1、.Ef5hhPCR!9Internet_s K1:研究了传染性法氏囊病病毒( IBDV) A 片断的cDNA 的结构,并使用PrimerDe-sign引物设计软件,在VP5 和VP2的重叠基因区设计了一对引物,各种IBDV 毒株在此部位有非常高的同源性,在对引物进行Internet 检索中得到了证实。经过对4种IBDV 的疫苗标准毒株、1种野毒株、1例病鸡标本试检测,验证引物设计符合要求,具有良好的适应性和较高的灵敏度。1oM:传染性法氏囊病病毒(IBDV) ;逆转录-聚合酶链式反应(RT -PCR) ;引物;Internet检索;诊断试剂ms|:S852.659.4;TP393.4DSM:A
2、.Ef5h(IBD)B$、(.%h,!1o。-h*ZEIc-TQ(RT-PCR)KrZEB。7NhMs,t!9?shr97?9。%B5,3IBDVy$!9B,Internet_sL%aZ|BtZ,C|T/。1IBDVys#!91.1IBDVysIBDVyFRNAF。l(B)IVP1,$RNAGRNA 2 ;v(A)c7b(ORF),BlORFIVP5 3 ,=vORFBI,VYV1V$FVP2 、VP4VP3h 4 。Mundt 3IBDVcDNA,lORFV97 534 bp,vORF5V131 3 169 bp。ORF131 534 bpWiB404 bpy。y1HVP5VP2I,Pyo。
3、D 5 84IB-DVAcDNAs,sYd9VP2 、VP3 、VP4 、VP5VP5VP2yuMsf(V1) 。V1Vn,Ay,7buMsK。Vu!9PCRKza。1.2PCR!9Sv3/j3/sIBDVGLSG l:1999-09-18:8BTe:.( 1959-),3,Fng,jSq,V3V1AMsd9Table 1Statisticsof nucleotide variation in segment Ay/bp9MsMs1/%VP5y449 8 1.78VP5、VP2y404 6 1.49VP2y1 355 106 7.82VP4y809 72 8.90VP3y869 39 4.49
4、 8 ,PPrimerDesign!9q 9!9。L1,3sYAVP5 、VP2 、VP4 、VP3yo!9。1.34#1G!94V,1IWu 10yp,H9ID 11, 124。(1)20 30 bpW;(2)A/TcG/CcV?L,H|M;(3)1&、W(ia,1&a?v3 bp ;W4a,3Wa,V7EC=8;(4)Kv,P?Wa;(5)as,EaQC,3V3GC,44Ma。8ZyInTK4VP5VP2yuB,51Hq。uMsa,9uMsa。A140 160 bp),/341 361 bp)。/:(1) 5ACAGATTGTTCCGTTCATACG3;/(2) 5TCGAACTTGTAG
5、TTCCCATTG 3。H|(56.1 ,GC:s(42.9 %,9vl222 bp。nm1。m1( IBDVPIVS)Figure 1Position of primers1.4SPromega,L-TE( 10 mmol/LTris -HCl, pH 8.5, 1 mmol/LEDTA )40 mol/LATiA,P-)dPP。2IBDVas2.1Internet_s|!912#=9uInternetBLAST 13GenBank+EMBL+DDBJ+PDB460 413_,9u_T24,(IB-DVO(,nV2 。2.25_s|4fSh、17、2hSL=_s,(!9M222 bp9。fh
6、f、.5hfrBvL(|T。_、+s+,r!91p。3IBDVBMsh,Msy9Va73L|T。L!9_W+。N+Internet_sL。HBtS、7、hEf5FLCr9。PIBDVPCR_VL7r,!9r。1yy!9VP5VP2yu=,u=yvo,P?MsiP9。NKv。50j2000MV2u9uInternet_sTTable 2Retrieval results of primer and amplifing regions in Internet %_#_|Primer 1Primer 29u=cDNA( 222 bp)dbj D00867 IBDSEGAC Cu-l 100 100
7、100dbj D00869 IBDSEGA 52/70 100 100 100dbj D10065 IBDLGVP2 - 100 100 99dbj D49706 D49706 OKYM 100 100 100dbj D83985 D83985 OKYMT 100 100 99emb X16107 IBDVA Cu-l 100 100 100emb X54858 AIBDVVP2 - 100 100 100emb X84034 IBDVORF OKYM 100 100 100emb X92760 IBDVVP523 UK661 100 100 99emb X95883 IBDVVP2A - 1
8、00 100 100gb AF051837 AF051837 GZ29112 100 100 99gb AF051838 AF051838 HK46 100 95 98gb AF051839 AF051839 HKL6 100 100 99gb AF092171 AF092171 - 100 95 99gb AF092943.1 AF092943 - 100 100 99gb AF109154.1 AF109154 - 100 100 99gb AF133904.1 AF133904 variant E 100 100 100gb AF140705.1 AF140705 - 100 95 99
9、gb AF165149.1 AF165149 k310 100 100 100gb AF165150.1 AF165150 KK1 100 100 100gb AF165151.1 AF165151 KSH 100 100 100gb L42284 IBDVKS KS 100 100 99gb M64285 IBDVP2 A 100 100 100gb M97346 IBDVPIV GLS 100 100 100ID: 1 Kibenge F S B,Russell R G.Biochemistry and immunology of infectious bursal disease vir
