CN104212918A - 一种h7n9禽流感患病概率预测试剂盒 - Google Patents
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Abstract
本发明提供了一种H7N9禽流感患病概率检测试剂盒，其特征在于，包含对于CPT2-chr1：53676448&53676401、CPT2-chr1：53679229&53679028、FCGR2A-chr1：161479745、IFITM3-chr11：320772、RPAIN-chr17：5326145和TLR3-chr4：187004074&187004217这5个基因9个位点进行检测的6对PCR引物。本发明发现了在H7N9患者中共有的9个位点的突变，可以用于H7N9禽流感感染患者及高危人群中检测，为H7N9感染疫情的防控提供一定的价值。本发明所确定的致病基因可能在H7N9禽流感感染的发病过程中起到一定作用，参与H7N9感染呼吸道上皮细胞，也可能为H7N9禽流感的易感基因。
Description
技术领域
本发明涉及一种H7N9禽流感患病概率检测试剂盒。
背景技术
2013年3月31日，上海和安徽两地发生3例人感染H7N9禽流感病毒的确诊病例，其中2例死亡，1例危重。此后疫情迅速蔓延，截止2013年6月，全国范围内共报告确诊病例132例，患者以严重肺炎和急性呼吸窘迫综合症(acuterespiratory distress syndrome，ARDS)为主要特征，已导致43人死亡，尚未发现人传人的确切证据。
H7N9禽流感病毒属于正粘病毒科甲型流感病毒，为有包膜分节段的单负链RNA病毒。禽流感病毒依据其外膜血凝素(H)和神经氨酸酶(N)蛋白抗原性不同，目前可分为16个H亚型(H1-H16)和9个N(N1-N9)亚型。H7N9为新型重配病毒，包含8个基因组片段。目前已经在禽类及其分泌物或排泄物中分流出H7N9禽流感病毒，与人感染H7N9禽流感病毒高度同源。传染源可能为携带H7N9禽流感病毒的禽类，尚无人际传播的确切证据。主要经呼吸道传播，也可通过密切接触感染的禽类分泌物或排泄物，或直接接触病毒感染。
感染后患者一般表现为流感样症状，如发热、咳嗽、少痰，可伴有头痛、肌肉酸痛和全身不适。重症患者病情发展迅速，多在5-7天出现重症肺炎，体温大多持续在39℃以上，呼吸困难，可伴有咯血痰；可快速进展为ARDS、脓毒症、感染性休克，甚至多器官功能障碍，部分患者可出现纵膈气肿、胸腔积液等。88.3％的病人淋巴细胞减少，73.0％的病人血小板降低，76.6％的病人进入了重症监护病房，27％死亡。
H7N9禽流感病毒结合位于人上呼吸道上皮细胞表面的唾液酸受体，同时可诱发细胞因子风暴，导致全身炎症反应，可出现ARDS，休克及多器官功能衰竭，但目前其确切的发病机制尚不明确。。虽然H7N9患病后致死率比较高，但是发病率极低，全国长期接触活禽的人群数以万计，但感染H7N9病毒的只有百例。这提示我们，这些H7N9患者可能由于自身基因的突变，使得他们与其他人在同样接触活禽的环境下，更易受到H7N9病毒的感染。为此，我们希望通过基因组测序找到H7N9感染的易感基因，并制作一个检测试剂盒，检测人群中是否有易感基因的突变，从而预测H7N9患病的概率。
最近，外显子组测序(exome sequencing)被成功地应用于发现稀有基因疾病的致病基因，如Freeman-Sheldon综合症的MYH3基因，Schinzel-Giedion综合征的SETBP1基因，以及严重大脑畸形的WDR62突变等(Ng SB，Turner EH，Robertson PD，et al，Targeted capture and massively parallel sequencingof 12human exomes.Nature 2009，461(7261)：272-276；Hoischen A，van BonBW，Gilissen C，et al.De novo mutations of SETBPl cause Schinzel-Giedionsyndrome.Nat 6enet 2010；42(6)：483-5；Bi lguvar K，Ozturk AK，Louvi A，etal，Whole-exome sequencing identifies recessive WDR62mutations in severebrain malformations.Nature 2010，467(7312)：207-210.)。全外显子测序技术已被证明为降低稀有单基因疾病候选基因甚至发现其致病基因的有力、有效手段，仅通过对几个很少的个体的全外显子进行测序来筛选与疾病相关的变异，其成功率大为提升。
本发明通过外显子组测序的方法确定了H7N9禽流感患者的突变基因，并制作了一个检测这些突变基因的试剂盒。该发明将为H7N9禽流感的发病机制研究奠定重要基础，有可能为H7N9禽流感疫情的防控提供重要的理论基础。
发明内容
本发明的目的是提供一种H7N9禽流感患病概率检测试剂盒，用于H7N9禽流感患者及高危人群中检测。
为了达到上述目的，本发明提供了一种H7N9禽流感患病概率检测试剂盒，其特征在于，包含对于CPT2-chr1：53676448&53676401、CPT2-chr1：53679229&53679028、FCGR2A-chr1：161479745、IFITM3-chr11：320772、RPAIN-chr17：5326145和TLR3-chr4：187004074&187004217这5个基因的9个位点进行检测的6对PCR引物。
优选地，所述的CPT2-chr1：53676448&53676401位点的PCR检测引物序列为：CCTGGTCAATGCGTATCCC和CTGGCTGGCTCTGTGGAGT；所述的CPT2-chr1：53679229&53679028位点的PCR检测引物序列为：ACCGACACTTGTTTGCTCTGC和ATTACAGGCATGAGCCACCAT；所述的FCGR2A-chr1：161479745位点的PCR检测引物序列为：ACCCTTGGAATCTATCCTTACAACT和CAACAGCCTGACTACCTATTACCTG；所述的IFITM3-chr11：320772位点的PCR检测引物序列为：CCTCTTTCCTCCCTCTCCTA和GTTCCCTTCTCACTTTCTGG；所述的RPAIN-chr17：5326145位点的PCR检测引物序列为：CAGAGGCCAAAACTGAGATTT和GGCAGGATGGTAGAAGAAACA；所述的TLR3-chr4：187004074&187004217位点的PCR检测引物序列为：ATTTGTTTTCTCACTCTTTGCACG和TATGTTGGCTATGTTGTTGTTGCT。
优选地，所述的CPT2-chr1：53676448&53676401位点的PCR退火温度为58±2℃，PCR产物长度为596bp。
优选地，所述的CPT2-chr1：53679229&53679028位点的PCR退火温度为60±2℃，PCR产物长度为400bp。
优选地，所述的FCGR2A-chr1：161479745位点的PCR退火温度为60±2℃，PCR产物长度为525bp。
优选地，所述的IFITM3-chr11：320772位点的PCR退火温度为58±2℃，PCR产物长度为795bp。
优选地，所述的RPAIN-chr17：5326145位点的PCR退火温度为58±2℃，PCR产物长度为758bp。
优选地，所述的TLR3-chr4：187004074&187004217位点的PCR退火温度为60±2℃，PCR产物长度为629bp。
本发明近一年来在国内收集到8例H7N9禽流感病例，全部为散发病例，多为老年男性，合并有多种基础疾病，如高血压，糖尿病等，多有活禽接触史。临床表现为发热，干咳，气促，乏力等。多伴有低氧血症，接受氧疗，无创通气甚至气管插管有创通气，最终均好转出院。
本发明用Agilent SureSelect Human All Exon Kit结合Solexa高通量测序技术对8例H7N9禽流感患者的外显子组序列进行了测序，具体如下：
1)将基因组DNA随机打断成150-200bp左右的片段，随后在片段两端分别连接上接头制备杂交文库(参见http：//www.illumina.com/提供的Illumina/Solexa标准建库说明书)。
2)文库经纯化后经过ligation-mediated PCR(LM-PCR)的线性扩增与SureSelect Biotiny lated RNA Library(BAITS)进行杂交富集，再经过LM-PCR的线性扩增后进行上机测序。测序平台为Illumina Hiseq 2000，读取长度为90bp，每个样本的平均测序深度最少为50×。
