PCR方法制备地高辛标记DNA探针检测中国对虾非包涵体型杆状病毒  

Development of Digoxigenin Labeled DNA Probe by PCR for Detection of Penaeus chinensis Non occluded Baculovirus (PcNOBV)

作　　者：徐洪涛[1] 朴春爱[2] 杨朵 王雷[1] 张新宇[1] 侯云德[2] 

机构地区：[1]中国科学院海洋研究所,山东青岛266071 [2]中国预防医学科学院病毒学研究所,北京100052 [3]西安医科大学,陕西西安710061

出　　处：《病毒学报》2000年第1期73-75,共3页Chinese Journal of Virology

基　　金：海洋 8 63 ( 819-0 2 -0 5 );病毒基因工程国家重点实验室客座课题

摘　　要：A pair of primers created from information of PmNOBⅢ genome DNA Sal I fragment produced a 355bp band by using Penaeus chinensis non occluded baculovirus (PcNOBV),the WSBV isolate from P.hinensis in China's Mainland,as the DNA template.The specific PCR product was cloned,sequenced and labeled with digoxigenin (DIG)DNA labeling kit(Boehringer Mannheim).The DIG labeled fragment was tested by dot blot hybridization for sensitivity and specificity with purified PcNOBV nucleocapsid,PcNOBV infected shrimp tissues and healthy shrimp tissues.The detection limit of the DNA probe is 6.8pg of purified PcNOBV DNA.No hybridization signals were observed using DNA from healthy shrimp as template.Healthy P.chinensis,artificially infected P.chinensis and pond reared adult P.chinensis were screened for PcNOBV infection by both PCR and the hybridization assay.The results showed a good relationship between PCR and the hybridization assay.These findings demonstrate that the DIG labeled probe can be used as a sensitive,specific and cost effective reagent for detection of PcNOBV.A pair of primers created from information of PmNOBⅢ genome DNA Sal I fragment produced a 355bp band by using Penaeus chinensis non occluded baculovirus (PcNOBV),the WSBV isolate from P.hinensis in China's Mainland,as the DNA template.The specific PCR product was cloned,sequenced and labeled with digoxigenin (DIG)DNA labeling kit(Boehringer Mannheim).The DIG labeled fragment was tested by dot blot hybridization for sensitivity and specificity with purified PcNOBV nucleocapsid,PcNOBV infected shrimp tissues and healthy shrimp tissues.The detection limit of the DNA probe is 6.8pg of purified PcNOBV DNA.No hybridization signals were observed using DNA from healthy shrimp as template.Healthy P.chinensis,artificially infected P.chinensis and pond reared adult P.chinensis were screened for PcNOBV infection by both PCR and the hybridization assay.The results showed a good relationship between PCR and the hybridization assay.These findings demonstrate that the DIG labeled probe can be used as a sensitive,specific and cost effective reagent for detection of PcNOBV.

关 键 词：中国对虾 非包涵体型杆状病毒 DNA探针 检测 

分 类 号：S945.463[农业科学—水产养殖]