Use of reverse transcriptase PCR to subtype N1 to N9 neurami

Use of Reverse Transcriptase PCR To

Subtype N1 to N9 Neuraminidase Genes of Avian Influenza Viruses

Kenji Tsukamoto, Takayoshi Ashizawa, Koji Nakanishi,Noriyuki Kaji, Kotaro Suzuki, Makiko Shishido, Masatoshi Okamatsu and Masaji Mase

J. Clin. Microbiol. 10.1128/JCM.02366-08.

2009, 47(7):2301. DOI:Published Ahead of Print 29 April 2009.

Updated information and services can be found at: http://doc.guandang.net/content/47/7/2301These include:

REFERENCES

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JOURNALOFCLINICALMICROBIOLOGY,July2009,p.2301–2303Vol.47,No.7

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UseofReverseTranscriptasePCRToSubtypeN1toN9

NeuraminidaseGenesofAvianIn uenzaViruses

KenjiTsukamoto,1*TakayoshiAshizawa,21KojiNakanishi,3NoriyukiKaji,41

KotaroSuzuki,1

MakikoShishido,MasatoshiOkamatsu,1andMasajiMaseResearchTeamforZoonoticDiseases,NationalInstituteofAnimalHealth,3-1-5Kannondai,Tsukuba,Ibaraki305-0856,

Japan1;TyuouLivestockHygieneServiceCenterofChibaPrefecture,Iwatomi,Sakura,Chiba285-0072,Japan2;LivestockHygieneServiceCenterofShigaPrefecture,Nishihongo,Oomi-hachiman,Shiga523-0813,Japan3;andLivestockHygieneServiceCenterofShimanePrefecture,Jinzaioki,Izumo,Shimane699-0822,Japan4

Received9December2008/Returnedformodi cation13March2009/Accepted4April2009

ReversetranscriptasePCRdesignedtoamplifytheN1toN9neuraminidase(NA)genesofavianin uenzavirusesdetected118ofthe119NAgenestested(99.2%)inasubtype-speci cmanner.Thistechniquesuccess-fullysubtypedall167recentavianin uenzavirusesisolatedfrombirds.Subtypespeci citywascon rmedbysequenceanalysesofall285PCRproducts.

ThespreadoftheH5N1highlypathogenicavianin uenzationwasperformedusingtheGeneAmpPCRSystem9700(AI)virusinAsia,Europe,andAfricaraisesconcernsthata(ABI,FosterCity,CA)andExTaqpolymerase(Takara,Shiga,pandemicwilloccur(1,15).MigratoryducksthataremoreJapan)(13).PCRproductsweresequenceddirectlyusingfor-resistanttotheH5N1viruscancarrytheviruslongdistances,wardandreverseprimers(ABI-3100sequencer;PEABI,Fos-whilemore-susceptibleswansandgeeseserveasindicatorsofterCity,CA)(13).

theinfectionsinwildaquaticbirdpopulations(3,4).ActiveThedetectionrateofNAgenesbytheNAsubtypingprimersurveillanceprogramshavebeeninstitutedworldwidetode-setswasdeterminedusingpreviouslyisolatedAIviruses(n terminethecurrentH5N1prevalenceamongwildaquatic119;isolatedbetween1946and2006).These119virusescon-birds(1,14).TheAIvirushastwoglycoproteinspikes,hem-sistedof83duckstrains,16chickenstrains,7turkeystrains,agglutinin(HA)andneuraminidase(NA).NAcomprisesaand13strainsfromotherbirds.Theyincluded77strainsfrommushroom-shapedtetramerandispidedserologicallyintoJapanand42strainsfromKorea,Thailand,Italy,TheNeth-ninesubtypesbyusinganNAinhibition(NI)test(8,12).NAerlands,theUnitedStates,orChile;alternatively,thevirusessubtypingusingPCRisusedforAIglobalsurveillance,sincecanbeviewedas92Eurasianand27Americanlineages.Thethistestcanbeperformedeasilyingeneraldiagnosticlabora-primersetsampli ed118NAgenes:12N1,32N2,20N3,6N4,tories.Incontrast,theNItestcanbeperformedonlyinrefer-12N5,17N6,3N7,5N8,and11N9genes.TheNAsubtypeencelaboratories.

speci citywascon rmedbysequenceanalysesofall118PCRComparisonof263completenucleotidesequencesofNAproductsbyusingthesameprimers.Onlyonegene,anN2genesdownloadedfromtheIn uenzaSequenceDatabasegene,wasnotampli edbyPCR.Fullgenomeanalysisshowed(http://www. http://doc.guandang.netnl.gov/)(10)revealedthatthevirusmem-thattheunampli edN2genebelongedtotheAmericanlin-braneanchorandstalkregionhaveagreatdealofnucleotideeage.Serial10-folddilutionsofallantoic uidscontainingninesequencevariability(8).ThisvariabilitycanbeexploitedtoAIviruses(onestrainforeachofsubtypesN1toN9)wereuseddistinguishthedifferentsubtypes.ForeachNAsubtype,wetocomparePCRsensitivitytoinfectivity,expressedas50%thusdesignedforwardprimersthatwerecomplementarytotheegg-infectivedose.ThedetectionlimitsofthePCRswere102.5membraneanchor(nucleotides 53to115)andreverseprim-to104.550%egg-infectivedoses.

erscomplementarytotheregioncarryingthestalktothe5 Thecross-reactivityoftheprimersetswastestedwith45AIendoftheheadregion(nucleotides 209to365)(Table1).viruses(5virusespersubtypefornineNAsubtypes).Homol-Thesesubtype-speci cprimerswerescreenedagainstprevi-ogousNAgeneswereampli edef ciently;faint,smeared,orouslyandrecentlyisolatedAIvirusesandfurthermodi edsize-differentPCRproductsthatweredistinguishablefromusingsequencedatafor41NAgenesthatwerenotdetectedspeci cproductsbytheirsizeorintensityonagarosegelswereinitiallybyPCR.

occasionallydetected.SequenceanalysesshowedthatsomeViralRNAwasextractedfromallantoic uidbyusingTrizolfaintbandswerecon rmedasrepresentingcross-reactivityorsolution(Invitrogen,Carlsbad,CA)andusedforcDNAsyn-nonspeci cproducts,whiletwoclearNAgenesinasinglethesiswithrandomprimers(6-mers)andPrimeScriptreversesamplewereindicativeofthepresenceoftwoAIviruses.Suchtranscriptase(Takara,Shiga,Japan)(13).ThePCRampli ca-faintbandsmightbeinevitableinourNAsubtypingPCR.TheNAsubtypingPCRcoulddetectadeletionintheNAstalk*Correspondingauthor.Mailingaddress:ResearchTeamforZoo-regionwhichwaspresentinsomeAIviruses,suchasA/noticDiseases,NationalInstituteofAnimalHealth,3-1-5Kannondai,chicken/Korea/ES/2004(H5N1)andA/turkey/Italy/4580/99Tsukuba,Ibaraki305-0856,Japan.Phone:81-29-838-7802.Fax:81-29-(H7N1).Itissurmisedthatthedeletionmightbeassociated838-7802.withtheadap …… 此处隐藏：10753字，全部文档内容请下载后查看。喜欢就下载吧 ……