医学RNA病毒属水平基因芯片筛查技术的研究
摘要
在人类传染病中,70%以上都是由病毒引起的,即病毒性传染病。目前已知的病毒有5400多种,其中与人类疾病相关的约有400多种。而在人类致病病毒即医学病毒中,RNA病毒占总数的80%以上,大部分烈性病毒性传染病由RNA病毒引起,如丝状病毒属中的埃博拉病毒及马尔堡病毒、沙粒病毒属中的拉沙热病毒、甲病毒属中的委内瑞拉马脑炎病毒等。因此,建立高通量、快速筛查及鉴定未知RNA病毒的技术平台具有重要的意义。基因芯片技术是一种大规模集成的固相杂交技术,其基本原理是核酸分子杂交,即依据DNA双链碱基互补配对、变性和复性的原理,以大量已知序列的oligo、cDNA或基因片段作探针,检测样品中哪些核酸序列与其互补,然后经过一定的检测系统对杂交信号进行检测,并配以计算机系统对每一杂交信号数据进行定性、定量分析和处理,从而迅速得出待测品的基因序列及表达的信息。
本研究的目的是建立能够在属水平对医学RNA病毒进行快速筛查的基因芯片技术,在出现新(突)发传染病疫情或公共卫生事件时,能利用该芯片,快速将病毒性病原体筛查到属水平,为进一步进行病原体的分离培养、鉴定及传染病疫情的防控提供有意义的参考。本研究的设计思路是在属水平设计医学RNA病毒的寡核苷酸(oligo)探针,针对每个病毒属设计10条左右探针,使探针序列能覆盖该病毒属中所有已知的病毒株,因此可将该芯片用于对目前已知的RNA病毒进行属水平的快速筛查。另外,由于同一病毒属内不同病毒株之间虽然碱基序列不完全相同,但存在相当程度的同源性,因此,通过采用较长的oligo探针(63mer),允许在杂交时模板与探针之间存在一定的碱基错配,而任何一种新发或未知病毒都必然与某个病毒属中的已知病毒存在一定的同源性,因此,利用该芯片也可对目前未知的新发病毒性病原体进行快速分类。
本课题的主要研究结果如下:
1.医学RNA病毒属水平oligo探针的设计及生物信息学验证
依据国际病毒分类委员会2005年发布的第八次分类报告中的病毒分类数据库(http://phene.cpmc.columbia.edu/ICTV/index.htm)及NCBI的病毒分类数据库(http://www.ncbi.nlm.gov/Taxomomy/Browser/Viruses),挑选对人类致病的RNA病毒,选取14个病毒科、32个病毒属的RNA病毒作为本课题拟检测的医学RNA病毒。然后从GenBank病毒数据库中下载拟检测病毒的基因组序列。如果一个病毒属已测基因组全序列≧10条的,下载已登录的所有病毒基因组全序列。如果一个病毒属已测基因组全序列≦10条的,除下载已登录的全序列之外,同时下载其它部分测序的序列。对于分段RNA病毒,需要下载所有片段的序列。共获得各种序列约15万条,用于医学RNA病毒属水平oligo探针的设计。利用Arraydesigner 4.0等软件并参考国外已发表的病毒属及种的oligo探针序列,针对每个病毒属设计10～30条探针,使探针序列能覆盖该病毒属的所有已知病毒株。探针长度均为63mer,Tm值为75±5℃,使所有探针尽量具有相近的杂交动力学参数。同时遵循oligo探针的其它设计原则,共设计了314条病毒属水平oligo探针及314条互补序列探针。杂交系统阳性对照探针是针对丙型肝炎病毒(Hepatitis C virus,HCV)基因组而设计,探针长度为63mer,共设计10条阳性对照探针。本课题中共设计了oligo探针638条。利用生物信息学技术,通过与所有已测序的病毒序列进行BLAST比较,进行了探针的特异性验证,包括病毒属内序列同源性及属间序列的特异性。
2.oligo探针的筛选与剔除及基因芯片杂交条件的优化
由于影响基因芯片杂交结果的因素很多,因此,在利用生物信息学方法完成探针特异性的验证之后,进一步利用实验手段对探针的特异性进行了验证。具体方法是在各个病毒属中选择一种病毒,设计该病毒的特异PCR扩增引物,使扩增片段中包含至少一条针对该病毒属的oligo探针序列。然后通过分析特异PCR产物与基因芯片的杂交结果,考核所设计的探针的特异性,并对特异性差的探针予以剔除。结果证实大多数探针具有良好的杂交特异性,但也有一些探针出现非特异阳性杂交信号,表明其特异性较差,通过实验验证共剔除了18条特异性较差的探针。另外,以日本乙型脑炎病毒作为模拟未知标本,采用锚定随机引物PCR法从病毒的培养上清中扩增样本核酸,通过与基因芯片杂交,进行了芯片点样后封闭、芯片杂交条件及杂交液盐离子浓度的优化。结果显示,点样后经过NaBH4封闭的杂交效果更好,可有效降低背景荧光。通过比较杂交温度分别为36℃、39℃、42℃、45℃、48℃时的基因芯片杂交结果,证实42℃是本芯片的最适杂交温度。通过比较杂交时间分别为2 hr、6 hr、8 hr及过夜杂交的结果,证实过夜杂交是本芯片的最适杂交时间。通过比较杂交液中甲酰胺浓度分别为25%、33%、40%、50%和60%时的杂交效果,证实50%是本芯片的最适甲酰胺浓度。
3.标本前处理方法的探索及未知病毒靶核酸扩增方法的建立
