性。对实际样品的检测表明该方法比已报道的套式PCR的检测灵敏度约高4倍。利用本方法对采集自江苏、海南和山东的3批凡纳滨对虾样品的肝胰腺组织DNA(Hp DNA)中的EHP SSU r DNA进行了q PCR检测,结果显示,EHP的载量指数与对虾生长速率呈负相关关系,肝胰腺中EHP载量在103 copies/(ng Hp DNA)时代表了较高的风险水平。本研究建立的q PCR方法具有特异、灵敏、快速、定量的优点,所建立的方法及检测数据可为EHP的防控提供技术参考。展开更多
Enterocytozoon hepatopenaei(EHP)infection has seriously affected prawn culture globally.The symptoms of the infection are not apparent,and traditional detection methods are time consuming and low in accuracy.We develo...Enterocytozoon hepatopenaei(EHP)infection has seriously affected prawn culture globally.The symptoms of the infection are not apparent,and traditional detection methods are time consuming and low in accuracy.We developed a new onsite rapid testing device(size 18.8×16.7×6.6 cm^(3))for EHP based on magnesium pyrophosphate precipitation and facilitated by loop mediated isothermal amplification(LAMP).The design and fabrication of the device enables efficient light absorbance.The device has a highly sensitive detector,high-precision thermal controller,and humanized touch screen.The temperature control precision of the device is 0.2-0.3℃ at 60℃,63℃,and 65℃.The coefficients of variation values(CVV)of the luminous power in one channel at light on and off were found to be 0.0097 and 0.0014,respectively,within 1 h.The CVV of the background,luminous power,and values of eight PCR tubes filled with pure water were all less than 5%.In the EHP experiment,eight samples(including seven positive and one negative)confirmed the effectiveness of the device,and four positive and four negative samples verified whether cross-contamination exists.Among them,the rise time of the curve was about 15 min.These results assert that the developed device exhibits enhanced stability and uniformity and has excellent performance with high sensitivity,good specificity,and low testing time.Moreover,the optimal and minimum absorbance range was 555-655 nm for monitoring the production of LAMP.展开更多
文摘根据Gen Bank中公布的虾肝肠胞虫(Enterocytozoon hepatopenaei)(EHP)SSU r DNA序列设计1对特异性引物,建立并优化了EHP的SYBR Green I实时荧光定量PCR(q PCR)检测方法。结果显示,该方法在60℃的退火温度时扩增效果最好,产物的熔解曲线为1个单峰,构建的方法对8.3×101–8.3×108 copies/μl的EHP SSU r DNA片段的检测响应具有良好的线性关系,扩增产物阈值循环数(Ct)与模板起始量的对数[log(Sq)]的关系为Ct=–3.369 log(Sq)+39.364(R2=0.992),扩增效率为98.1%,检测灵敏度下限为8.3×101 copies/μl,在线性范围内具有良好的组内和组间重复性。对实际样品的检测表明该方法比已报道的套式PCR的检测灵敏度约高4倍。利用本方法对采集自江苏、海南和山东的3批凡纳滨对虾样品的肝胰腺组织DNA(Hp DNA)中的EHP SSU r DNA进行了q PCR检测,结果显示,EHP的载量指数与对虾生长速率呈负相关关系,肝胰腺中EHP载量在103 copies/(ng Hp DNA)时代表了较高的风险水平。本研究建立的q PCR方法具有特异、灵敏、快速、定量的优点,所建立的方法及检测数据可为EHP的防控提供技术参考。
基金supported by the grant from the National Natural Science Foundation of China(Nos.61901168,61971187,61871180,82002405)Hunan Key Research and Development Program(No.2021SK2003)+1 种基金Zhuzhou Innovative City Construction Project(No.2020-020)China Postdoctoral Science Foundation(No.2018M630498)。
文摘Enterocytozoon hepatopenaei(EHP)infection has seriously affected prawn culture globally.The symptoms of the infection are not apparent,and traditional detection methods are time consuming and low in accuracy.We developed a new onsite rapid testing device(size 18.8×16.7×6.6 cm^(3))for EHP based on magnesium pyrophosphate precipitation and facilitated by loop mediated isothermal amplification(LAMP).The design and fabrication of the device enables efficient light absorbance.The device has a highly sensitive detector,high-precision thermal controller,and humanized touch screen.The temperature control precision of the device is 0.2-0.3℃ at 60℃,63℃,and 65℃.The coefficients of variation values(CVV)of the luminous power in one channel at light on and off were found to be 0.0097 and 0.0014,respectively,within 1 h.The CVV of the background,luminous power,and values of eight PCR tubes filled with pure water were all less than 5%.In the EHP experiment,eight samples(including seven positive and one negative)confirmed the effectiveness of the device,and four positive and four negative samples verified whether cross-contamination exists.Among them,the rise time of the curve was about 15 min.These results assert that the developed device exhibits enhanced stability and uniformity and has excellent performance with high sensitivity,good specificity,and low testing time.Moreover,the optimal and minimum absorbance range was 555-655 nm for monitoring the production of LAMP.
