Rt pcr amplification of enterovirus belonging to the a, b, c. We describe an improvement of an earlier reported realtime rt pcr assay for the detection of enterovirus rna, based on the 5 exonuclease digestion of a duallabeled fluorogenic probe by taq dna polymerase. Enterovirus is the leading cause of meningitis and is. Usefulness of published pcr primers in detecting human. Diagnosis of enterovirus infection by genusspecific pcr and. Nasopharyngeal, dermal, rectal, or other sites clinically indicated.
All 14 sampled farms were positive for pev9 and the overall prevalence of infection in the studied pigs was 8. A commercial enterovirus pcr test the enterovisiontm pcr detection kit dna technology as, aarhus, denmark was used as described by the. The amplified products were detected by 2% agarose gel electrophoresis to identify a 93 base pair bp fragment for rhinovirus or a 154 bp fragment for enterovirus. A different extraction method, realtime rt pcr instrument and primer set were evaluated. Enterovirus, molecular detection, pcr, varies essentia. A tool for rapid species identification of uncharacterized enteroviruses. Vipr uses phyml guindon and gascuel, 2003 and offers multiple evolutionary models to infer phylogenies. The enterovirus d68 2014 realtime rt pcr assay evd68 2014 rrt pcr is intended for the in vitro qualitative detection of rna from the enterovirus d68 evd68 strains detected in north america. Coxsackievirus a6 and hand, foot, and mouth disease, finland.
Nonpolio enteroviruses and parechoviruses can be detected in stool or rectal swabs and. Detection of human enterovirus 71 reverse transcription. Enterovirus, molecular detection, pcr, varies asante lab. The pcr uses biotinylated primers targeted to the 5 utr of the viral genome. We identified an unusual enterovirus type, coxsackievirus a6 cva6, as the causative agent. Revealing enterovirus infection in chronic human disorders. In rapid cycle realtime pcr methods and applications. Cva6 infections may be emerging as a new and major cause of epidemic hfmd.
B efficiency of near fulllength ev genome pcr tested on vigr clinical stool samples n 68 compared. Collect specimen from the affected site using the flocked swab. Detection of enterovirus nucleic acid by pcr is also the most sensitive diagnostic method for the diagnosis of cns infection caused by these viruses. Prevalence of porcine enterovirus 9 in pigs in middle and. Pcr primers and probes were obtained from the department of microbiology, shenzhen center for disease control and prevention in china. Speciesspecific rt pcr amplification of human enterovirus. An internal control is added to ensure the extraction was performed correctly and the pcr reaction was not inhibited. A sensitive and quantitative singletube realtime reverse transcriptase pcr for detection of enteroviral rna.
The first step detects picornaviruses ev2 and ev3 primers, and the second step distinguishes human rhinovirus from human enterovirus with specific primers ccrv3ccrv4 to hrv and ev3evnc1 to hev. Mar 28, 20 little information on the epidemiology and pathogenicity of porcine enterovirus 9 pev9 is available. Rhinovirus and enterovirus by realtime pcr accu reference. We carry a diverse portfolio of bacterial, viral, fungal, and protozoan pathogen primers. As part of our evaluation of the outbreak, we sequenced and published the. Alternatively, enterovirus pcr has become standard practice due to. This assay provides a more specific identification of evd68 strains allowing better diagnosis. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype a total of 193 isolates. Our data show that the optimized assay yields a higher sensitivity and reproducibility and. This pcr assay has been optimized to detect a target sequence in the polyprotein region. The triplex rt pcr was developed and optimized using a mixture of ev71, ca16, evs and ic primers and their corresponding probes. A new realtime reverse transcriptionpcr assay for detection. Export image and legend, or download trees as newick or phyloxml files for other viewing software.
Primers and probe sequence design are among the most critical factors in realtime polymerase chain reaction pcr assay optimization. Thus, viral culture is not an optimal test in the evaluation of meningitis. The genotypetool accepts up to 20000 sequences at a time. The amplified products were detected by 2% agarose gel electrophoresis to. Analysis of the pcr amplification and probe melting curves is accomplished through the use of lightcycler software. Alternatively, enterovirus pcr has become standard practice due to its muchimproved sensitivity and turnaround time. In addition, the previously published primers were used in assays divided between those using 1step rt pcr and those using a separate rt cdna synthesis step. Original article detection of human enterovirus 71. Pcr amplification was significantly affected by c t value, with successful amplification resulting from c t values ranging between 15. Codehop is suited for cases where the protein sequences are distant from each other and degenerate primers are needed. In particular, accu reference medical lab has revolutionized testing for respiratory and gastrointestinal diseases, which allows for a significantly earlier detection of pathogens than any other technology. Routine testing from respiratory swabs, and rectal swabsstool is not recommended since enterovirus can shed in these sites and detection of enteroviral rna may not represent an active infection.