10、us J .J Gen Virol, 1988, 69:1757 1775. 2 Azad A A,Barrett S A, Fahey K J.Thecharacterization and molecularcloning of the double-stranded RNA genomeof anAustralian strain of infectious bursal disease virus J .Virology,1985, 143:35 44. 3 Mundt E,Beyer J,Mller H.Identification of a novel viral protein
11、in infectious bursal diseasevirus-infected cells J .GenVirol, 1995,76:437 443. 4 Azad A A,Jagadish M N,Brown M A.Deletion mapping and expression in Escherichia coli of the large genomicsegmentof abirnavirus J .Virology,1987,161:145 152. 5 Mundt E,Mueller H.Complete nucleotidesequences of 5- and 3-no
12、ncoding regions of both genomesegmentsof differ-ent strains infectious bursal disease virus J .Virology, 1995, 209:10 18. 6 Brown M D,SkinnerM A.Coding sequencesof both genomesegments of a European very virulent infectious bursaldiseasevirus DB .Submitted to the EMBL/GenBank/DDBJ databases in 03-NOV
13、-1995. 7 Spies U.Nuclotide sequence of infectiousbursal diseasevirus genome segment A delineates two major open reading frames J .NucleicAcids Res, 1989, 17:7982. 8 Vakharia V N,He J,Ahamed B, et al .Molecular basis of antigenic variation in infectious bursal disease virus J .VirusRes, 1994, 31:265
14、273. 9 http:/www .chemie.uni-marburg.de/ CP .511.:.Ef5hhPCR!9Internet_s 10 Wu D Y,Ugozzoli L,Pal B K.Laboratory methods:The effect of temperature and oligonucleotide primer length on thespecificity and efficiency of amplification by the polymerasechain reaction J .DNA and Cell Biology, 1991,10:23323
15、8. 11 林万明,杨瑞馥,黄尚志,等.PCR技术操作和应用指南 M .北京:人民军医出版社,1992.15 23. 12 Lowe T, Sharefkin J, YangS Q.A computerprogram forselection of oligonucleotideprimers forpolymerasechain reac-tions J .Gene, 1990,93:125 128. 13 http:/www.ncbi.nlm.nih.gov/blast/ CP .Design of Infectious Bursal Disease Virus PCR Primer an
16、d Internet RetrievalRONG Jun, YU Long-jiang( School of Life Science at Huazhong University of Science and Technology, Wuhan, Hubei 430074, China)Abstract:Based on the study of segment A of theinfectious bursal disease virus( IBDV) cDNA, apairof primers was designed with the PrimerDesign software.Thi
17、s pair of primers is in theoverlapping areaofVP5 and VP2 where the sequence is highly homologous, which was verified when searching them in the In-ternet .Four standard virus strains, one wild strain and one IBDV strain from the cloacal bursa of diseasedchicken had been amplified successfully with t
18、he primers.Two other viruses, which could result in otherdisease of chicken and one healthy chicken s cloacal bursa had given negative results.This proved that theprimers accord with the demand of theamplification of IBDV,and havegood adjustability and high sensitiv-ity .Key words:infectious bursal disease virus( IBDV) ;reverse transcriptase-polymerase chain reaction( RT-PCR) ;primer;internet retrieval;diagnosis reagent52j2000M