宿主细胞基因组成分的干扰及如何对未知病毒核酸进行有效扩增是制约高通量病毒基因芯片技术发展的主要因素。本研究对标本前处理方法及未知病毒核酸的扩增方法进行了探索与比较。结果表明,在提取病毒RNA之前,对标本进行离心、过滤及DNA酶I/RNA酶的双酶预处理等步骤可有效降低宿主细胞基因组成分对芯片杂交结果的干扰。而对于如何有效扩增未知病毒核酸,从RNA的逆转录及序列非依赖性扩增两个步骤进行了比较。针对RNA的逆转录步骤,比较了病毒偏性引物逆转录法、随机引物逆转录法及锚定随机引物逆转录法,结果表明病毒偏性引物逆转录法更能提高病毒RNA的逆转录水平。针对序列非依赖性扩增,比较了PVA引物扩增法、多重置换扩增法、锚定随机引物扩增法及限制性显示PCR扩增法,证实采用锚定随机引物法,并以多重置换扩增法作为补充进行病毒核酸的扩增,可实现对未知病毒核酸的有效及均衡扩增,提高基因芯片检测的敏感性及特异性。
4.基因芯片筛查技术的建立
综合以上的研究结果,初步建立了医学RNA病毒属水平筛查基因芯片技术,并对基因芯片的检测敏感性及特异性进行了考核与验证。以日本乙型脑炎病毒为模拟未知标本,证实本芯片对该病毒培养上清的检测敏感性约为100个病毒粒子。然后分别利用已知病毒的细胞培养上清、新分离未知标本的培养上清及模拟临床标本,对本芯片进行了检测特异性的考核。结果显示,对已知病毒的细胞培养上清、新分离未知标本的培养上清,本芯片均获得了特异性较好的筛查结果。对于模拟临床标本,虽然杂交结果中存在一些非特异信号,但阳性信号绝对值最高的是待检测标本,因此本芯片也能对模拟临床标本进行快速筛查。
总之,本研究首先在医学RNA病毒属水平设计oligo探针,对探针的特异性进行了生物信息学及实验手段的验证,剔除了部分特异性差的探针。然后对基因芯片杂交条件进行了优化,选择了针对本芯片的最适杂交温度、杂交时间及盐离子浓度。在此基础上,进行了标本前处理方法的摸索及未知病毒核酸扩增方法的优化,初步建立了医学RNA病毒属水平筛查基因芯片技术。应用该技术,可对细胞培养上清中的未知病毒进行属水平的快速筛查。在出现传染病疫情时,多数时候通过观察病原体对抗生素的敏感性并结合临床症状等,可快速判定病原体是否为病毒。但即使是在敏感细胞中观察到了细胞病变,由于是未知病毒,对病原体的鉴定仍是一项非常困难的工作。本研究为解决该问题提供了一种高通量的快速筛查技术。将未知病毒快速筛查到属水平后,可能利用病毒属通用PCR并结合序列测定技术等,实现对未知病毒的最终鉴定。
More than 70%of human infectious diseases are caused by viruses,which are termed as viral infectious disease.Among 5400 known viruses,about 400 viruses are related with human diseases.And more than 80%of human pathogenic viruses are RNA viruses.Most of the high pathogenic viral diseases are infected by RNA viruses,such as Ebola and Marburg virus in Filovirus,Lassa virus in Arenavirus and Venezuelan equine encephalitis virus in Alphavirus.It is extremely important to establish methods for high throughput and parallel screening and identification of unknown RNA viruses. Microarray(genechip) is a large scale integrated solid-phase hybridization technique.Its fundamental principle is the hybridization of nucleic acid molecules.Based on the principle of complementarily match of double-strand DNA,oligonucleotides,cDNA or gene fragments with known sequences are printed on chip as probes,and hybridized with target nucleic acids in tested sample.The results could further be interpreted by computer program or software qualitatively and quantitively.
