大鼠细小病毒 H-1株和 KRV 株双重 PCR检测方法的建立及应用 .pdfVIP

大鼠细小病毒H-1株和KRV株双重PCR检测方法的建立及

应用

李晓波;付瑞;王吉;卫礼;王淑菁;岳秉飞;贺争鸣

【摘要】ObjectiveTodevelopaduplexPCRassayfordetectionofrat

parvovirusH-1andKRVanditsapplication.MethodsTodesignspecific

primersonthebasisofH-1(NC_001358)andKRV(U790330)genome

sequencespublishedinNCBIandestablishaduplexPCRassayusingH-1

andKRVDNAastemplates.Toverifythesensitivityandspecificityofthe

methodafteroptimizingPCR.Theratswereinfectedbyoral

inoculation.Theratsweredividedintothreegroups:H-1infection,KRV

infectionandmixedinfectiongroups.Tocollectfecesatthe4th,6th,

8thnbsp;and10thdayspostinfection.Ratswereeuthanizedonthe10th

dayandsamplesfromheart,liver,spleen,lung,kidneyandcecalcontents

werecollectedfromeachrat,thenallthesampleswerescreenedwiththe

duplexPCR.ResultsThe183bpand302bpbandswereamplifiedusingH-

1andKRVastemplates.ThesensitivitytestshowedthatthePCRmethod

candetectaslowas3.8pg/mLH-1and0.73pg/mLKRV.Therewereno

bandsamplifiedwhenmouseminusvirus,canineparvovirusandfeline

parvoviruswereusedastemplates,showingthatthespecificityofthe

duplexPCRassayisverygood.ThenucleicacidsofH-1orKRVwere

detectedinallratfecesonthe2thdaypostinfectionandtherewasno

obviousclinicalsymptomsinalltheinfectedrats.ThepositiveratesofH-1

wereasfollows:50%(4/8)hearttissues,50%(4/8)livertissues,62.5%(5/8)

spleentissues,50%(4/8)lungtissues,37.5%(3/8)kidneytissuesand

62.5%(5/8)cecumcontents,andthepositiverateofsingleinfectiongroup

washigherthanthatofmixedinfectiongroup.ThepositiveratesofKRV

wereasfollows:0(0/8)hearttissues,25%(2/8)livertissues,87.5%(7/8)

spleentissues,12.5%(1/8)lungtissues,25%(2/8)kidneytissuesand

62.5%(5/8)cecumcontents,andthepositiverateofmixedinfectiongroup

washigherthanthatofs