荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究.pdfVIP

维普资讯 理杂志 ChineseJournalofP 20o7．23(8)：1623—1626 · 1623 · [ArticleID] 1000—4718I2007)08—1623—04 Identificationofrecombinantbaculovirusanddeterminationof virustiterwithfluorescencequantitativePCR assay TONG Xia—sheng ‘MENG Zhe—feng△ ． Nationa1． CenterforAIDS／STDControlandPreventoin，ChineseCenterforDiseaseControland Preventoin，Bejiing100002，China．E—mail：zfin863@yahoo．corn．ca) [ABSTRACT] AIM：Todevelopareal—timePCRassaybasedonTaqMantechnologyfortheidentificationofre— combinantbaculovirusanddeterminationofvirusphysical titersinBac—to—Bacsystem．METHODS：Therecombinant baculoviurscontaininghumna IL一18genewas producedusingBac—to—Bacsystem．A 10一foldseriallydilutedprimary viralstockwasusedforplaqueassaynadDNAextraction．Bacmid(baculovirusplasmid)was 10一foldseriallydilutednad esrvedas standards．Real—timePCR amplificationofhteIL一18genewas perfomr de intriplicateforeachdilutde reeomb i- nantviurs．Athtesametime，plaqueassayswere perfomr de usingovedayagarosemehtod．RESULTS：Th estandardline— arrnagef10’to10copies)ofrqunatitationwas achievedwit}Ihtestandardcurve．Wealsofindhtatthe“vs／mL”titerval- ueisgenerallyabout10timeshtna “pfu／mL”titerof htesamereocmbinantviursstock．CONCLUSION ：ATaqMna real— timePCRmehtodisestablishedtoidentifythereocmbinnatbaculoviursnaddetemrinethe“vS／mL”titerofviurs．Th emeth— odisrapidnaduq natitativeoverawidernageofviurstiters． [KEYwonDs] FluorescenceuqnatiattivePCR；Identificationofrecomb innatbaculoviurs：Viurstiter [CLCum ber] R363 D『ocumentcode] A Recombinantbaculoviruseshavebeen extensivel