The purpose of this study was to establish a microarray method to screen human pathogenic RNA viruses on genus levels.By using this high throughput method, hopefully,the unknown RNA viral pathogen could be screened to a certain viral genus during the outbreak of viral infectious disease or other public health events.This would be helpful for latter isolation and identification of the pathogen and for further control of the epidemic.The principle of this study was to design the oligo probes on genus levels. For each viral genus,about 10 oligos should be designed to cover all the known viruses in this certain genus.By using these oligos,the known RNA viruses could be quickly identified to its belonged genus.In addition,due to the fact that different viral strains in a certain genus have different sequences but possessed certain amount of same conserved region,by employing long oligos(63mer),certain mismatch of base pairs are permitted.As any new viral strains must have some homology with the sequences in a certain genus,so this method could also be applied for the rapid classification of the novel virus.
The chief results of this study are listed as following:
1.The designing of oligo probes on genus levels and their validation by bioinformatics
According to the database of the eighth report of ICTV submitted in 2005 (http://phene.cpmc.columbia.edu/ICTV/index.htm) and the taxonomy database of NCBI(http://www.ncbi.nlm.gov/Taxomomy/Browser/Viruses),32 RNA viral genus in 14 viral families were selected for this study.Viral genome sequences were downloaded from Genbank.For those genus more than 10 whole genomes were sequenced,all of the whole genome were downloaded.For those less than 10 whole genome were sequenced, apart from downloading the whole genome,other partial sequenced fragments were also downloaded.As for segmented RNA viruses,all of the segments were downloaded. Totally,more than 150000 sequences were obtained for oligo probe designing. Arraydesigner 4.0 and other softwares were employed for the designing.The probe sequenced published by other groups were also taken into consideration.10～30 63-mer oligos were designed for each genus and the Tm value of these oligos were 75±5℃.This was necessary to ensure all the oligos had similar hybridization dynamics.Totally,314 viral probes and 314 complementary probes were designed at the first step.10 positive control probes were designed specifically according to the sequence of Hepatitis C virus. Bioinformatics was employed for the BLAST validation of these oligos.The sequence homology inside the genus and the specificity among different genus were analyzed.
2.Selection of oligo probes and the optimization of hybridization conditions Lots of factors could affect the microarray hybridization results.Due to the above reason,after validation of the oligos by bioinformatics,experiments were carried out to validate the specificity of the oligos.The method was to select a virus in a certain genus and to design the specific RT-PCR primers for this virus,and to ensure that the amplified PCR product must contain at least one sequence of the oligo in this genus. Then the PCR product was hybridized with the microarray.By analyzing the hybridization result,the specificity of the oligo probe was evaluated and the other probes hybridized with the PCR product non-specifically was deleted in later experiments.The validation results showed that most of the oligos tested had good specificity,whereas some of the probes continuously gave positive signals.Totally 18 of these probes were deleted by experimental validation.In addition,Japanese B encephalitis virus(JEV) was selected as simulated unknown virus for optimization of hybridization conditions.The nucleic acid was amplified by anchored random-PCR and hybridized with the microarray.Hybridization time,hybridization temperature and concentration of formamide were optimized.The results showed that after printing on chip,the probes were blocked with NaBH4 could decrease the background fluorescence.By comparing the hybridization results under different conditions,hybridization at 42℃for overnight and at 50%formamide in hybridization buffer were selected as the optimal conditions.
