摘要
麻疹病毒属在病毒学分类上位于单负链RNA病毒目，副粘病毒科，副粘病毒亚科。根据国际病毒学委员会第8次分类会议的病毒分类目录，犬瘟热病毒、麻疹病毒、小反刍兽疫病毒、牛瘟病毒、海豹瘟热病毒和海豚瘟热病毒等六种病毒属于麻疹病毒属，这六种病毒分别在各自的宿主中引起重要的疾病，造成严重的公共卫生危害、社会影响和经济损失。上述病毒宿主范围广泛，致病性强，病死率高，已经引起了研究人员的重视，RT-PCR，实时定量PCR以及免疫学和血清学检测等多种方法被应用于上述病毒检测，但是这些方法多数是检测一种病毒或区分两种病毒，目前没有一种方法能同时检测麻疹病毒属上述六种重要病毒。本文研究目的是建立麻疹病毒属6种重要病毒的快速、准确、灵敏、高通量的基因芯片检测技术平台，同时完成麻疹病毒、犬瘟热病毒、小反刍兽疫病毒、牛瘟病毒、海豹瘟热病毒、海豚瘟热病毒等麻疹病毒属病毒的种特异性检测，为疫病临床诊断、人畜传染病防控及反恐怖活动等提供有效的诊断手段。
在NCBI网站下载上述病毒基因组序列信息，利用生物学软件对6种病毒下载的序列进行比对，获得每种病毒长度在200bp以上的种保守性特异核苷酸序列，以此作为设计oligo探针的备选序列，利用分子生物学软件PrimerPremier5.0设计特异性好、长度均一、Tm值相近的寡核苷酸探针。待检样品经荧光引物扩增后与基因芯片在优化的条件下杂交，激光扫描荧光信号分布和强度，Genepix软件分析结果；麻疹病毒属种特异性检测基因芯片制备后，用参比样品检验本基因芯片的敏感性、特异性、重复性和稳定性。麻疹病毒属种特异性基因芯片检测方法建立后，对来自吉林、辽宁和黑龙江的38例疑似犬瘟热病毒感染的临床病料进行了检测。在建立麻疹病毒属核酸检测基因芯片基础上，利用生物素-链霉亲和素的结合，引入胶体金显色及银染放大系统，建立麻疹病毒属的目视化基因芯片检测系统，使用目视化基因芯片对东北三省的48例临床病料进行了检测，共检测到38例犬瘟热病毒阳性样品，基因芯片方法和PCR方法的检测符合率为94.7%。
本实验主要完成了以下四部分工作
一、麻疹病毒属病毒种特异性寡核苷酸探针的设计
从NCBI网站的Genome数据库下载麻疹病毒、犬瘟热病毒、小反刍兽疫病毒、牛瘟病毒、海豹瘟热病毒、海豚瘟热病毒的基因组序列，从该网站的Oligonucleotide数据库下载上述病毒的核酸序列信息。每种病毒的下载序列分别比对后得到种特异性病毒保守区，在各自保守区内以Primer Premier5.0软件设计种特异性寡核苷酸探针及对应的PCR引物。遵循探针的设计原则，探针长度为25士5mer，TM值为48士5℃，使所有探针具有相近的杂交动力学参数。利用生物信息学技术在线比对，通过与公开发表的所有已知病毒序列进行比较，进行了病毒保守区和寡核苷酸探针的特异性验证，包括病毒属内序列同源性、种间序列的特异性、种内毒株的保守性。经过筛选与验证，从一千余条备选探针中确定了6条麻疹病毒属种特异性鉴定短寡核苷酸探针。
二、麻疹病毒属病毒多重PCR扩增方法的建立
上述实验确定了各病毒探针，在探针的两端分别设计麻疹病毒属各病毒特异性PCR扩增上下游引物，在上游引物的5′端标记HEX荧光染料，该染料可以被基因芯片扫描仪检测呈现绿色荧光。经优化各病毒引物比例，建立了麻疹病毒属多重PCR扩增方法。以细胞毒、合成模拟毒、质粒和病料为模板，验证了该多重PCR方法的特异性。实验结果显示，所建立的多重反应体系对麻疹病毒、犬瘟热病毒、小反刍兽疫病毒、牛瘟病毒、海豹瘟热病毒、海豚瘟热病毒等均能扩增出与预期设计大小一致的特异条带，而对犬副流感病毒、VERO细胞和健康犬组织等无特异扩增，该结果与特异PCR方法检测结果一致。
三、麻疹病毒属病毒种特异性基因芯片检测方法的建立及初步应用
探针用MG2-610型点样仪通过机械手臂分别以夹缝针接触式点样方式印迹于醛基玻片上，探针浓度为40pmol/μl，点样温度为25℃，点样湿度为70%，点样后经NaBH4封闭。经优化杂交条件，初步建立了麻疹病毒属种水平鉴定基因芯片。基因芯片检测犬瘟热病毒、麻疹病毒、海豹瘟热病毒、海豚瘟热病毒、小反刍兽疫病毒和牛瘟病毒的细胞毒和质粒结果均为阳性，而检测犬副流感病毒、VERO细胞和健康犬组织的结果为阴性，证明本基因芯片具有良好的麻疹病毒属种检测特异性。将TCID50为10-4的以犬瘟热病毒细胞毒10倍比稀释，分别用基因芯片和PCR方法梯度检测，基因芯片能检测到10-2TCID50的犬瘟热病毒，检测灵敏度是PCR方法100倍。分别使用同一批次和不同批次的多张芯片检测犬瘟热病毒，结果均为阳性，证明本芯片具有良好的重复性。制备的基因芯片放置1年以上仍然具有良好的检测活性。
