25. RT-Nested PCR diagnosis of Mouse Hepatitis Virus Contamination in Immunocompromised mouse Population
Hiromi Takahashi-Omoe, Satoru Matsushita and Akihiro Kawano
Keywords: mouse hepatitis virus, immunocompromised mouse, RT-nested PCR
Mouse hepatitis virus (MHV) is a positive-strand RNA virus in the family Coronaviridae and is known to be the most prevalent virus infecting laboratory mice. MHV infection is a serious problem, because MHV is a fatal pathogen for immunocompromised mice, such as nude mice and severe combined immunodeficient (Scid) mice.
MHV infection in a mouse population is usually diagnosed serologically by such methods as enzyme linked immunosorbent assay (ELISA) and immunofluorescent staining. These methods, however, cannot be applied to the detection of MHV infection in immunocompromised mice, since they do not develop normal antibody responses. When serologic methods are not applicable, the infectious viruses or viral genes should be directly detected. Therefore, we have developed a reverse transcription plus nested polymerase chain reaction (RT-nested PCR) system combined with southern blot analysis of PCR. The first and second PCRs were designed to amplify 989 bp and 575 bp DNA fragments from the coding region of MHV mRNA7, and the southern blot was designed to confirm the specificity of the first PCR product. Using these methods, mouse samples dissected from necrotic foci on the liver of thirteen C.B-17/Icr-scid/scid mice and six BALB/c-nu/nu mice were tested. The results gained from RT-nested PCR combined with southern blot analysis demonstrated that no tested mice were infected by MHV. We suggest that the methods employed in this study are useful for MHV detection in specimens that are obtained from immunocompromised mice.
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