Chemical degradation of tobacco mosaic virus followed by infectivity assay, reverse transcription-polymerase chain reaction and gel electrophoresis

In order to determine the detection limit for chemically treated virions by gel electrophoresis, reverse transcription-polymerase chain reaction (RT-P CR) and infectivity assay, tobacco mosaic virus (TMV) exposed to various co ncentrations of chemicals was studied. When virions were exposed to 0.2 N H Cl for 30 mins, partially degraded TMV particles were observed by gel elect rophoresis. Under the same exposure, a major RT-PCR amplified DNA product c orresponding to the target size of 806 bp, which decreased as a function of time, could be detected for up to 60 mins of exposure. When virions were t reated with NaOH (0.02 N or higher normality) for 5 mins, partially degrade d virions were detected by gel electrophoresis, exhibiting multiple band pa tterns. Exposure of the virions to 0.1 N NaOH for 5 mins revealed severely degraded viral RNA, but disappearance of the amplified RT-PCR products was apparent during 30-60 mins of exposure. Therefore, these data showed clearl y the difference in the detection limit of gel electrophoresis and that of RT-PCR for the degraded viral RNA. In addition, the infectivity assay showe d that the number of local lesions in Nicotiana rustica were significantly reduced by more than 95% when the virus was exposed to 0.2 N HCl for 15 min s or 0.1 N NaOH for 10 mins. From these results we conclude that loss of in fectivity was not related to that of PCR product. Other chemical disinfecta nts such as phenol or formalin were also found to be effective to reduce th e virus infectivity, but a corresponding degradation of viral RNA was detec ted by neither gel electrophoresis nor RT-PCR.