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Journal of Clinical Microbiology, July 2003, p. 3133-3141, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3133-3141.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evaluation of Real-Time PCR versus PCR with Liquid-Phase Hybridization for Detection of Enterovirus RNA in Cerebrospinal Fluid

K. Kay-Yin Lai,1 Linda Cook,1 Sharon Wendt,1 Lawrence Corey,1,2,3 and Keith R. Jerome1,3*

Departments of Laboratory Medicine,1 Medicine, University of Washington Medical Center, Seattle, Washington 98195,2 Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, Washington 981093

Received 27 January 2003/ Returned for modification 17 March 2003/ Accepted 28 April 2003

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 (r2 = 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 µl 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.

* Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, D3-100, Seattle, WA 98109. Phone: (206) 667-6793. Fax: (206) 667-4411. E-mail: kjerome{at}fhcrc.org.

Journal of Clinical Microbiology, July 2003, p. 3133-3141, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3133-3141.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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