We developed and validated an improved real-time RT-QPCR assay for WNV. Detection with SYBR green is as sensitive as probe-based real-time PCR without the need for a specialized probe (Fig. ). All samples that yielded a fluorescence signal at least five times that of the nontemplate control reaction in the SYBR green-based assay also produced an easily discernible band upon agarose gel electrophoresis. Hence, these samples would be considered positive by conventional end-point RT-PCR. Using a pipetting robot, we were able to detect WNV in field samples with a 15-μl RT-PCR. Using previously published WNV env
primers, we achieved an SE for CT
values of less than 1% in a 96-sample repeat experiment (Fig. and data not shown). Lanciotti and colleagues introduced the TaqMan primers and probe for WNV used here and also, more recently, nucleic acid sequence-based amplification (NASBA) for WNV (21
). Their comparison of TaqMan real-time RT-QPCR, NASBA, and conventional RT-PCR revealed a strong concordance for all three methods, except for operator-based interpretations of threshold settings for NASBA and real-time RT-QPCR at very low target levels (≤0.1 PFU/ml). We found that real-time quantitative PCR with SYBR green also has a comparable performance profile. Comparisons at the extreme lower end of the linear range between different amplification methods are difficult to interpret, since different primer pairs with associated different amplification efficiencies (27
) and different reagents (13
) have been used. Previous studies estimated the limit of detection for the probe-based TaqMan RT-QPCR assay for WNV to be ≤0.1 PFU/ml (2
), well below the detection limit for culture-based assays. Using serial dilutions on Vero cells (Fig. ), we confirmed these observations for SYBR green-based real-time quantitative PCR.
The possibility of false-negative test results poses a substantial problem in diagnostic TaqMan assays because mutations within the probe-binding site can prevent annealing of the probe and subsequent detection. While this problem has been documented for herpes simplex virus (3
), it is a particular concern for WNV, severe acute respiratory syndrome virus, human immunodeficiency virus, hepatitis C virus, and other RNA viruses, since they exhibit higher sequence variability than DNA viruses and since it is not always possible to identify regions of the genome which are highly conserved. By synthesizing and testing every possible point mutation in the target region for the widely used WNV TaqMan assay, we encountered a potential false-negative rate of 47% for the TaqMan assay due to sequence variations in the probe-binding site. In comparison, the SYBR green-based assay produced a false-positive rate of 0% (Fig. ). We did not explore the effects of mutations within the binding sites for the forward and reverse primers, since these would affect the TaqMan assay and the SYBR green-based assay (as well as gel-based PCR assays) in the same way. The high-stringency annealing temperature used in real-time quantitative PCR (60°C) for the two outer primers leaves open the possibility that viral variants with mutations in the forward or reverse primer-binding sites may escape detection. Although not tested here, it is likely that the frequency of such false-negatives can be reduced by the use of multiple primer sets for each virus.
Could a SYBR green-based real-time PCR assay be used to screen for novel WNV isolates? A number of studies investigated WNV sequence variability (2
). For this flavivirus, the average sequence variability did not exceed 3% at the nucleotide level, and the majority of mutations did not lead to amino acid substitutions. For the 1999 outbreak in New York, Connecticut, and New Jersey, 99% sequence identity was reported over a 1,278-nucleotide region in 13 isolates from avian species, humans, and mosquitoes. Many of the same mutations were shared among isolates from mosquitoes, birds, and humans. Presumably, the need to enter and replicate in cells of avian as well as arthropod origins places severe constraints on the variability of this virus. In contrast, human retroviruses, such as human immunodeficiency virus type 1 (15
), exhibit amino acid variability of up to 20% within the same subtype. We found that a preliminary measure of natural variability can be obtained by Tm
or dissociation profile analysis (Fig. ). SYBR green-based real-time quantitative PCR incorporates dissociation profile analysis for each sample at no extra cost. This feature constitutes a considerable improvement over gel-based or TaqMan-based PCR, neither of which can identify the presence of single nucleotide mutations or small (<5-bp) insertions or deletions. In the TaqMan assay, these alterations led to a complete loss of signal (false-negative) or produced signals that were indiscernible from the wild-type signal (Fig. ); in the gel-based assay, the signals produced by these mutations were indiscernible from the wild-type signal. Since real-time quantitative PCR amplicons are small (<100 bp), even a single base-pair change can result in a distinguishable change in the Tm
), although in this study only changes of >3 nucleotides were identified with 100% accuracy. Currently, we are trying to improve upon the bioinformatics tools for this analysis. While not as reliable as sequence analysis, dissociation profile analysis has the potential to be used for rapid initial screening for the identification of viral mutants.