CAJ | 학술논문

采集一疑似犬细小病毒( CPV)患犬的粪便,采用胶体金试纸条和PCR方法对病料进行CPV检测,并应用猫肾细胞F81进行病毒分离培养,分别采用免疫过氧化物酶单层细胞染色法( IPMA)和PCR检测病毒在F81细胞中的增殖动态,采用无血清培养和有血清培养2种方法进行体外培养,IPMA测定病毒滴度。利用PCR扩增分离病毒的VP2基因,经序列测定后采用DNAStar 7.0和MEGA 5.0软件进行生物信息学分析。结果表明,胶体金试纸条检测为CPV抗原阴性, PCR检测结果为CPV核酸阳性,病料接种到F81细胞培养后出现明显细胞病变( CPE ),血清学鉴定为CPV抗原阳性,将分离得到的病毒命名为CPV-NY130615。 IPMA可从感染后6 h的 F81细胞中检出病毒,PCR能从感染后6 h培养上清中检出CPV核酸。血清同步接种培养法培养和无血清单层接种培养法培养的第15代病毒滴度分别为1×108.06 TCID50/mL和1×107.65 TCID50/mL。序列测定分析表明,该毒株VP2基因开放阅读框( ORF)为1755 bp,编码584 aa。进化分析显示,该毒株属于CPV-2 a型。
채집일의사견세소병독( CPV)환견적분편,채용효체금시지조화PCR방법대병료진행CPV검측,병응용묘신세포F81진행병독분리배양,분별채용면역과양화물매단층세포염색법( IPMA)화PCR검측병독재F81세포중적증식동태,채용무혈청배양화유혈청배양2충방법진행체외배양,IPMA측정병독적도。이용PCR확증분리병독적VP2기인,경서렬측정후채용DNAStar 7.0화MEGA 5.0연건진행생물신식학분석。결과표명,효체금시지조검측위CPV항원음성, PCR검측결과위CPV핵산양성,병료접충도F81세포배양후출현명현세포병변( CPE ),혈청학감정위CPV항원양성,장분리득도적병독명명위CPV-NY130615。 IPMA가종감염후6 h적 F81세포중검출병독,PCR능종감염후6 h배양상청중검출CPV핵산。혈청동보접충배양법배양화무혈청단층접충배양법배양적제15대병독적도분별위1×108.06 TCID50/mL화1×107.65 TCID50/mL。서렬측정분석표명,해독주VP2기인개방열독광( ORF)위1755 bp,편마584 aa。진화분석현시,해독주속우CPV-2 a형。
The faeces samples were collected from a dog suspected infected by canine parvovirus( CPV) , CPV in the samples was detected by colloidal gold test strip and polymerase chain reaction(PCR),then the samples were innoculated into F81 cells to isolate CPV,propagation dynamics characterization of CPV in F81 cells were determined by immunoperoxidase monolayer assay( IPMA) and PCR,samples that cul-tured with or without serum were titrated by IPMA. VP2 gene of this virus strain was amplified by PCR and sequenced then analyzed by software DNAStar 7 . 0 and MEGA 5 . 0 . Results of colloidal gold test strip test showed CPV Ag negative in the samples,however,PCR detection results showed positive. The sample inoculated into F81 cells showed significant cytopathic effect( CPE) . Serologic test results showed that the culture samples were CPV Ag positive,the isolated virus strain was named CPV-NY130615. CPV Ag in the F81 cells 6 hours post infected( hpi) with CPV-NY130615 strain was successfully detected by IPMA, as well as the culture supernant was CPV nucleotied positive detected by PCR. The fifteenth passages vi-rus titers of two different culture method,synchronization inoculated culture with serum and monolayer in-oculated culture without serum were 1 × 108. 06 TCID50/mL and 1 × 107. 65 TCID50/mL respectively. Nucle-otid sequences showed that the open reading frame of VP2 gene was 1 755 bp encoding 584 aa. Phyloge-netic analysis showed that CPV-NY130615 strain belonged to the genotype CPV-2a.