两种PCR方法对检测A组轮状病毒膜芯片杂交结果的影响  

Two PCR Amplification Methods Influence the Membrane Chip Hybridizations Results for Detection of Group A Rotavirus

作　　者：黄海燕[1] 韩金祥[1] 王健伟[2,3] 朱波[1] 

机构地区：[1]山东省医药生物技术研究中心卫生部生物技术药物重点实验室 [2]山东省病毒学研究所山东省医学病毒学重点实验室 [3]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《生物技术》2005年第3期40-43,共4页Biotechnology

基　　金："十五"国家重大科技专项项目资助("食品安全关键技术研究";2001BA804A22)

摘　　要：A组轮状病毒(rotavirus,RV)是导致全世界婴幼儿腹泻的最主要病原,危害巨大。拟用RT-巢式PCR技术对A组RV的保守序列进行高度扩增,通过同本室自制的膜芯片杂交,实现对该病毒的检测。分别采用对称PCR和不对称PCR扩增,均可得到扩增的目的片段。对称式扩增产物杂交结果不理想。而不对称式扩增得到了大量待检单链产物,同膜芯片杂交获得了理想的杂交结果。显著地提高了对A组RV杂交检测的灵敏度。表明不对称式PCR扩增是一种制备用于芯片杂交大量单链产物的理想方法,尤其是针对富含AT的核酸检测区域。Group A rotavirus was the most important etiological agents of severe diarrhoeal illness of infants and young children worldwide.In this study,a reverse transcirptase-polymerase seminested chain reaction (RT-seminested PCR) using digoxigenin(dig)-labeled up-prime and oligoprobes distinctive to group A RV using chemiluminescence has been developed to detect group A RV.Two different PCR methods,seminested symmetry PCR and seminested asymmetry PCR,were adopted respectively.The results suggested both methods could produce the aimed size nucleotide sequences according to the agarose gel electrophoresis but different hybridization signals.The hybridization of seminested symmetry PCR product didnt show the ideal signals.While the asymmetry PCR could produce adequate single-strand nucleotide and display ideal hybridization signals , remarkably improved the detection sensitivity for group A RV. This study indicated that the seminested asymmetry PCR was a robust method to obtain abundant single-strain nucleotide for the use of chip hybridization,especially for the sequences contain of AT-rich.

关 键 词：不对称PCR 轮状病毒 A组 芯片 杂交 

分 类 号：R373.2[医药卫生—病原生物学]