3.The exploring of sample pretreatment and target nucleic acids amplification of unknown virus
The interference of host genome and effectively amplification of target nucleic acids from unknown virus were the main obstacles of developing high throughput virus microarray.In this study,the pretreatment of samples by differents ways and different non-sequence relyed amplification methods were explored and compared.The results showed that by sample centrifuge,filtration and treatment with DNaseI/RNaseT1 before RNA extraction,the interference of host genome could significantly be decreased.As for viral nucleic acid amplification,virus-bias primer,random primer and anchored random primer were employed during the reverse transcript of RNA,and random PVA primer PCR,anchored-random primer PCR,mutiple displacement amplification and modified restrict display PCR were compared for their amplification results.The results showed that by using virus-bias primer as reverse transcrip primer and anchored-random primer PCR combined with mutiple displacement amplification for latter amplification,viral nucleic acid could be effectively and balancedly amplified.These pretreatment and optimal amplification could significantly increase the sensitivity and specificity of this microarray.
4.The establishment of microarray screening technique
Taking all the above results together,the medical RNA viruses microarray on genus level was extablished.The testing sensitivity and specificity of the array were evaluated and validated.By testing the cultured supernatants of JEV,the sensitivity of this microarray to JEV was evaluated as about 100 virus copies.Then the cultured supernatants of known viruses and newly isolated sample and simulated clinical sample were used for validation of the microarray's specificity.The results showed that the microarray gave good hybridization results for both cultured cultured supernatants of known viruses and newly isolated sample.For simulated clinical sample,although there existed some non-specific positive singals,the positive singal density of detected virus was higher enough to determine the screening results.
In summary,the oligo probes were designed on genus level for human pathogenic RNA viruses and the specificity of the probes was validated both by bioinformatics and experiments.Some of the poor specified probes were deleted.Hybridization conditions were optimized and optimal temperature,time and salt ion concentration were selected for this microarray to ensure the same hybridization dynamics of different oligos.Based on these results,the pretreatment of samples and optimal amplification of unknown virus were also explored.The microarray technique for parallel screening of human pathogenic RNA viruses was primarily established.By using this microarray,the viral pathogens in culture supertatants of both known virus and new isolated sample could be rapidly screened to genus level.Further epxeriments would be taken out to increase the specificity of the microarray and to validate the screen and detect effecicy of the microarray on clinical samples.
本研究的目的是建立能够在属水平对医学RNA病毒进行快速筛查的基因芯片技术,在出现新(突)发传染病疫情或公共卫生事件时,能利用该芯片,快速将病毒性病原体筛查到属水平,为进一步进行病原体的分离培养、鉴定及传染病疫情的防控提供有意义的参考。本研究的设计思路是在属水平设计医学RNA病毒的寡核苷酸(oligo)探针,针对每个病毒属设计10条左右探针,使探针序列能覆盖该病毒属中所有已知的病毒株,因此可将该芯片用于对目前已知的RNA病毒进行属水平的快速筛查。另外,由于同一病毒属内不同病毒株之间虽然碱基序列不完全相同,但存在相当程度的同源性,因此,通过采用较长的oligo探针(63mer),允许在杂交时模板与探针之间存在一定的碱基错配,而任何一种新发或未知病毒都必然与某个病毒属中的已知病毒存在一定的同源性,因此,利用该芯片也可对目前未知的新发病毒性病原体进行快速分类。
本课题的主要研究结果如下:
1.医学RNA病毒属水平oligo探针的设计及生物信息学验证
依据国际病毒分类委员会2005年发布的第八次分类报告中的病毒分类数据库(http://phene.cpmc.columbia.edu/ICTV/index.htm)及NCBI的病毒分类数据库(http://www.ncbi.nlm.gov/Taxomomy/Browser/Viruses),挑选对人类致病的RNA病毒,选取14个病毒科、32个病毒属的RNA病毒作为本课题拟检测的医学RNA病毒。然后从GenBank病毒数据库中下载拟检测病毒的基因组序列。如果一个病毒属已测基因组全序列≧10条的,下载已登录的所有病毒基因组全序列。如果一个病毒属已测基因组全序列≦10条的,除下载已登录的全序列之外,同时下载其它部分测序的序列。对于分段RNA病毒,需要下载所有片段的序列。共获得各种序列约15万条,用于医学RNA病毒属水平oligo探针的设计。利用Arraydesigner 4.0等软件并参考国外已发表的病毒属及种的oligo探针序列,针对每个病毒属设计10～30条探针,使探针序列能覆盖该病毒属的所有已知病毒株。探针长度均为63mer,Tm值为75±5℃,使所有探针尽量具有相近的杂交动力学参数。同时遵循oligo探针的其它设计原则,共设计了314条病毒属水平oligo探针及314条互补序列探针。杂交系统阳性对照探针是针对丙型肝炎病毒(Hepatitis C virus,HCV)基因组而设计,探针长度为63mer,共设计10条阳性对照探针。本课题中共设计了oligo探针638条。利用生物信息学技术,通过与所有已测序的病毒序列进行BLAST比较,进行了探针的特异性验证,包括病毒属内序列同源性及属间序列的特异性。
2.oligo探针的筛选与剔除及基因芯片杂交条件的优化
由于影响基因芯片杂交结果的因素很多,因此,在利用生物信息学方法完成探针特异性的验证之后,进一步利用实验手段对探针的特异性进行了验证。具体方法是在各个病毒属中选择一种病毒,设计该病毒的特异PCR扩增引物,使扩增片段中包含至少一条针对该病毒属的oligo探针序列。然后通过分析特异PCR产物与基因芯片的杂交结果,考核所设计的探针的特异性,并对特异性差的探针予以剔除。结果证实大多数探针具有良好的杂交特异性,但也有一些探针出现非特异阳性杂交信号,表明其特异性较差,通过实验验证共剔除了18条特异性较差的探针。另外,以日本乙型脑炎病毒作为模拟未知标本,采用锚定随机引物PCR法从病毒的培养上清中扩增样本核酸,通过与基因芯片杂交,进行了芯片点样后封闭、芯片杂交条件及杂交液盐离子浓度的优化。结果显示,点样后经过NaBH4封闭的杂交效果更好,可有效降低背景荧光。通过比较杂交温度分别为36℃、39℃、42℃、45℃、48℃时的基因芯片杂交结果,证实42℃是本芯片的最适杂交温度。通过比较杂交时间分别为2 hr、6 hr、8 hr及过夜杂交的结果,证实过夜杂交是本芯片的最适杂交时间。通过比较杂交液中甲酰胺浓度分别为25%、33%、40%、50%和60%时的杂交效果,证实50%是本芯片的最适甲酰胺浓度。
3.标本前处理方法的探索及未知病毒靶核酸扩增方法的建立
宿主细胞基因组成分的干扰及如何对未知病毒核酸进行有效扩增是制约高通量病毒基因芯片技术发展的主要因素。本研究对标本前处理方法及未知病毒核酸的扩增方法进行了探索与比较。结果表明,在提取病毒RNA之前,对标本进行离心、过滤及DNA酶I/RNA酶的双酶预处理等步骤可有效降低宿主细胞基因组成分对芯片杂交结果的干扰。而对于如何有效扩增未知病毒核酸,从RNA的逆转录及序列非依赖性扩增两个步骤进行了比较。针对RNA的逆转录步骤,比较了病毒偏性引物逆转录法、随机引物逆转录法及锚定随机引物逆转录法,结果表明病毒偏性引物逆转录法更能提高病毒RNA的逆转录水平。针对序列非依赖性扩增,比较了PVA引物扩增法、多重置换扩增法、锚定随机引物扩增法及限制性显示PCR扩增法,证实采用锚定随机引物法,并以多重置换扩增法作为补充进行病毒核酸的扩增,可实现对未知病毒核酸的有效及均衡扩增,提高基因芯片检测的敏感性及特异性。
4.基因芯片筛查技术的建立
综合以上的研究结果,初步建立了医学RNA病毒属水平筛查基因芯片技术,并对基因芯片的检测敏感性及特异性进行了考核与验证。以日本乙型脑炎病毒为模拟未知标本,证实本芯片对该病毒培养上清的检测敏感性约为100个病毒粒子。然后分别利用已知病毒的细胞培养上清、新分离未知标本的培养上清及模拟临床标本,对本芯片进行了检测特异性的考核。结果显示,对已知病毒的细胞培养上清、新分离未知标本的培养上清,本芯片均获得了特异性较好的筛查结果。对于模拟临床标本,虽然杂交结果中存在一些非特异信号,但阳性信号绝对值最高的是待检测标本,因此本芯片也能对模拟临床标本进行快速筛查。
总之,本研究首先在医学RNA病毒属水平设计oligo探针,对探针的特异性进行了生物信息学及实验手段的验证,剔除了部分特异性差的探针。然后对基因芯片杂交条件进行了优化,选择了针对本芯片的最适杂交温度、杂交时间及盐离子浓度。在此基础上,进行了标本前处理方法的摸索及未知病毒核酸扩增方法的优化,初步建立了医学RNA病毒属水平筛查基因芯片技术。应用该技术,可对细胞培养上清中的未知病毒进行属水平的快速筛查。在出现传染病疫情时,多数时候通过观察病原体对抗生素的敏感性并结合临床症状等,可快速判定病原体是否为病毒。但即使是在敏感细胞中观察到了细胞病变,由于是未知病毒,对病原体的鉴定仍是一项非常困难的工作。本研究为解决该问题提供了一种高通量的快速筛查技术。将未知病毒快速筛查到属水平后,可能利用病毒属通用PCR并结合序列测定技术等,实现对未知病毒的最终鉴定。
More than 70%of human infectious diseases are caused by viruses,which are termed as viral infectious disease.Among 5400 known viruses,about 400 viruses are related with human diseases.And more than 80%of human pathogenic viruses are RNA viruses.Most of the high pathogenic viral diseases are infected by RNA viruses,such as Ebola and Marburg virus in Filovirus,Lassa virus in Arenavirus and Venezuelan equine encephalitis virus in Alphavirus.It is extremely important to establish methods for high