摘要
马流感是由马流感病毒(EIV)引起的一种马属动物呼吸道传染病。近年来,全球马流感的流行呈现流行范围广、发病频次高、发生年度连续性强的特点。我国由于受周边国家马流感疫情的影响,2007年马流感暴发、流行,直接威胁着2008年北京奥运马术的比赛。本研究从我国新疆地区马流感暴发、流行的的疑似马流感病例分离到一株马流感病毒,经鉴定为H3N8亚型,命名为A/equine/Xinjiang/3/0(7H3N8),并用该毒株开展了HA基因序列分析以及马流感双重RT-PCR和TaqMan荧光定量PCR的研究,这些HA基因序列分析和诊断技术的建立对于马流感防治与监测起到了重要作用。
从Genbank上下载了世界各地多个典型H3N8亚型马流感毒株HA基因,在编码区以外相对保守区设计了一对特异性引物,通过RT-PCR方法扩增出本分离株的HA基因,将HA基因克隆测序。序列分析发现该毒株的HA基因扩增片段全长1725 bp。本毒株HA基因序列与Kentucky/02(H3N8)株的同源性最高,HA基因遗传进化树分析结果表明本毒株属美洲株佛罗里达支系。
传统的马流感检测方法和亚型鉴定方法操作比较复杂,不能对病毒进行快速诊断,不利于马流感的及时有效防控。为此,建立了两种分子生物学检测方法用于马流感的快速诊断:
(一)根据马流感病毒的M基因和HA基因的保守序列,设计了两对引物,MF和MR为通用引物,对H3N8和H7N7亚型EIV均能够扩增出大小为227bp目的片段,HF和HR为H3N8亚型特异性引物,目的片段596bp。利用这两对引物,通过对双重RT-PCR扩增条件的优化,建立了快速检测鉴别H3N8亚型EIV的双重RT-PCR技术。该方法具有很好的特异性,对EIAV、EAV、EHV等主要马属动物病毒扩增均为阴性。对该方法的敏感性分析结果表明,其最低核酸检出浓度为5.4ng/mL。由于该方法可以在几个小时内一次性完成鉴定工作,非常有利于EIV的快速诊断和亚型鉴定,为马流感的有效控制争取宝贵时间。
(二)针对H3N8亚型马流感病毒HA基因高度保守序列设计并合成了两对引物和一条TaqMan荧光探针,建立了TaqMan荧光定量PCR方法。经用TaqMan荧光定量PCR、RT-PCR和病毒分离方法分别检测新疆等省135份疑似马流感病马鼻拭子样品,其结果表明:三种方法的马流感检出率分别为54.07%、37.78%、0.89%;TaqMan荧光定量PCR可检出马流感病毒基因组RNA的灵敏度可达10拷贝/反应,且与其他马呼吸道病毒均无交叉反应,具有明显的特异性、敏感性和良好的重复性。该方法为H3N8亚型马流感的早期快速诊断及分子流行病学调查等提供了一种新的快速、准确的定量检测技术。
Equine influenza (EI) is an acute contagious respiratory infectious disease which is common for horses. In recent years,Equine Influenza showed those characters,wide prevalence,high frequency of outbreak, short interval epidemic time. In 2007,Equine influenza outbreak and spread quickly in China because influenced by these neighbour countries.The EI threat to the equestrian competition directly in 2008 Beijing Olympic Games. In this study,we successfully isolated an strain of equine influenza from suspected EI horse in Xinjiang , identified H3N8 subtype and named A/equine/Xinjiang/3/07(H3N8).Based on this isolated strain, HA gene was cloned ,sequenced and sequencing analyzed,and two molecular biology diagnosis method were established to detection EIV which were duplex RT-PCR and real-time PCR.Those study play an very important role to prevention and control.
HA gene sequences of some typical strains of equine influenza virus subtype H3N8 all over the world was downloaded from Genbank,then design a pair of specific primers in a relatively conservative region out of the coding region. Amplified the HA gene of the isolates by RT-PCR,cloned and sequenced the HA gene.Sequence analysis revealed that the amplified fragment length of HA gene was 1725 bp. By comparing with some strains of the same subtype published in GeneBank,there is the highest percent homology of nucleotide with American strain Kentucky/02(H3N8).The result of phylogenetic analysis of HA gene showed that this isolated strain belonged to Florida subline of American line.
The conventional tests for EIV diagnosis were timely and uneffective.In order to well preventation and control of EIV,the establishment of the two detection methods in molecular biology for the rapid diagnosis of EIV:
(A) Two pairs of primers were designed based on the highly conserved region of M gene and HA gene of equine virus. The two pairs of primers were able to detecte EIV and identify the H3N8 EIV.By optimized the reaction parameters,the duplex RT-PCR was developed to detect EIV and identify the subtype. The sensitivity and specificity tests were used to judge the validity of the RT-PCR.
(B) A real-time fluorescent quantitative PCR assay based on TaqMan probe was developed to rapidly detect equine influenza virus subtype H3N8. The assay was based on primers and TaqMan probes selected from highly conserved regions of the hemagglutinin gene of equine influenza virus subtype H3N8.A total number of 135 nasopharyngeal swabs suspected equine influenza which were collected from Xinjiang and other provinces were tested by real-time PCR, RT-PCR and viruses isolation. The results showed that the positive detection rates of those thee methods were 0.89%,37.78%,54.07%,respectively. The detection limit of the assay was 10 copies per reaction. This assay had obvious sensitivity,specificity and repeatability and had no cross-reaction with other equine respiratory disease.This real-time PCR assay is an excellent method suitable for rapid and quantitative detection of equine influenza virus subetype H3N8 under clinical conditions and molecular epidemiological investigation.
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