犬瘟热病毒能够感染犬科、鼬科、浣熊科、大熊猫科、猫科等多种动物，且传染性强、致死率高，对我国家养犬类、宠物、经济动物、野生动物、动物园以及实验动物等危害极大。在基因芯片制备完成后，使用基因芯片对吉林、辽宁和黑龙江等省送检的犬、狐狸和貉子病料进行检测，38例标本中，普通PCR方法和基因芯片方法检测犬瘟热病毒均是阳性结果的为24例，而单独基因芯片方法检测为犬瘟热病毒阳性结果的有2例，两种方法的检测符合率为92.7%。
四、麻疹病毒属目视化基因芯片检测方法的建立及其初步应用
根据上述实验得到的各病毒种特异性探针，在5’端连接短臂后按照规律的阵列点样在玻璃片上，水合之后形成可以长期保存的芯片。在PCR引物的5’端引入生物素，于胶体金上引入链霉亲和素，利用生物素-链霉亲和素反应使胶体金在点样探针上特异性的结合与聚集，最后利用银染放大信号，形成肉眼可见的结果。该检测平台特异性好，通量高，灵敏度与基因PCR方法相仿，能应用于临床样品，与麻疹病毒属核酸检测基因芯片相互补充，更加适用于基础实验室及临床检测。
本实验建立了四步法探针设计方法，建立了麻疹病毒属6种重要病毒探针数据库，制备了能同时检测12份样品、每份样品能同时检测6种病毒的基因芯片。在探针两端设计PCR扩增引物，建立了麻疹病毒属6种重要病毒多重PCR扩增方法，在此基础上，建立了麻疹病毒属种特异性鉴定扫描芯片和目视化芯片技术，并初步应用于犬瘟热病毒临床病料检测，该技术具有良好的灵敏度和特异性，在出现传染病疫情时，结合临床症状及流行病学特征等，可在种水平上快速鉴定上述六种病毒。
Morbillivirus belongs to Mononegavirales,Paramyxoviridae,Paramyxovirinae.According to the8thof international classification of Virology Committee meeting,there are six major viruses in morbillivirus. They are canine distemper virus,measles virus, Peste des petits ruminants virus, rinderpest virus, phocine distempervirus and dolphin morbillivirus virus. All of them may cause serious viral disease intheir respective host. Morbillivirus may cause public health danger, social impactand economic loss seriously. They had broad-spectrum Virus host, highpathogenicity and high mortality which have attracted the attention of researchers.There are some method such as ordinary RT-PCR, real-time quantitative PCR，serological and immunological means that have been applied to scan morbillivirus,but most of them are designed to test one or two viruses. There is not one processcan identify all the six virus in a route. The purpose of this study is to establish arapid, accurate and sensitive high-throughput microarray platform to detect measlesvirus, canine distemper virus, Peste des petits ruminants virus, rinderpest virus,phocine distemper virus and dolphin morbillivirus virus in one assay. Thismicroarray could be a rapid and effective diagnostic tool for clinical diagnosis,prevention and control of infectious diseases and anti-terrorism.