As described in our previous study 22, the vp1 gene of enterovirus was used to distinguish enterovirus serotypes and the primers were designed based on sequence infor mation obtained from genbank. Virus pathogen database and analysis resource vipr. Primers gibcobrl, merelbeke, belgium and probes eurogentec, seraing, belgium were designed using primer express 1. Attempts to identify an etiologic agent in cerebrospinal fluid by using reverse transcription pcr showed positivity to enterovirus ev in 66 21. All specimens were tested for human glyceraldehyde3phosphate dehydrogenase to confirm rna integrity and control for rt pcr inhibition.
The third protocol was a duplex seminestedrtpcr involving two pcr steps with 5utr target primers. Using taqman probes we have previously established a real time rtpcr method for coxsackievirus a10 and other enterovirus detection 1. Specimens grossly contaminated with blood may inhibit the pcr and produce false negative results. Enterovirus, molecular detection, pcr, varies united.
A sensitive and quantitative singletube realtime reverse. The blast results are then automatically analyzed to avoid primer pairs that can cause amplification of targets other than the. Wellcharacterized primer sets for other respiratory virus pathogens unpublished data, including human respiratory syncytial virus, parainfluenzaviruses 1, 2, and 3, influenzaviruses a and b, adenovirus, and picornavirus rhinovirus and enterovirus, were also used to test clinical specimens in this study primers available on request. Impact of a diagnostic cerebrospinal fluid enterovirus. To evaluate the ability to simultaneously detect different pathogens of hfmd, an ev71c4 virus strain js06523 and a ca16b1 virus strain 3927 at the same concentration of 1 tcid 50 ml were mixed, and the. Detection of rhinovirus and enterovirus in upper respiratory. Our approach includes the utilization of proprietary design tools and ongoing sequence surveillance studies to provide the highest quality in pcr reagents. Pdf detection of enteroviruses from water samples using. Enterovirus d68 evd68 2014 outbreak strainspecific realtime reverse transcription polymerase chain reaction rrt pcr assay instructionsversion 10142014 the cdc picornavirus laboratory from midseptember to midoctober developed and evaluated the evd68specific rrt pcr assay. Pcr were used to amplify vp1 gene of ev71 with a size of 1082 bp. Proper handling and storage is very important to prevent the destruction of the target rna by rnases that can be introduced by human hands. Collect specimen from the affected site using the flocked swab provided with red top utm collection kit. The primers and the probe table 1 were designed with.
Laboratory test directory share rhinovirus and enterovirus by realtime pcr m127. In the multiplex assay, the first pcr reaction used the same set of primers. Primer blast was developed at ncbi to help users make primers that are specific to the input pcr template. Source information should include main anatomical site of collection. During fall 2008, an outbreak of hand, foot, and mouth disease hfmd with onychomadesis nail shedding as a common feature occurred in finland. The realtime rt pcr primers and probes were obtained from the department of microbiology, shenzhen center for disease control and prevention in china. Enterovirus, molecular detection, pcr, plasma saint. Diasorin molecular has an extensive and growing menu of over 50 analyte specific reagents asrs for the amplification and detection of relevant viral, bacterial and fungal targets as well as common genetic singlenucleotide polymorphisms. The sequences of the primers and their locations were shown in table 1 and fig. The lightcycler instrument can rapidly 3040 minutes detect amplicon development through stringent aircontrolled temperature cycling in capillary cuvettes. Enterovirus detection by pcr polymerase chain reaction. Enterovirus, molecular detection, pcr, plasma essentia. Enterovirus pcr, p overview useful for aids in diagnosing enterovirus infections method name realtime polymerase chain reaction pcr rna probe hybridization ny state available yes specimen specimen type plasma edta specimen required submit a raw clinical sample not a culture isolate for enterovirus pcr. Context enterovirus ev infection, the most common cause of aseptic meningitis, can be rapidly diagnosed with an evspecific reverse transcriptase polymerase chain reaction evpcr test.
We carry a diverse portfolio of bacterial, viral, fungal, and protozoan pathogen primers for global laboratory professionals. However, no studies have examined evpcr in a clinical context in which it is routinely used. Realtime rt pcr assay for rapid, highly sensitive and specific detection of human enterovirus d68 evd68 human enterovirus d68 evd68 is a nonpolio enterovirus that can cause mild to severe respiratory illness, especially in infants and children with asthma. Designs pcr polymerase chain reaction primers from protein multiplesequence alignments. Development and evaluation of an enterovirus d68 realtime. The aim of this study was to determine the applicability of reverse transcriptionpcr rtpcr with universal panenterovirus primers and enterovirus 71 ev71 typespecific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. Enterovirus, molecular detection, pcr, varies duke. In addition, the previously published primers were used in assays divided between those using 1step rtpcr and those using a separate rt cdna synthesis step. Mgb alert enterovirus primers are designed to amplify nucleic acid sequences from enteroviruses universal cycling conditions run multiple assays simultaneously meltcurve capable increase specificity, detect genetic variants streamlined workflow optimize staff utilization open platform runs on most realtime pcr instruments expert technical support committed to maximize your investment for additional information on ordering mgb alert.
