PCR和生物素探针对HFRSV的检测和分型的探讨

Detecting and Typing of HFRSV with PCR and Biotinylated Probes

分析比较肾综合征出血热病毒（ＨＦＲＳＶ）７６／１１８株和Ｒ＿（２２）株的核苷酸序列，根据引物设计原则及检测分型的目的，设计并合成了３对引物。１对引物取于两毒株间的高同源区段，作为共同引物和外引物；另两对引物取于两毒株间的低同源区段，分别作为野鼠型引物、家鼠型引物和内引物。建立了ＤＮＡ聚合酶链反应（ＰＣＲ）和ＮｅｓｔＰＣＲ方法，并用ＮｃｓｔＰＣＲ合成了两种型特异的生物素探针。ＰＣＲ检测７６／１１８、Ａ＿９、陈、Ｒ＿２、Ｒ＿２２５个毒株，用外引物时均扩增出１条约３００ｂｐ的条带；用内引物的野鼠型引物时，除Ｒ＿（２２）株之外，其余４株均扩增出１条约７０ｂｐ的条带。斑点杂交试验证实了ＰＣＲ检测分型的准确性。ＮｅｓｔＰＣＲ和生物素探针斑点杂交试验可以测出１－１０ｂｇ的目的ｃＤＮＡ。

y analysis and comparison of nucleotides sequences of HFRSV 76/118 and R_(22) strains,tlireepairs of primers were designed and synthesised,one pair of primer lying in the high homologous regionbetWeen 76/18 strain and R_(22) strain was used as common and outer primers;the other two pairs ofprimers were in the low homologous region,as the type-specific and inner primers.Using above primers and RT-PCR techneque,we measured five strains of HFRSV,76/118,A9,Chen,R_2,and R_(22).When using the outer primers,all of the five strains produced one DNA lane of 300bp;using the field-rat type inner primers all strains but R_(22) strain preduced one DNA Iane of 70bpand using the hom-rat type inner primers,only R_(22) strain produced one DNA lane of 70bp.Part of M fragment cDNA of 76/118 and R_(22) strains was used respectively as template,two type-specific biotinylated probed were synthesised by nest PCR techneque,the probes were used to hybridization with the RT-PCR preducts of the five strains,the results showed:RT-PCR techneque may beused to the detecting and typing of HFRSV,and had great accuracy,the sensitivity of dot hybridization with biotinylated probe was 1-10pg of cDNA.