Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis.
Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed. The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis.
Casas
I
I
Department of Pathological Sciences, University of Manchester, United Kingdom.
Klapper
P E
PE
Cleator
G M
GM
Echevarría
J E
JE
Tenorio
A
A
Echevarría
J M
JM
D000208:Acute Disease; D000328:Adult; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D004770:Enterovirus / Q000235:genetics* / Q000302:isolation & purification; D005243:Feces / Q000821:virology; D006801:Humans; D008582:Meningitis, Aseptic / Q000134:cerebrospinal fluid / Q000821:virology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000134:cerebrospinal fluid*; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured; D014709:Vero Cells
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase