中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2008年
11期
1030-1034
,共5页
刘杨%田宏%陈锦英%苏旭%杨东靖%万丽霞%雷玥%田永琴
劉楊%田宏%陳錦英%囌旭%楊東靖%萬麗霞%雷玥%田永琴
류양%전굉%진금영%소욱%양동정%만려하%뢰모%전영금
麻疹病毒%反转录-聚合酶链反应%限制性片段长度多态性分析%基因型%序列分析
痳疹病毒%反轉錄-聚閤酶鏈反應%限製性片段長度多態性分析%基因型%序列分析
마진병독%반전록-취합매련반응%한제성편단장도다태성분석%기인형%서렬분석
Measles virus%Reverse transeription-polymerase chain reaction(RT-PCR)%Restric-tion fragment length polymorphism (RFLP)%Genotype%Sequence analysis
目的 建立RT-PCR-RFLP方法 用于天津地区2002-2008年流行的麻疹野病毒基因型研究.方法 采集疑似麻疹患者的尿标本和咽拭子,传代细胞分离病毒.提取病毒液中的RNA,用一步RT-PCR法扩增麻疹病毒核蛋白(nucleoprotein,N)基因C端594个核苷酸片段,扩增产物经Bcn I酶切后琼脂糖凝胶电泳进行限制性片段多态性分析(RFLP),同时与序列分析进行对比验证.根据结果 构建基因系统发生树进行亲缘关系和遗传距离分析.结果 2002-2008年189份标本,分离获得69株麻疹病毒,N基因C端RT-PCR检测全部阳性,RFLP分析H1a是优势流行亚型,占98.55%(68/69),仅1株(1.45%)为H1b基因亚型,分型结果 与序列测定完全一致.系统发生树分析显示H1a亚型毒株分为2个亚枝,变异范围为0.2%～3.8%,存在不同病毒株引起的传播链共循环.结论 基于Bcn I限制性内切酶的RT-PCR-RFLP方法 能够特异性区分A基因型、H1a、H1b基因亚型,具有快速、简便、准确和经济的优点,更适用于国内大规模麻疹病毒的监测.
目的 建立RT-PCR-RFLP方法 用于天津地區2002-2008年流行的痳疹野病毒基因型研究.方法 採集疑似痳疹患者的尿標本和嚥拭子,傳代細胞分離病毒.提取病毒液中的RNA,用一步RT-PCR法擴增痳疹病毒覈蛋白(nucleoprotein,N)基因C耑594箇覈苷痠片段,擴增產物經Bcn I酶切後瓊脂糖凝膠電泳進行限製性片段多態性分析(RFLP),同時與序列分析進行對比驗證.根據結果 構建基因繫統髮生樹進行親緣關繫和遺傳距離分析.結果 2002-2008年189份標本,分離穫得69株痳疹病毒,N基因C耑RT-PCR檢測全部暘性,RFLP分析H1a是優勢流行亞型,佔98.55%(68/69),僅1株(1.45%)為H1b基因亞型,分型結果 與序列測定完全一緻.繫統髮生樹分析顯示H1a亞型毒株分為2箇亞枝,變異範圍為0.2%～3.8%,存在不同病毒株引起的傳播鏈共循環.結論 基于Bcn I限製性內切酶的RT-PCR-RFLP方法 能夠特異性區分A基因型、H1a、H1b基因亞型,具有快速、簡便、準確和經濟的優點,更適用于國內大規模痳疹病毒的鑑測.
목적 건립RT-PCR-RFLP방법 용우천진지구2002-2008년류행적마진야병독기인형연구.방법 채집의사마진환자적뇨표본화인식자,전대세포분리병독.제취병독액중적RNA,용일보RT-PCR법확증마진병독핵단백(nucleoprotein,N)기인C단594개핵감산편단,확증산물경Bcn I매절후경지당응효전영진행한제성편단다태성분석(RFLP),동시여서렬분석진행대비험증.근거결과 구건기인계통발생수진행친연관계화유전거리분석.결과 2002-2008년189빈표본,분리획득69주마진병독,N기인C단RT-PCR검측전부양성,RFLP분석H1a시우세류행아형,점98.55%(68/69),부1주(1.45%)위H1b기인아형,분형결과 여서렬측정완전일치.계통발생수분석현시H1a아형독주분위2개아지,변이범위위0.2%～3.8%,존재불동병독주인기적전파련공순배.결론 기우Bcn I한제성내절매적RT-PCR-RFLP방법 능구특이성구분A기인형、H1a、H1b기인아형,구유쾌속、간편、준학화경제적우점,경괄용우국내대규모마진병독적감측.
Objective To establish RT-PCR-RFLP method for studying the genotype of wild mea-sles virus strains isolated from Tianjin area from 2002 to 2008. Methods Isolations of measles virus were carried out by tissue culture method from urine and throat swab specimens collected from suspected cases. RNA were extracted from the virus specimens. The 594 bp fragment of C terminal of the N (nucleoprotein) gene was amplified by one-step RT-PCR, then the PCR products were digested with Bcn I , separated on agarose gel electrophoresis and then analyzed by the method of RFLP (restriction fragment length polymor-phism). In addition, above results were compared with DNA sequencing. Phylogenetic tree was plotted based on the results for the genetic relationship and distance analysis. Results Sixty-nine measles virus strains were isolated from 189 specimens from 2002 to 2008, of which the C terminals of N gene were all de-tected positive. Among the 69 strains of measles virus isolates, 98.55% (68/69) belonged to Hla sub-geno-type which was the predominant sub-genotype, and only one strain (1.45%) belonged to H1b sub-genotype by RFLP analysis which was in accordance with the results by DNA sequencing method. Phylogenetic tree analysis indicated the H1a sub-genotype measles virus strains should be further divided into 2 clades, and the variation fluctuated between 0.2% and 3.8%. There were transmission chains caused by different virus strains co-cireulation. Conclusion A genotype, H1a and H1b sub-genotype can be identified by RT-PCR-RFLP assay specically based on the restriction enzyme Bcn I .The RT-PCR-RFLP assay can be a rapid, simple, accurate and efficient method for large-scale surveillance of measles virus strains in China.
