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-PCR) and infectivity assay, tobacco mosaic virus (TMV) exposed to various concentrations of chemicals was studied. When virions were exposed to 0.2 N HCl for 30 mins, partially degraded TMV particles were observed by gel electrophoresis. Under the same exposure, a major RT-PCR amplified DNA product corresponding 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 treated with NaOH (0.02 N or higher normality) for 5 mins, partially degraded virions were detected by gel electrophoresis, exhibiting multiple band patterns. 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 clearly the difference in the detection limit of gel electrophoresis and that of RT-PCR for the degraded viral RNA. In addition, the infectivity assay showed 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 mins or 0.1 N NaOH for 10 mins. From these results we conclude that loss of infectivity was not related to that of PCR product. Other chemical disinfectants such as phenol or formalin were also found to be effective to reduce the virus infectivity, but a corresponding degradation of viral RNA was detected by neither gel electrophoresis nor RT-PCR.
Choi
C W
CW
Department of Biology, Pai Chai University, Taejon 302-735, South Korea. choicw@mail.paichai.ac.kr
Park
S H
SH
Choi
J K
JK
Ryu
K H
KH
Park
W M
WM
D004587:Electrophoresis, Agar Gel; D005557:Formaldehyde / Q000494:pharmacology; D006851:Hydrochloric Acid / Q000494:pharmacology; D019800:Phenol / Q000494:pharmacology; D010935:Plant Diseases / Q000821:virology; D010947:Plants, Toxic; D012367:RNA, Viral / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012972:Sodium Hydroxide / Q000494:pharmacology; D013997:Time Factors; D014026:Nicotiana / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000187:drug effects* / Q000472:pathogenicity / Q000528:radiation effects; D014771:Virion / Q000302:isolation & purification / Q000378:metabolism
D012367:RNA, Viral; D005557:Formaldehyde; D019800:Phenol; D012972:Sodium Hydroxide; D006851:Hydrochloric Acid