一种基于新冠病毒特异性逆转录引物的建库方法及运用
技术领域
本发明涉及基因建库技术领域，尤其涉及一种基于新冠病毒特异性逆转录 引物的建库方法及运用。
背景技术
新型冠状病毒是一种具有包膜的、不分节段的正链单股RNA病毒，颗粒呈 圆形或椭圆形，直径约80～120nm，属于网巢病毒目冠状病毒科Betacoronavirus。 病毒粒子被宿主细胞提供的脂质双层所包裹，其中含有核酸及核衣壳蛋白，有 三种主要蛋白：包膜蛋白(E蛋白)、膜蛋白(M蛋白)和刺突蛋白(S蛋白)。 此病毒每组基因组长度约三万个核苷酸左右，基因序列显示SARS-CoV-2属于 乙型冠状病毒属谱系β(Betacoronavirus Lineage β,Sarbecovirus)进化树中分支 较长的一种病毒，与中华菊头蝠中发现的冠状病毒相似，例如MERS-CoV或 SARS-CoV。对病毒的生物遗传学分析显示，同属人类冠状病毒的SARS病毒分离株AY274119相比MERS病毒分离株KC164505、JX869059等在亲缘关系上 距离SARS-CoV-2病毒更近。
病毒潜伏期平均大约3-7天，最长不超过14天。大多数患者的表现以下呼 吸道症状为主，常见临床表现包括发热、四肢乏力、干咳等症状，其他表现包 含鼻塞、流鼻涕、头痛、咽痛、咳血，咳痰、或腹泻等。有部分患者仅表现为 低热、轻微乏力等，无肺炎表现。还有部分患者无任何临床表现。病毒感染严 重后，会引发多种并发症包含急性呼吸窘迫综合征(ARDS)、脓毒症休克、全 身炎症反应综合征、难以纠正的代谢性酸中毒、急性心肌损伤，和出凝血功能 障碍等。
针对新型冠状病毒引发的疫情，目前确诊主要是基于荧光PCR的技术，利 用引物探针去扩增病毒靶序列，根据目前中国疾控中心出的诊疗方案中提供的 三个靶点，有任何两个靶点呈现阳性，都可以断定病毒为新型冠状病毒 2019-nCov。检测结果若呈阳性就会呈现出一条曲线；阴性就是一条平线。
除荧光PCR检测技术外，基于检测样本的宏基因组测序(Meta)、探针捕 获测序(Capture)和多重PCR扩增子测序(Amplicon)等方法可以提供更加精 确的检测。受限于早期口腔拭子等样本病毒载量非常低，提取的样本核酸往往 是降解，且总量低，病毒核酸的比例往往在万分之一以下，需要测大数据量的 宏基因组数据，才能进行病毒的序列分析。该方法采用dT引物与随机引物进行 逆转录，获得所有的RNA的逆转录信息，没有选择性，同时后续建库也需要合 成双链cDNA后，经打断、末端修复、加A与加接头构建测序文库，对DNA总量要求至少在50ng以上。
由于新冠病毒序列一致性高，不同病人之间的病毒序列具有高度的一致性， 但是病毒样本往往属于超微量，常规的构建测序文库方式无法满足。目前尚未 有研究基于新冠病毒一致性序列设计特异性逆转录引物，并利用特异性切割 DNA-RNA杂合链的酶获得几百倍至几千倍的病毒数据，同时满足超微量样本的 建库测序方法。
发明内容
针对上述技术问题，本发明提出了一种基于新冠病毒特异性逆转录引物的 建库方法及运用，提供了一种适用于新冠病毒的特异性逆转录引物，该引物可 以特异性的结合病毒RNA进行逆转录反应，富集病毒序列的比例，同时结合特 异性的转座酶酶切DNA-RNA杂合链的方法，可以实现低至1ng以下的超微量 新冠病毒测序。
为了达到上述目的，本发明提出了一种新冠病毒特异性逆转录引物，引物 设计步骤为：通过多条新冠病毒序列，处理并获得完整的新冠病毒一致性序列， 基于一致性序列设计病毒特异性逆转录引物，引物覆盖整个新冠病毒基因组。
进一步地，所述一致性序列如SEQ ID NO.1所示；
进一步地，所述逆转录引物的位置基于基因组的保守区域设计；
进一步地，所述逆转录引物为15条，逆转录引物在新冠病毒基因组上共有 106个结合位点；
进一步地，所述逆转录引物在病毒基因组上逆转录引物结合位点最小距离 为3bp，最大距离为757bp；
进一步地，所述逆转录引物核苷酸序列如SEQ ID NO.2-SEQ ID NO.16所示。
本发明另外一个目的在于提出了一种基于新冠病毒特异性逆转录引物的建 库方法，步骤为：将15条引物等体积混合后，与环境样本RNA进行DNA-RNA 杂合链合成，再利用Tn5 VR转座酶建库。
其中，所述DNA-RNA杂合链合成步骤为：取环境RNA与引物混合物、dNTP 混合液混合后离心、第一次孵育后冷却，再与5X逆转录反应缓冲液、RNA酶 抑制剂、PrimeScript逆转录酶混合后离心，第二次孵育后离心、冷却；
进一步地，所述环境RNA样本浓度为0.01～0.15ng/uL，总量为0.1～1.5ng；
进一步地，所述第一次孵育为65℃孵育5min，所述第二次孵育为30℃保 持10min，42℃保持60min，70℃保持15min。
进一步地，所述利用Tn5 VR转座酶建库步骤为：
I.取所述DNA-RNA杂合链合成所得反应液，与Tagment缓冲液、Tn5 VR 转座酶混合后，离心、第三次孵育后冷却后，再加入终止液进行第四次孵育；