throughput and parallel screening and identification of unknown RNA viruses. Microarray(genechip) is a large scale integrated solid-phase hybridization technique.Its fundamental principle is the hybridization of nucleic acid molecules.Based on the principle of complementarily match of double-strand DNA,oligonucleotides,cDNA or gene fragments with known sequences are printed on chip as probes,and hybridized with target nucleic acids in tested sample.The results could further be interpreted by computer program or software qualitatively and quantitively.
The purpose of this study was to establish a microarray method to screen human pathogenic RNA viruses on genus levels.By using this high throughput method, hopefully,the unknown RNA viral pathogen could be screened to a certain viral genus during the outbreak of viral infectious disease or other public health events.This would be helpful for latter isolation and identification of the pathogen and for further control of the epidemic.The principle of this study was to design the oligo probes on genus levels. For each viral genus,about 10 oligos should be designed to cover all the known viruses in this certain genus.By using these oligos,the known RNA viruses could be quickly identified to its belonged genus.In addition,due to the fact that different viral strains in a certain genus have different sequences but possessed certain amount of same conserved region,by employing long oligos(63mer),certain mismatch of base pairs are permitted.As any new viral strains must have some homology with the sequences in a certain genus,so this method could also be applied for the rapid classification of the novel virus.
The chief results of this study are listed as following:
1.The designing of oligo probes on genus levels and their validation by bioinformatics
According to the database of the eighth report of ICTV submitted in 2005 (http://phene.cpmc.columbia.edu/ICTV/index.htm) and the taxonomy database of NCBI(http://www.ncbi.nlm.gov/Taxomomy/Browser/Viruses),32 RNA viral genus in 14 viral families were selected for this study.Viral genome sequences were downloaded from Genbank.For those genus more than 10 whole genomes were sequenced,all of the whole genome were downloaded.For those less than 10 whole genome were sequenced, apart from downloading the whole genome,other partial sequenced fragments were also downloaded.As for segmented RNA viruses,all of the segments were downloaded. Totally,more than 150000 sequences were obtained for oligo probe designing. Arraydesigner 4.0 and other softwares were employed for the designing.The probe sequenced published by other groups were also taken into consideration.10～30 63-mer oligos were designed for each genus and the Tm value of these oligos were 75±5℃.This was necessary to ensure all the oligos had similar hybridization dynamics.Totally,314 viral probes and 314 complementary probes were designed at the first step.10 positive control probes were designed specifically according to the sequence of Hepatitis C virus. Bioinformatics was employed for the BLAST validation of these oligos.The sequence homology inside the genus and the specificity among different genus were analyzed.
