摘要
目的建立粪便中腺病毒的PCR-毛细管电泳-激光诱导荧光和PCR-微流控芯片电泳-激光诱导荧光快速检测方法。方法采用试剂盒法提取粪便中腺病毒DNA,选择腺病毒六邻体基因保守区域进行PCR扩增反应。PCR产物分别用高灵敏度的SYBR Gold和SYBR Orange核酸荧光染料进行标记,在优化的毛细管电泳和微流控芯片电泳条件下分离,采用激光诱导荧光检测特异性PCR扩增产物。结果腺病毒扩增产物在优化的毛细管电泳和微流控芯片电泳条件下可分别于9min和6min内完成检测。将PCR扩增产物测序,并与NCBI基因库中核酸序列比对,结果表明所选用的扩增序列特异性良好。商品化DNA Marker毛细管电泳和微流控芯片电泳对目标DNA片段bp值求得的日内精密度分别为1.14%-1.34%和1.18%-1.48%,日间精密度分别为1.27%-2.76%和2.85%-4.06%。毛细管电泳-激光诱导荧光法和微流控芯片电泳-激光诱导荧光法检测腺病毒的灵敏度分别为2.33×10^2 copies/mL和2.33×10^3 copies/mL。两种方法用于实际粪便样品测定,准确度高。结论本研究建立的PCR-毛细管电泳-激光诱导荧光和PCR-微流控芯片电泳-激光诱导荧光检测方法能够快速、灵敏、准确地检测出粪便中的腺病毒。
Objective To establish a rapid and sensitive method based on polymerase chain reaction（PCR）combined with capillary electrophoresis-laser induced fluorescence（CE-LIF）and microchip capillary electrophoresislaser induced fluorescence（MCE-LIF）for detecting adenoviruses in fecal samples. Methods The DNA of adenovirus in fecal samples were extracted by the commercial kits and the conserved region of hexon gene was selected as the target gene and amplified by PCR reaction.After labeling highly sensitive nucleic acid fluorescent dye SYBR Gold and SYBR Orange respectively,PCR amplification products were separated by CE and MCE under the optimized condition and detected by LIF detector.Results PCR amplification products could be detected within 9min by CE-LIF and 6 min by MCE-LIF under the optimized separation condition.The sequenced PCR product showed good specificity in comparison with the prototype sequences from NCBI.The intraday and inter-day relative standard deviation（RSD）of the size（bp）of the target DNA was in the range of 1.14%-1.34% and 1.27%-2.76%,respectively,for CE-LIF,and 1.18%-1.48% and 2.85%-4.06%,respectively,for MCE-LIF.The detection limits was 2.33×10^2 copies/mL for CE-LIF and 2.33×10^3 copies/mL for MCE-LIF.The two proposed methods were applied to detect fecal samples,both showing high accuracy.Conclusion The two proposed methods of PCR-CE-LIF and PCR-MCE-LIF can detect adenovirus in fecal samples rapidly,sensitively and specifically.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2015年第4期615-618,627,共5页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(青年基金)项目(No.81102162)资助