II.所得反应液与扩增预混液、PCR引物混合物混合后离心、经第五次孵育 冷却；
III.所得反应液加入N5端引物和N7端引物混合后离心，进行扩增反应， 经纯化、洗脱后获得测序文库；
进一步地，所述第三次孵育为55℃孵育15min；所述第四次孵育为20℃孵 育5min；所述第五次孵育为55℃保持5min，60℃保持5min，95℃保持5min进 行孵育反应；
进一步地，所述扩增预混液为VAHTS HiFi Amplification Mix，所述PCR引 物混合物为PCR Primer Mix 3 for Illumina；
进一步地，所述扩增反应为98℃保持3min；98℃保持20s、60℃保持15s、 72℃保持30s，共18个循环；72℃保持5min。
本发明还有一个目的在于，提出了一种基于新冠病毒特异性逆转录引物的 建库方法的运用，在新冠病毒样本测序过程中采用上述建库方法。
与现有技术相比，本发明的有益效果为：
1)针对新冠病毒的序列保守性特点设计了15条病毒特异性逆转录引物， 该引物可以和病毒上的特定互补序列选择性结合，可以避免宿主RNA的污染， 可以将病毒数据比例提高几百倍至几千倍。
2)在使用特异性逆转录引物合成病毒DNA-RNA杂合链后，使用特异性切 割DNA-RNA杂合链的转座酶方法进行建库，可以避免样本中存在的宿主RNA、 DNA对文库造成污染，同时该方法建库极大的简化了建库流程，将二代建库由 10个小时左右缩短至仅需要3个小时即可完成文库的构建，极大的提高了建库 效率。
3)该方法从酶切打断至PCR扩增步骤中间不需要额外的纯化步骤与酶反 应，样本零损失，可以用于低于1ng的超微量样本的建库，即在实际运用中可 以测序到低于1ng的超微量样本。
附图说明
图1为特异性逆转录引物分布示意图。
图2为建库示意图。
图3为文库检测峰图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限 于此。
实施例1：
1.病毒特异性逆转录引物设计
在NCBI上搜索新冠病毒，共下载获得74条新冠病毒序列，通过检查序列 完整性，删除长度在20k以下的序列，共获得了43条完整的新冠病毒序列。所 有43条完整基因组序列经比对后获得一致性序列，新冠病毒基因组一致性序列 如SEQ ID NO：1所示。
基于一致性序列，共设计15条病毒特异性逆转录引物，这15条逆转录引 物在新管病毒基因组上共有个106个结合位点，均匀的覆盖整个新冠病毒基因 组。设计的这15个逆转录引物在病毒基因组上平均283.8bp距离就存在一个逆 转录引物结合位点，其中最小距离为3bp，最大距离为757bp。理论上，在进行 逆转录时，一个逆转录结合位点可以获得2kb左右的逆转录cDNA。
所述15条特异性逆转录引物分布情况如图1所示，所述特异性逆转录引物 核苷酸序列如表1所示：
表1：病毒特异性逆转录引物
该病毒特异性逆转录引物在新冠病毒基因组上的结合位置如表2所示。
表2：
2.新冠病毒建库测序
本实施例中新冠病毒建库示意图如图2所示。
设计的新冠特异性逆转录引物统一稀释至10uM，将15个引物等体积混合 为新冠特异性引物混池，命名为CoVSP。一份6个疑似新冠病毒环境RNA样本 的项目实施例操作如下：
2.1 环境样本RNA的质检
该环境样本使用Nanodrop进行初步定量与纯度检测，样本浓度非常低。使 用电泳检测均不可见电泳条带，说明总量极低，经过Qubit定量检测确定，样本 浓度在0.01～0.15ng/uL之间，总量在0.1～1.5ng之间。
2.2 DNA-RNA杂合链合成
配置表3中的反应。
表3：
混合均匀后瞬时离心，65℃孵育5min后，迅速置于冰上冷却。
按表4配制下一步反应。
表4：
充分混匀，瞬时离心，30℃保存10min，42℃保持60min，70℃保持15min 进行孵育反应。反应完成后，瞬时离心，置于冰上，完成DNA-RNA杂合链的 合成。
2.3 Tn5 VR转座酶建库
1)室温解冻-20℃的试剂，混匀后配制如表5中反应：
表5：
充分混匀，瞬时离心，55℃孵育15min。反应结束后置于冰上，加入入2μL 的TStopSolution(Vazyme)，震荡混匀，20℃孵育5min。
2)室温解冻试剂，轻弹混匀，配制如表6中反应。
轻弹混匀，瞬时离心，充分混匀，瞬时离心，55℃保持5min，60℃保持 5min，95℃保持5min进行孵育反应。
表6：
3)应完成后，瞬时离心，置于冰上，配制表7中反应。
表7：
轻弹混匀，瞬时离心，在PCR仪上进行如下反应：
98℃保持3min；98℃20s、60℃15s、72℃30s，共18个循环；72℃保持5min。
扩增反应完成后，使用0.8X的AMPure磁珠进行磁珠纯化，最后使用20μL 洗脱缓冲液洗脱，获得测序文库。取1μL文库进行进行安捷伦2100的片段大小 分析，结果如图3所示，文库大小在350bp左右，说明制备的文库合格。
实施例2：测序数据对比
以下采用了6个样本，使用了常规的宏转录组建库测序方法与使用实施例1 中的方法作为对比，测序数据对比如表8。
