SENSITIVE DETECTION OF GROUP-A ROTAVIRUSES BY IMMUNOMAGNETIC SEPARATION AND REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

被引:31
作者
GRINDE, B [1 ]
JONASSEN, TO [1 ]
USHIJIMA, H [1 ]
机构
[1] INST PUBL HLTH, MINATO KU, TOKYO 108, JAPAN
关键词
ROTAVIRUS; POLYMERASE CHAIN REACTION (PCR); IMMUNOMAGNETIC SEPARATION; ENVIRONMENTAL DETECTION;
D O I
10.1016/0166-0934(95)00070-X
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
An immunomagnetic separation (IMS) method was developed for concentrating rotaviruses from environmental samples, as well as a reverse transcription-polymerase chain reaction (RT-PCR) for the sensitive and specific detection of group A rotaviruses. Magnetic beads were coated with monoclonal antibodies directed against the group-specific, inner capsid protein (VP6) and subsequently used to capture and purify the virus with the help of a magnet. The genome was made available for RT by heat-disrupting the viral particles. A single 40-cycle PCR was as sensitive as a nested PCR, both detecting 0.005 PFU of the Wa strain, corresponding to approximately 5 particles as indicated by EM. The nested PCR was positive for all the group A strains tested, but negative for group C rotaviruses and other RNA viruses. The IMS-RT-PCR method functioned satisfactorily with virus seeded out in fresh water samples; with sea water, the IMS removed most, but not all, inhibiting activity.
引用
收藏
页码:327 / 338
页数:12
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