Applications and challenges of realtime pcr for the clinical microbiology laboratory. A sensitive and quantitative singletube realtime reverse transcriptasepcr for detection of enteroviral rna nahla mohameda, amal elfaitouria, jan fohlmanb, goran frimanb, jonas blomberga. Rhinovirus detection using different pcrbased strategies. Isolation of members of a rapidly evolving family of novel cytosine methyltransferase homologs from diverse plants demonstrated his practical utility. Enzymelinked immunosorbent assays elisas that detect heterotypical antibodies against enterovirus immunoglobulin m igm, iga, and igg were compared with reverse transcriptionpcr by using primers specific to the 5. Primers and probe design and precision assessment of the. Speciesspecific rtpcr amplification of human enterovirus. Enterovirus, molecular detection, pcr, plasma duke. Specimen collection and processing instructions for medical laboratory test enterovirus, pcr csf at geisinger medical laboratories. In addition, this approach recognized more divergent evd68 subtypes and was not crossreactive with non enterovirus strains. Analytical and performance characteristics are not established. Extraction of enterovirus nucleic acid from specimen, followed by reverse transcription of viral rna, then amplification and detection of cdna using realtime, quantitative pcr.
The data presented in this article show the conserved regions for primers and probe design using multiple sequence alignment fig. The region chosen for the vzv design open reading frame orf 38 gene is conservative and does not impose any restrictions on primer design. Nonpolio enterovirus for health care professionals. A method for rapid detection of enteroviruses in water sample was established by using rt pcr with universal primers with pv1, pv2, pv3 and cvb3 as the reference viruses.
The present study investigated the prevalence of pev9 in pig populations in middle and eastern china using reverse transcriptase rt pcr. National jewish health advanced diagnostic laboratories in colorado, is a leading iso 15189 accredited clinical laboratory with central laboratory services. Cdc investigators have developed a realtime rtpcr reverse transcriptase polymerase chain reaction taqman assay using primers and probes specific for evd68 viral protein 1 nucleic acid. Accu reference medical lab is a regional leader in the fields of toxicology, pharmacogenetics and molecular testing, in addition to routine blood and urine testing. Genotyping was conducted by using rtpcr of virion protein vp 12a and vp1 regions and sequencing 7, 8. Jun 10, 2015 we have developed and evaluated a realtime reverse transcriptase pcr rt pcr assay for the detection of human enterovirus d68 evd68 in clinical specimens. In comparison, realtime pcr allows sameday detection. Enterovirus infections characteristically take place during summerfall in areas of the north temperate zone.
Quick tree produces an accurate topology for 1,000 sequences. Enterovirus genomic rna is first transcribed to cdna by reverse transcriptase, followed by amplification of the cdna product. Primers and probe design and precision assessment of the real. Enterovirus, molecular detection, pcr, plasma saint francis. This data article contains data related to the research article entitled rapid detection of enterovirus and coxsackievirus a10 by a taqman based duplex onestep real time rtpcr assay chen at al. It uses primer3 to design pcr primers and then submits them to blast search against userselected database. First, two nested rtpcr with specific primers in the 5. The published study confirmed these primers could detect evd68 in clinical samples with a higher degree of sensitivity 10100x compared to the commercially available tests. Decoration options let you color tree leaves by metadata. Submit a raw clinical sample not a culture isolate for enterovirus pcr. Table 1 describes the locations and sequences of the primers used in the assays. Utr of enterovirus and rhinovirus respectively, were tested hyypia et al. Traditional cell culture methods require 6 days, on average, for enterovirus detection.
The aim of the present study was to develop and validate an evd68speci. Sequencing and phylogenetic analyses of pcr products from 59 89. As part of our evaluation of the outbreak, we sequenced and published the genome. Geisinger medical laboratoriesgeisinger proven diagnostics. Luminex offers a broad menu of analyte specific reagents asr products based on our unique, patented multicodertx technology for the identification and quantification of various pathogens. This test will detect enterovirus, but will not differentiate viruses in this family or provide serotyping information. Nonpolio enteroviruses and parechoviruses can be detected in stool or rectal swabs and respiratory specimens including from the throat. This assay was developed in response to the unprecedented 2014 nationwide evd68 outbreak in the united states associated with severe respiratory illness. The evd68 rrtpcr primer and probe set targets the coding region for the viral protein 1 vp1 gene of the enterovirus d68 and shows. Realtime rtpcr assay for rapid, highly sensitive and. This is called the melting temperature tm of that virus.
First, two nested rt pcr with specific primers in the 5. Enterovirus dna pcr regional medical lab test directory. The sensitivity of viral culture of cerebrospinal fluid csf for enterovirus is 30 to 75% and an average of 7 days is required for isolation. The second round of pcr was carried out with the specific rhinovirus and enterovirus primer sets.
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