Comparative evaluation of Taqman real-time PCR and semi-nested VP1 PCR for detection of enteroviruses in clinical specimens
Molecular diagnostic tests to detect enterovirus in clinical specimens usually target highly conserved sites in the 5'-non-translated region, allowing detection of all members of the genus. The sequences in the 5'-NTR do not correlate with serotype, but PCR and sequencing of the VP1 region can be used for typing; this system has largely replaced traditional antigenic typing.
To investigate the relative performance of two common enterovirus assays.
We compared the relative sensitivities of Taqman real-time RT-PCR (rRT-PCR) and a VP1 semi-nested PCR (RT-snPCR) assay in which sequencing the VP1 amplicon also provides typing information.
There was 89% concordance between the two methods among the 371 clinical specimens tested (74 positive in both assays and 257 negative in both assays). Twenty-seven rRT-PCR-negative/VP1-positive specimens were confirmed positive by sequencing; 13 specimens were rRT-PCR-positive/VP1-negative.
The results suggest that either assay can produce satisfactory results, but that using both assays in parallel should provide the highest sensitivity for clinical diagnostic testing.
Available from: Seiha Yen
- "Enterovirus RNA was detected using a previously described assay (Oberste et al., 2010) and the SuperScript III Platinum One- Step System (Life Technologies, Carlsbad, CA, USA). Each 25 L reaction contained 5 L of RNA, 12.5 L of 2X Invitrogen Reaction Mix, 0.5 L of 50mM MgSO 4 , 0.5 L of SuperScript ® III RT/Platinum ® Taq Mix, 0.1 M probe and 0.4 M primers. "
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ABSTRACT: The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.
Journal of virological methods 09/2013; 195. DOI:10.1016/j.jviromet.2013.08.035 · 1.78 Impact Factor
Available from: Xiaoyan Lu
- "Development of real-time RT-PCR assays that can detect all RV and EV strains and distinguish between both groups is challenging due to the extensive sequence diversity within each group and sequence similarity between some EV and RV strains. These data confirmed previous experience with the in-house EV and RV assays: both assays cross-react with some RV and EV strains, particularly if present in high copy number (Lu et al., 2008; Oberste et al., 2010). "
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ABSTRACT: Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI=0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation.
Journal of virological methods 07/2012; 185(2):259-66. DOI:10.1016/j.jviromet.2012.07.010 · 1.78 Impact Factor
Available from: Lai zhen-rung
- "Real-time RT-PCR was performed with specific primers, SYBR green PCR Master Mix, and SYBR green I dsDNA binding dye. Oligonucleotides for VP1 were forward primer 5′-GGCCCCTGAATGCGGCTAATCC-3′ (nt 458–480) and reverse primer 5′-GCGATTGTCA CCATWAGCAGYCA-3′ (nt 603–581) as described in the report of Oberste et al. . PCR product level was monitored with an ABI PRISM 7000 sequence detection system (Applied Biosystems). "
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ABSTRACT: Pandemic infection or reemergence of Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) occurs in tropical and subtropical regions, being associated with hand-foot-and-mouth disease, herpangina, aseptic meningitis, brain stem encephalitis, pulmonary edema, and paralysis. However, effective therapeutic drugs against EV71 and CVA16 are rare. Kalanchoe gracilis (L.) DC is used for the treatment of injuries, pain, and inflammation. This study investigated antiviral effects of K. gracilis leaf extract on EV71 and CVA16 replications. HPLC analysis with a C-18 reverse phase column showed fingerprint profiles of K. gracilis leaf extract had 15 chromatographic peaks. UV/vis absorption spectra revealed peaks 5, 12, and 15 as ferulic acid, quercetin, and kaempferol, respectively. K. gracilis leaf extract showed little cytotoxicity, but exhibited concentration-dependent antiviral activities including cytopathic effect, plaque, and virus yield reductions. K. gracilis leaf extract was shown to be more potent in antiviral activity than ferulic acid, quercetin, and kaempferol, significantly inhibiting in vitro replication of EV71 (IC(50) = 35.88 μg/mL) and CVA16 (IC(50) = 42.91 μg/mL). Moreover, K. gracilis leaf extract is a safe antienteroviral agent with the inactivation of viral 2A protease and reduction of IL-6 and RANTES expressions.
Evidence-based Complementary and Alternative Medicine 05/2012; 2012(6):503165. DOI:10.1155/2012/503165 · 1.88 Impact Factor
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