| 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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