SYBR Green-Based Real-Time Quantitative PCR Assay for Detection of West Nile Virus Circumvents False-Negative Results Due to Strain Variability

Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Journal of Clinical Microbiology (Impact Factor: 3.99). 05/2004; 42(4):1511-8. DOI: 10.1128/JCM.42.4.1511-1518.2004
Source: PubMed


Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus
(WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them
undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point
mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect
47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than
two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR
green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region
variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result
from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed

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    • "Single mismatches were detected in the forward and reverse primers or in the probe hybridization sequences of 12 strains (Table 2). While single mismatches have been described to cause failure in the detection of respiratory syncytial virus (Whiley and Sloots, 2006) and West Nile virus (Papin et al., 2004), it has also been demonstrated that single SNP may not completely prevent amplification, although they may cause inefficient annealing and amplification and underestimation of the copy number (Lefever et al., 2013). In four strains (HE800529, KM115677, KM115678, KM115679) the single mismatch can affect extension since it occurs at three bases from the 3 end of the reverse primer. "
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    • "Viral load was determined by RT-qPCR as per our published procedures [51] with primers: WNV Env F: 5′-TCAGCGATCTCTCCACCAAAG-3′; WNV Env R: 5′-GGGTCAGCACGTTTGTCATTG-3′. The following oligo was diluted to generate a standard curve to determine copy number: WNV Env Oligo: TCAGCGATCTCTCCACCAAAGCTGCGTGCCCGACCATGGGAGAAGCTCACAATGACAAACGTGCTGACCC. "
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    ABSTRACT: The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.
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