All the six virus genome sequence were download from NCBI website,meaningful species-specific sequence of each virus were found by using biologicalsoftware clustal w which is important for oligo probe design. Probe ofspecies-specific, uniform length, similar Tm values were designed using molecularbiology software Primer Premier5.0. Samples were hybridized to microarray afterbeing amplified by fluorescent primers. Pictures were scanned by laser and datawere analyzed by Genepix4100A. Sensitivity, specificity, reproducibility andstability of microarray were proved by both cultured virus and clinical specimens.38clinical specimens with suspected disease of canine distemper were detected bygene chip of Jilin, Liaoning and Heilongjiang province.
The chief results of the study were listed as following:
1. The designment of morbillivirus oligo probes on species level and theirvalidation by bioinformatics.
Species-specific sequence was selected after comparing the downloadedgenome of morbillivirus. Six25mer probes were designed by Primer Premier5.0forevery virus with Tm value about48±5℃. All probes have similar hybridizationparameters. The validation of conserved sequence and species-specific probedesignation were tested by bioinformatics. At last, six species-specific oligo probeswere designed and selected.
2. Multiplex PCR amplification for morbillivirus
Primers for the six major morbillivirus were designed according to the sameconserved sequence with probe. All six pare of primers were mixed to detectcultured virus, clinical specimens and synthesized virus in one assay. There werepositive signal for CDV, MV, DMV, PDV, PPRV and RpV while the signals ofcontroled Vero, CPIV and healthy dog were not detected.
3. The establishment of microarray for morbillivirus species-specific detection.
Microarray was prepared by MG2-610. Probes of40pmol/μl were spotted ontothe aldehyde slides at25℃and70%humidity. Before it can be used forhybridization, the chip must be disposed by NaBH4. Hybridization condition wasoptimized and microarray for species-spefic morbillivirus identification was set up.To test the species-specificity of the probes in the microarray, the target CDV,PPRV, CPIV, Vero, synthesized MV, RPV, PDV, DMV, and tissue of healthy dogwere prepared according to the method mentioned above and hybridized onto themicroarray. The probes only hybridized with their respective target viruses andnon-specific or cross-reactive signal was not observed CDV with TCID50as10-4wasdiluted by10folds and detected by microarray and PCR, and microarray can detectCDV as a minimum level as10-6TCID50which is103times higher than PCRmethord.
4. Application of microarray
CDV can infect dogs, ferret, Raccoon, giant panda, cat and other animals. Itwas a great harm for domestic dogs, pets, economic animals, wild life, zoo andlaboratory animals. Microarray were used to detect22simulated unknown clinical specimens, and the detection ration of chip is100%. Clinical specimens of dog, foxand raccoon which were sent from Jilin, Liaoning and Heilongjiang province weretested for CDV by the microarray. Among the38specimens there were24samplewhich were tested as CDV positive by both microarray and PCR methord.26weredetected CDV positive by microarray only，and it is the same with virus isolation.
5. Establishment of visual microarray
According to the above experimental, the short arm are connected to the5'endof virus species-specific probes,then probes are spotted on the glass, visualdetection chip could be long-term preservati after hydration. biotin were introducedto5'end of the PCR primers. the colloidal gold-streptavidin-avidin biotin reactionsystem are used to get specific binding and visual signal. Finally use silver stainingto amplify the signal. The detection platform are specific, high-throughput, highsensitivity.The visual microarray and the usual chip of virus are complement to eachother.The former are more suitable for the basic laboratory and clinical testing
In short, six25mer oligo probes were designed on species level formorbillivirus and the specificity of them was validated by both bioinformatics andexperiments. Then low-density micorarray was prepared using the oligo probes.PCR primers were designed within the same conservation that has been used to planprobes, and multiplex PCR assay was launched to amplify the six majormorbillivirus. Microarray for morbillivirus species-specific detection was initiated.Cell culture supernatants and clinical specimen could be detected by the microarrayto test morbillivirus rapidly. Morbillivirus could be detected on species levelreferring to clinical symptoms and epidemiological characteristics when infectiousdisease outbreaks.
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