Detection of mouse hepatitis virus by the polymerase chain reaction and its application to the rapid diagnosis of infection.
Eight different strains of mouse hepatitis virus (MHV) were analyzed by the polymerase chain reaction (PCR) to see whether two sets of oligonucleotides, which were synthesized based on the published nucleotide sequence of MHV-JHM mRNAs 6 and 7, could be used as universal primers for amplification. Total RNA extracted from virus-infected cells or virus-infected culture fluids was transcribed into cDNA by using reverse transcriptase and oligo(dT) as primer, then the cDNA transcripts were amplified by PCR. The MHV-specific fragments of 199-bp and 241-bp were obtained from all eight strains irrespective of nucleotide differences in the primer regions. The same fragments were also amplified from RNA derived from the liver and brain of MHV-JHM-infected mice as soon as day 1 after intraperitoneal injection, even from the liver from which the virus was not detected. Results of PCR amplification from the liver RNA extracts became positive when more than 10(-2)PFU of MHV-JHM was contained in the PCR reaction mixture. In contrast, anti-MHV antibody was not detected by enzyme-linked immunosorbent assay until day 6 after inoculation. These results suggest that PCR is a very sensitive method to identify a variety of MHV infections in laboratory animals, especially at the early phase of infection.
Yamada
Y K
YK
Division of Experimental Animal Research, National Institute of Health, Tokyo, Japan.
Yabe
M
M
Yamada
A
A
Taguchi
F
F
D000818:Animals; D001483:Base Sequence; D018352:Coronavirus Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012376:Rodent Diseases / Q000175:diagnosis* / Q000382:microbiology
D017931:DNA Primers; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral