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. 2008 Aug;46(8):2519-24.
doi: 10.1128/JCM.00277-08. Epub 2008 Jun 4.

Detection of all known parechoviruses by real-time PCR (VSports)

W Allan Nix  1 , Kaija Maher, E Susanne Johansson, Bo Niklasson, A Michael Lindberg, Mark A Pallansch, M Steven Oberste
Affiliations

Detection of all known parechoviruses by real-time PCR

W Allan Nix et al. J Clin Microbiol. 2008 Aug.

Abstract

The Parechovirus genus of the Picornaviridae family contains two species, Human parechovirus (HPeV) and Ljungan virus (LV). The HPeVs (including the former echoviruses 22 and 23, now HPeV type 1 (HPeV1) and HPeV2, respectively) cause a wide spectrum of disease, including aseptic meningitis, gastroenteritis, encephalitis, acute respiratory illness, and neonatal sepsis-like disease. The LVs were isolated from bank voles in Sweden during a search for an infectious agent linked to fatal myocarditis cases in humans. Because of the decline in use of cell culture and neutralization to investigate enterovirus-like disease, very few laboratories currently have the capability to test for parechoviruses. We have developed a real-time reverse transcription-PCR (RT-PCR) assay for detection of all known members of the genus Parechovirus. The assay targets the conserved regions in the 5' nontranslated region (5'NTR) of the parechovirus genome and can detect both HPeVs and LVs, unlike other published parechovirus 5' NTR assays, which only detect known HPeVs or only LVs. HPeV and LV can be differentiated by sequencing the 5'NTR real-time RT-PCR amplicon, when needed. The assay is approximately 100 times more sensitive than cell culture and may be used to test original clinical specimens VSports手机版. The availability of a broad-specificity PCR method should facilitate the detection of new human parechoviruses, as well as new parechoviruses in other mammalian species, and provide an opportunity to investigate the role of these viruses in human and animal disease. .

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Figures

FIG. 1.
FIG. 1.
(A) Schematic representation of the parechovirus genome, showing the location of the 5′NTR. (B) 5′NTR nucleotide sequences of HPeV1 to -6 and four LVs were aligned with Pileup (Wisconsin sequence analysis package, version 11.1; Accelrys, San Diego, CA). The average similarity across the entire alignment was plotted using PlotSimilarity (Wisconsin Package) with a window of 5 nucleotides. A part of this similarity plot is shown. Similarity scores of 100% indicate nucleotide sequence identity among all 14 aligned parechovirus sequences. (C) The minimal sequence heterogeneity among the aligned HPeVs and LVs is shown along with the locations of the primer and probe sites. The numbering of the primer/probe site nucleotide positions is relative to the published sequence of HPeV1-Harris and is for orientation only. (D) The degenerate primer and probe sequences are shown with primer orientations and probe labeling. Ambiguity codes: R, A or G; Y, C or T; W, A or T; and S, C or G. GenBank sequences aligned for primer and probe design included the following: HPeV1, S45208 and EF051629; HPeV2, AJ005695; HPeV3, AB084913 and AJ889918; HPeV4, AM235750 and DQ315670; HPeV5, AM235749 and AF055846; HPeV6, AB252582; and LV, AF327920, AF327921, AF327922, and AF538689.
FIG. 2.
FIG. 2.
Amplification of dilution series for HPeV1 and LV viral RNAs and synthetic RNAs, run in triplicate. The insets show the standard curves for each of the standard dilution series. Positive reaction CT values were averaged before standard curve plotting, using the least-mean-squares curve-fitting algorithm of the Stratagene Mx4000 software (version 4.2). (A) Real-time PCR amplification of 10-fold serial dilutions of HPeV1 viral RNA, containing the equivalent of 103 to 10−3 CCID50 per reaction mixture. (B) Real-time PCR amplification of 10-fold serial dilutions of LV viral RNA, containing the equivalent of 102 to 10−2 CCID50 per reaction mixture. (C) Real-time PCR amplification of 10-fold serial dilutions of HPeV1 sRNA, containing the equivalent of 106 to 100 RNA copies per reaction mixture. (D) Real-time PCR amplification of 10-fold serial dilutions of LV sRNA, containing the equivalent of 106 to 100 RNA copies per reaction mixture.
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