10种猪常见病毒基因芯片检测方法的建立及初步应用

Research on DNA Microarray for Detecting Ten Different Virus from Swine

旨在建立一种可同步检测猪10种常见疫病病毒,猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(classical swine fever virus,CSFV)、猪伪狂犬病病毒(pseudorabies virus,PRV)、猪圆环病毒2型(porcine circovirus 2,PCV2)、猪细小病毒(porcine parvovirus,PPV)、猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)、猪传染性胃肠炎病毒(transmissible gastroenteritis virus of swine,TGEV)、猪流感病毒(swine influenza virus,SIV)、猪巨细胞病毒(porcine cytomegalovirus,PCMV)和口蹄疫病毒(food-and-mouth disease virus,FMDV)的基因芯片检测技术,为临床病料的初步诊断及流行病学调查提供一个新的可靠方法。对10种病毒基因的序列进行分析,设计引物对靶基因进行PCR扩增,将纯化后的靶基因点制成芯片,通过Cy3标记特异性引物扩增病毒得到具有荧光标记的探针,然后与芯片进行杂交,扫描分析结果,建立其基因芯片检测方法,并对该方法的特异性、敏感性和稳定性进行检测。应用建立的基因芯片检测方法和普通PCR/RT-PCR方法对1 006份样品进行符合检测。试验结果表明,基因芯片检测10种病毒特异性好、灵敏度高,最低检测限度可达10^(5 )copies·μL^(-1),检测中,基因芯片与普通PCR/RT-PCR对1 006份临床样品的检测符合率为96.79%以上。初步研究表明,该基因芯片技术可同时同步检测10种猪疫病病毒,可应用于临床病料的初步诊断。

To develop a new reliable method for initial diagnosis and epidemiological investigation of ten common diseases of pig,an DNA microarray was established for simultaneous detection of porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),pseudorabies virus(PRV),porcine circovirus 2(PCV2),porcine parvovirus(PPV),porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis of swine(TGEV),swine influenza virus(SIV),porcine cytomegalovirus(PCMV)and food-and-mouth disease virus(FMDV).According to the similarity of the ten viral sequences in the GenBank database,ten pairs of specific primers were designed and ten kinds of virus-specific conservative regions were amplified by PCR,respectively.Then the purified target genes were fixed on the amino group modified slides.The probes labeled with Cy3 were amplified from samples by PCR and hybridized with the cDNA array on the slides.The hybridized results were scanned by scanner.The gene chip detection method was established and its specificity,sensitivity and repeatability were detected.The coincidence test between cDNA chip and PCR/RT-PCR was performed in 1 006 samples.The results showed that this cDNA array was very specific and sensitive,the minimum sensitivity was 10 5 copies·μL-1.The coincidence rate between cDNA chip and PCR/RT-PCR was 96.79%.In all,the gene chip detection technology for ten kinds of common swine pathogenic viruses was successfully developed and can be applied to swine pathogens detection in clinic.