Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid

Abstract

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.

FIG. 1.

Enterovirus sequence alignments for 5′ primer, 3′ primer, and probe regions. The 14 most clinically important enterovirus serotypes were determined, and sequences from the primer and probe binding sites were aligned using GeneDoc version 2.6.2. Shown for each region is the consensus sequence, any serotypes varying from the consensus sequence, and the alignment and sequences of primers and probes previously described in the literature. , identical base; ▪, not available; ∗, base deletion; X, insertion of A prior to this base. Superscript numbers are reference numbers. Superscript letters: a, numbering based on Cox A16 sequence, GenBank accession no. 458298; b, consensus sequence was present in Echo 30 (accession no. 14139951 and 17224872), Echo 7 (1199448, 15788480, and 1199449), Cox B2 (7263148 and 7263147), Echo 6 (17530514 and 15788479), Cox B1 (15788477 and 323417), Echo 25 (1154653 and 4539509), Cox A9 (12802345 and 221214), Echo 16 (15788484), Echo 18 (15788486), Cox B3 (17148519, 10863164, 5833884, and 5833880), Echo 11 (17530521, 17530520, and 17530519), enterovirus 71 (4753701, 4753698, 4753699, 4753694, and 18158530), and Cox B4 (61031 and 914055); c, consensus sequence was present in Echo 30 (14139951, 14139942, and 17224872), Cox B1 (914052), Echo 7 91199448), Cox B2 (7263148 and 7263147), Echo 6 (17530514 and 509558), Echo 11 (17530521, 17530520, and 17530519), enterovirus 71 (18158530), Echo 9 (769799 and 509559), Cox B2 (16555707, 16555709, and 16555708), and Echo 25 (4572558). d, consensus sequence was present in Echo 30 (14139951), Cox B2 (7263148 and 7263147), Echo 6 (17530514 and 509558), Echo 25 (1154653 and 4572558), Cox A9 (221214), Cox B1 (323417), Cox B3 (10863164, 5833884, and 5833880), Echo 7 (1199449), Echo 11 (17530520 and 17530519), Echo 9 (769799 and 509559), and Cox B4 (61031 and 914055).

FIG. 1.

Enterovirus sequence alignments for 5′ primer, 3′ primer, and probe regions. The 14 most clinically important enterovirus serotypes were determined, and sequences from the primer and probe binding sites were aligned using GeneDoc version 2.6.2. Shown for each region is the consensus sequence, any serotypes varying from the consensus sequence, and the alignment and sequences of primers and probes previously described in the literature. , identical base; ▪, not available; ∗, base deletion; X, insertion of A prior to this base. Superscript numbers are reference numbers. Superscript letters: a, numbering based on Cox A16 sequence, GenBank accession no. 458298; b, consensus sequence was present in Echo 30 (accession no. 14139951 and 17224872), Echo 7 (1199448, 15788480, and 1199449), Cox B2 (7263148 and 7263147), Echo 6 (17530514 and 15788479), Cox B1 (15788477 and 323417), Echo 25 (1154653 and 4539509), Cox A9 (12802345 and 221214), Echo 16 (15788484), Echo 18 (15788486), Cox B3 (17148519, 10863164, 5833884, and 5833880), Echo 11 (17530521, 17530520, and 17530519), enterovirus 71 (4753701, 4753698, 4753699, 4753694, and 18158530), and Cox B4 (61031 and 914055); c, consensus sequence was present in Echo 30 (14139951, 14139942, and 17224872), Cox B1 (914052), Echo 7 91199448), Cox B2 (7263148 and 7263147), Echo 6 (17530514 and 509558), Echo 11 (17530521, 17530520, and 17530519), enterovirus 71 (18158530), Echo 9 (769799 and 509559), Cox B2 (16555707, 16555709, and 16555708), and Echo 25 (4572558). d, consensus sequence was present in Echo 30 (14139951), Cox B2 (7263148 and 7263147), Echo 6 (17530514 and 509558), Echo 25 (1154653 and 4572558), Cox A9 (221214), Cox B1 (323417), Cox B3 (10863164, 5833884, and 5833880), Echo 7 (1199449), Echo 11 (17530520 and 17530519), Echo 9 (769799 and 509559), and Cox B4 (61031 and 914055).

FIG. 2.

Rhinovirus sequences with similarity to enterovirus 5′ primer, 3′ primer, and probe regions. All rhinovirus sequences containing the 5′ untranslated region bound by our enterovirus real-time primers and probe were aligned with sequences from our primers and probe using GeneDoc version 2.6.2. Shown for each region is the consensus sequence, any sequences varying from the consensus sequence, and the alignment and sequences of our selected real-time primer-probe set. Superscript a: numbering based on rhinovirus 1B sequence, GenBank accession no. 221708.

FIG. 3.

Comparison of LightCycler versus TaqMan results with patient-derived specimens. Thirty-eight enterovirus-positive patient-derived samples submitted to the clinical laboratory between June 2001 and December 2002 were evaluated using the two-step LightCycler and TaqMan methods. The correlation between the two methods is shown.

FIG. 4.

Quality control data for two-step TaqMan method. Shown are quality control data obtained from 19 runs of the two-step TaqMan method over a 1-month period for high (8.7 × 10⁷ copies/ml), intermediate (8.7 × 10³ copies/ml), and low (8.7 × 10² copies/ml) standards (A) and high positive (3.3 × 10⁶ copies/ml) and low positive (2.2 × 10³ copies/ml) controls (B).