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《Chinese Jouranl of Endemiology》 2002-06
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Anti-contamination PCR-microplate hybridization-enzyme immunoassay for detecting Brucella spp

GUO Zhao biao,ZHANG Min li,SONG Ya jun,WANG Jin,WANG Xiao hui,YANG Rui fu (Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China)  
Objective To establish an anti contamination PCR microplate hybridization enzyme immunoassay (EIA) for detecting Brucella spp.Methods The specific primers were designed based on two genes, encoding 31 KD heat shock protein and outer membrane protein (OMP)2B. UDG was used in PCR system for digesting the carry over amplicons to preventing contamination, and the microplate hybridization EIA used for verifying the specific amplicons.Results A duplex PCR system was developed by using the primers located on two genes of Brucella spp, which was used to detect the two genes simultaneously to prevent the false negative results caused by gene mutation. Our results proved that the primer sequences had a great influences on the sensitivity of PCR. 0.5 U of UDG can digest 10 8 molecules contaminated in the PCR system. The sensitivity showed by PCR microplate hybridization is 10 times higher than that demonstrated by PCR agarose electrophoresis. The anti contamination PCR microplate hybridization EIA system could detect 2 to 200 bacteria in the PCR system.Conclusions This technique overcomes the problems for conventional PCR, that is inconvenience for reagent's transportation, amplicon's carry over and lack of objective evaluation of results by electrophoresis. This is a powerful method for rapid detecting Brucella spp.
【Fund】: 解放军总后卫生部基金 (19990 1)
【CateGory Index】: R446.5
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