Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid.
A LightCycler and two TaqMan real-time PCR assays were evaluated against an older PCR with liquid-phase hybridization method for the detection of enterovirus RNA in 74 patient samples. The two-step LightCycler and the two-step TaqMan formats correlated well with each other (r(2) = 0.90) and were equally sensitive compared to the liquid-phase hybridization method, whereas the one-step recombinant Tth DNA polymerase format was rather insensitive, detecting enterovirus RNA in only about one-half of those patient samples previously positive by liquid-phase hybridization. The two-step TaqMan method was optimized utilizing 10 micro l of cDNA and demonstrated the highest degree of analytical sensitivity among the methods evaluated in our study, being able to reproducibly quantify down to 510 copies of enteroviral RNA/ml of cerebrospinal fluid. This new assay can be performed in 4 h, is much less labor intensive, and showed less cross-reactivity with rhinovirus than the liquid-phase hybridization assay. Thus, the two-step TaqMan assay should prove useful in the diagnosis of enteroviral meningitis versus bacterial meningitis, thereby resulting in timely and appropriate clinical management that can amount to significant cost savings to the patient and health care system.
Lai
K Kay-Yin
KK
Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington 98195, USA.
Cook
Linda
L
Wendt
Sharon
S
Corey
Lawrence
L
Jerome
Keith R
KR
D019295:Computational Biology; D017931:DNA Primers; D004770:Enterovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000821:virology; D006801:Humans; D008587:Meningitis, Viral / Q000821:virology*; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000134:cerebrospinal fluid*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D017931:DNA Primers; D012367:RNA, Viral; D019914:Taq Polymerase