2.Selection of oligo probes and the optimization of hybridization conditions Lots of factors could affect the microarray hybridization results.Due to the above reason,after validation of the oligos by bioinformatics,experiments were carried out to validate the specificity of the oligos.The method was to select a virus in a certain genus and to design the specific RT-PCR primers for this virus,and to ensure that the amplified PCR product must contain at least one sequence of the oligo in this genus. Then the PCR product was hybridized with the microarray.By analyzing the hybridization result,the specificity of the oligo probe was evaluated and the other probes hybridized with the PCR product non-specifically was deleted in later experiments.The validation results showed that most of the oligos tested had good specificity,whereas some of the probes continuously gave positive signals.Totally 18 of these probes were deleted by experimental validation.In addition,Japanese B encephalitis virus(JEV) was selected as simulated unknown virus for optimization of hybridization conditions.The nucleic acid was amplified by anchored random-PCR and hybridized with the microarray.Hybridization time,hybridization temperature and concentration of formamide were optimized.The results showed that after printing on chip,the probes were blocked with NaBH4 could decrease the background fluorescence.By comparing the hybridization results under different conditions,hybridization at 42℃for overnight and at 50%formamide in hybridization buffer were selected as the optimal conditions.
3.The exploring of sample pretreatment and target nucleic acids amplification of unknown virus
The interference of host genome and effectively amplification of target nucleic acids from unknown virus were the main obstacles of developing high throughput virus microarray.In this study,the pretreatment of samples by differents ways and different non-sequence relyed amplification methods were explored and compared.The results showed that by sample centrifuge,filtration and treatment with DNaseI/RNaseT1 before RNA extraction,the interference of host genome could significantly be decreased.As for viral nucleic acid amplification,virus-bias primer,random primer and anchored random primer were employed during the reverse transcript of RNA,and random PVA primer PCR,anchored-random primer PCR,mutiple displacement amplification and modified restrict display PCR were compared for their amplification results.The results showed that by using virus-bias primer as reverse transcrip primer and anchored-random primer PCR combined with mutiple displacement amplification for latter amplification,viral nucleic acid could be effectively and balancedly amplified.These pretreatment and optimal amplification could significantly increase the sensitivity and specificity of this microarray.
4.The establishment of microarray screening technique
Taking all the above results together,the medical RNA viruses microarray on genus level was extablished.The testing sensitivity and specificity of the array were evaluated and validated.By testing the cultured supernatants of JEV,the sensitivity of this microarray to JEV was evaluated as about 100 virus copies.Then the cultured supernatants of known viruses and newly isolated sample and simulated clinical sample were used for validation of the microarray's specificity.The results showed that the microarray gave good hybridization results for both cultured cultured supernatants of known viruses and newly isolated sample.For simulated clinical sample,although there existed some non-specific positive singals,the positive singal density of detected virus was higher enough to determine the screening results.
In summary,the oligo probes were designed on genus level for human pathogenic RNA viruses and the specificity of the probes was validated both by bioinformatics and experiments.Some of the poor specified probes were deleted.Hybridization conditions were optimized and optimal temperature,time and salt ion concentration were selected for this microarray to ensure the same hybridization dynamics of different oligos.Based on these results,the pretreatment of samples and optimal amplification of unknown virus were also explored.The microarray technique for parallel screening of human pathogenic RNA viruses was primarily established.By using this microarray,the viral pathogens in culture supertatants of both known virus and new isolated sample could be rapidly screened to genus level.Further epxeriments would be taken out to increase the specificity of the microarray and to validate the screen and detect effecicy of the microarray on clinical samples.
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2.Wang D,Urisman A,Liu YT,et al.Viral discovery and sequence recovery using DNA microarrays.PLoS Biol.2003;1(2):17.
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5.Elnifro,E.M.,Ashshi,A.M.,Cooper,R.J.& Klapper,P.E.(2000) Clin.Microbiol.Rev.13,559-570.
6.Boriskin Y S,Rice P S,Stabler R A,et al.DNA Microarrays for Virus Detection in Cases of Central Nervous System Infection.J.Clin.Microbiol.2004;42(12):5811-5818
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