表8：
从以上数据对比看出，当样本起始量降低至1ng以下时，常规的病毒建库 方法无法构建出合格的文库，文库峰型异常，但按照实施例1的方法可构建低 至1ng以下起始量的合格文库。在测序数据中病毒的比例方面，按照实施例1 的方法可以将病毒序列比例提高几百倍，因此本专利方法适用于超低起始量的 病毒样本的建库测序。
序列表
<110>武汉菲沙基因信息有限公司
<120>一种基于新冠病毒特异性逆转录引物的建库方法及运用
<141>2020-06-04
<160>16
<170>SIPOSequenceListing 1.0
<210>1
<211>29899
<212>RNA
<213>新冠病毒一致性序列(2019-nCoV)
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aaaugaaucu cucaucgauc uccaagaacu uggaaaguau gagcaguaua uaaaauggcc 25200
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cacucaacau ggcaaggaag accuuaaauu cccucgagga caaggcguuc caauuaacac 28500
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cagaggcggc agucaagccu cuucucguuc cucaucacgu agucgcaaca guucaagaaa 28860
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caucaaauug gaugacaaag auccaaauuu caaagaucaa gucauuuugc ugaauaagca 29340
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ugaugaaacu caagccuuac cgcagagaca gaagaaacag caaacuguga cucuucuucc 29460
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aaaaaaaaaa aaaaaaaaa29899
<210>2
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>2
tttgtcat8
<210>3
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>3
aacaccat8
<210>4
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>4
aacaattt8
<210>5
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>5
aatcacca8
<210>6
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>6
caaaaggt8
<210>7
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>7
cagcttct8
<210>8
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>8
tttaacaa8
<210>9
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>9
acagcatc8
<210>10
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>10
agcagcaa8
<210>11
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>11
gtgtaact8
<210>12
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>12
ttcataag8
<210>13
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>13
ttcgttta8
<210>14
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>14
ctttttag8
<210>15
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>15
tttctaca8
<210>16
<211>8
<212>DNA
<213>人工序列(Artificial Sequence)
<400>16
aaacctag8
