Comparison of methods to detect enteroviral genome in frozen and fixed myocardium by polymerase chain reaction.
BACKGROUND
Understanding the pathogenesis of viral myocarditis is linked to the availability of sensitive assays to detect viral genome in clinical material. Recent advances in molecular techniques permit detection of viral-specific RNA in tissue samples. We describe here a comparison of different methods for RNA extraction, reverse transcription, and gene amplification of Coxsackievirus B3 virus in fixed and frozen mouse myocardium.


METHODS
Homogenized Coxsackie B3 virus-infected myocardium was assayed for virus titer and formalin fixed, paraffin-embedded sections from the same heart were subjected to RNA extraction by 3 different methods. Optimal conditions were determined for a one-step assay combining reverse transcription and the polymerase chain reaction to detect enteroviral genome in RNA extracted by these different methods. The presence of amplifiable cDNA was confirmed by amplification of porphobilinogen deaminase mRNA as a positive control.


RESULTS
Extraction of RNA from paraffin-embedded myocardium after overnight digestion with proteinase K (200 micrograms/ml) and 0.5% sodium dodecyl sulfate was the most efficient of the three methods compared. With our optimized polymerase chain reaction assay, in which the cDNA synthesis and amplification steps are combined, we detected as little as 10 TCID50 of virus from frozen viral stocks and tissue homogenates and 1.0 TCID50 of virus from fixed tissue.


CONCLUSIONS
This sensitive polymerase chain reaction assay will facilitate examination of archival clinical samples to establish a retrospective diagnosis of enterovirus infection.
Department of Pediatrics, University of California, San Diego, School of Medicine, La Jolla.
H
H
Shimizu
D P
DP
Schnurr
J C
JC
Burns
D000818:Animals; D001483:Base Sequence; D003384:Coxsackievirus Infections / Q000175:diagnosis; D015925:Cryopreservation; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D004770:Enterovirus / Q000235:genetics*; D029822:Enterovirus B, Human / Q000235:genetics; D005784:Gene Amplification; D016679:Genome, Viral; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D016707:Tissue Fixation
D004279:DNA, Viral; D012367:RNA, Viral