Black rockfish Sebastes schlegelii juveniles （30-40 mm total length） were immersed in a range of calcein （CAL） solutions at concentrations ranging from 50 to 250 mg/L and alizarin red S （ARS） solutions at concen...Black rockfish Sebastes schlegelii juveniles （30-40 mm total length） were immersed in a range of calcein （CAL） solutions at concentrations ranging from 50 to 250 mg/L and alizarin red S （ARS） solutions at concentrations ranging from 100 to 500 mg/L in filtered seawater （salinity 30） for 24 h. Fluorescent marks were detected in otoliths （sagittae, asteriscus）, scales, fin rays （dorsal, pectoral, ventral, anal, and caudal fin rays）, and fin spines （dorsal, ventral, and anal fin spines） after a 60-d growth experiment. With the exception of 50-100 rng/L CAL, acceptable marks were produced in the otoliths and fin spines by all concentrations of CAL and ARS. In particular, marks were clearly visible under normal light in the sagittae, asteriscus, and fin spines offish immersed in 200 500 mg/L, 300-500 rag/L, and 200-500 mg/LARS, respectively. Scales and fin rays had acceptable marks at much higher concentrations （≥50 mg/L CAL, ≥300 mg/L ARS for scales and ≥50 mg/L CAL,≥200 mg/L ARS for fin rays）. The mark quality was highest （i.e., acceptable marks were observed in all sampled structures after immersion marking） in fish immersed in 150-250 mg/L CAL or 300-500 mg/LARS. In addition, there was no significant difference in survival and growth of marked fish compared with controls 60 d post-marking （P〉0.05）.展开更多
【目的】建立基于荧光显色的可视化禽传染性支气管炎病毒(IBV)环介导等温扩增(LAMP)检测体系,为IBV的快速临床检测提供有力的技术支持。【方法】根据NCBI中收录的IBV 5a+5b基因的8个区域设计了3对LAMP引物,通过对反应体系各组分和条件...【目的】建立基于荧光显色的可视化禽传染性支气管炎病毒(IBV)环介导等温扩增(LAMP)检测体系,为IBV的快速临床检测提供有力的技术支持。【方法】根据NCBI中收录的IBV 5a+5b基因的8个区域设计了3对LAMP引物,通过对反应体系各组分和条件的优化,建立IBV LAMP检测方法,并对该方法的特异性和敏感性进行验证。分别用SYBR Green I和一定浓度比的钙黄绿素/锰离子混合物作为显色剂,对IBV LAMP检测结果进行可视化判定。应用该检测体系和PCR方法对临床送检的60份样品同时进行检测,比较2种方法的阳性检出率。【结果】成功建立了IBV LAMP检测方法,该方法能够在等温(63℃)条件下1h内完成反应,具有良好的敏感性和特异性,最低可以检出1拷贝/μL的阳性重组质粒,比普通PCR方法敏感100倍,而对其他病毒检测结果均为阴性。应用SYBR Green I和钙黄绿素/锰离子显色时,阴阳性样品均有强烈的颜色对比,其中0.05mmol/L钙黄绿素与0.6mmol/L锰离子混合液可以用于LAMP扩增产物的判断,其结果与琼脂糖凝胶电泳结果一致。在对60份临床样本的检测中,LAMP和PCR方法检测出的阳性样本数量分别为49份和42份。【结论】建立了基于荧光显色的IBVLAMP检测方法,该方法具有在科研机构、基层实验室特别是检测现场推广和应用的潜力。展开更多
基金Supported by the Special Fund for Agro-scientific Research in the Public Interest(No.201003068)the National Natural Science Foundation of China(Nos.31172447,41176117)
文摘Black rockfish Sebastes schlegelii juveniles （30-40 mm total length） were immersed in a range of calcein （CAL） solutions at concentrations ranging from 50 to 250 mg/L and alizarin red S （ARS） solutions at concentrations ranging from 100 to 500 mg/L in filtered seawater （salinity 30） for 24 h. Fluorescent marks were detected in otoliths （sagittae, asteriscus）, scales, fin rays （dorsal, pectoral, ventral, anal, and caudal fin rays）, and fin spines （dorsal, ventral, and anal fin spines） after a 60-d growth experiment. With the exception of 50-100 rng/L CAL, acceptable marks were produced in the otoliths and fin spines by all concentrations of CAL and ARS. In particular, marks were clearly visible under normal light in the sagittae, asteriscus, and fin spines offish immersed in 200 500 mg/L, 300-500 rag/L, and 200-500 mg/LARS, respectively. Scales and fin rays had acceptable marks at much higher concentrations （≥50 mg/L CAL, ≥300 mg/L ARS for scales and ≥50 mg/L CAL,≥200 mg/L ARS for fin rays）. The mark quality was highest （i.e., acceptable marks were observed in all sampled structures after immersion marking） in fish immersed in 150-250 mg/L CAL or 300-500 mg/LARS. In addition, there was no significant difference in survival and growth of marked fish compared with controls 60 d post-marking （P〉0.05）.
文摘【目的】建立基于荧光显色的可视化禽传染性支气管炎病毒(IBV)环介导等温扩增(LAMP)检测体系,为IBV的快速临床检测提供有力的技术支持。【方法】根据NCBI中收录的IBV 5a+5b基因的8个区域设计了3对LAMP引物,通过对反应体系各组分和条件的优化,建立IBV LAMP检测方法,并对该方法的特异性和敏感性进行验证。分别用SYBR Green I和一定浓度比的钙黄绿素/锰离子混合物作为显色剂,对IBV LAMP检测结果进行可视化判定。应用该检测体系和PCR方法对临床送检的60份样品同时进行检测,比较2种方法的阳性检出率。【结果】成功建立了IBV LAMP检测方法,该方法能够在等温(63℃)条件下1h内完成反应,具有良好的敏感性和特异性,最低可以检出1拷贝/μL的阳性重组质粒,比普通PCR方法敏感100倍,而对其他病毒检测结果均为阴性。应用SYBR Green I和钙黄绿素/锰离子显色时,阴阳性样品均有强烈的颜色对比,其中0.05mmol/L钙黄绿素与0.6mmol/L锰离子混合液可以用于LAMP扩增产物的判断,其结果与琼脂糖凝胶电泳结果一致。在对60份临床样本的检测中,LAMP和PCR方法检测出的阳性样本数量分别为49份和42份。【结论】建立了基于荧光显色的IBVLAMP检测方法,该方法具有在科研机构、基层实验室特别是检测现场推广和应用的潜力。