3)测序后获得的原始数据由Illumina basecalling Software 1.7进行处理，经过过滤去污染、使用SOAPaligner 2.20(Li R，Li Y，Kristiansen K，etal，SOAP：short oligonucleotide alignment program.Bioinformatics 2008.24(5)：713-714；Li R，Yu C，Li Y，ea al，SOAP2：an improved ultrafast toolfor short read alignment.Bioinformatics 2009，25(15)：1966-1967.)比对参考基因组，获得比对到基因组上的Unique mapped reads。靶区域的基因型由SOAPsnp(Li R，Li Y，Fang X，Yang H，et al，SNP detection for massivelyparallei whole-genome resequencing.Genome Res 2009，19(6)：1124-1132.)确定。
本发明通过HuGE Navigator搜索关键词“流感”，选取了40个可能与禽流感相关的基因(如表1所示)，在这40个基因中发现在其中的27个基因上有89个单核苷酸多态性(SNPs)。由于外显子组测序存在一定程度的假阳性，本发明通过查阅文献重点选取了可能具有致病意义的5个基因10个位点，利用Sanger测序方法对其进行验证，结果有9个SNP为真正的de novo SNP位点，其中1个是假阳性的(如表3所示)，也就是说，这1个位点，要么父母一方携带了(但是外显子组测序没检测出来)，要么就是患者并没有这个突变，而外显子组测序因为假阳性的问题认为它是突变。
与现有技术相比，本发明的有益效果是：
本发明发现了9个位点的突变，可以用于H7N9禽流感患者及高危人群中检测，为H7N9感染疫情的防控提供一定的价值。本发明所确定的致病基因可能在H7N9禽流感感染的发病过程中起到一定作用，参与H7N9感染呼吸道上皮细胞，也可能为H7N9禽流感的易感基因。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1：样品制备及sanger法测序验证
分别对8名H7N9禽流感患者的5个基因、10个位点进行检测，将这10个位点分为6个片段(如表2所示)，针对每个片段的外显子序列设计引物，通过PCR扩增，产物纯化，sanger测序的方法获得每个片段的序列，根据序列测定结果对比属于突变型还是野生型，验证每个基因与H7N9禽流感之间的相关性。具体方法步骤如下：
步骤1：DNA提取：
采集8例H7N9禽流感患者的外周血，利用omegabiotek生产的sQ Blood DNAKit II(货号：D0714-250)抽提外周血白细胞中的基因组DNA，利用NanoDrop2000以及琼脂糖凝胶电泳检测基因组DNA的浓度及纯度，所得的每个标本基因组DNA的OD260/OD280均位于1.8-2.0之间，浓度不少于300ng/ul，总量不少于30μg。
步骤2：引物设计及PCR反应
a)引物序列：引物设计参考人类基因组序列数据库hg19，具体见表2。
b)反应体系：25μL
| 模板(μl) | 1 |
| 正向引物(μl) | 1 |
| 反向引物(μl) | 1 |
| dNTP 10mM(μl) | 0.5 |
| Taq Buffer(μl) | 2.5 |
| 25mM MgCl2(μl) | 2 |
| Taq酶(μl)5U/μl | 0.5 |
| 水(μl) | 16.5 |
c)PCR反应条件
步骤3：将步骤2中获得的PCR扩增产物直接进行DNA测序：
如表3所示，测序结果显示其中的9个位点为真正的de novo SNP位点，1个位点(IFITM3，11号染色体320649位)是假阳性的。
实施例2：H7N9禽流感患病概率检测试剂盒及其制备方法
一种H7N9禽流感患病概率检测试剂盒，包含对于CPT2-chr1：53676448&53676401、CPT2-chr1：53679229&53679028、FCGR2A-chr1：161479745、IFITM3-chr11：320772、RPAIN-chr17：5326145和TLR3-chr4：187004074&187004217这5个基因9个位点进行检测的6对PCR引物。
含有检测这9个位点的6对PCR引物序列如下：
按照实施例1中步骤1所述的方法提取待测者DNA，以所提取的DNA为模板与上述引物按照实施例1中步骤2所述的PCR条件进行PCR反应，按照本领域常规方法对PCR产物进行纯化，将纯化的产物进行sanger测序。观察测序所得到的序列是否有这9个位点的突变。若含有这9个位点的突变，说明该待测者在接触感染了H7N9病毒的活禽后，较其他人更容易患H7N9禽流感。
表1：40个可能与禽流感相关的基因
表2：引物序列及PCR反应条件
表3：8例患者的测序结果
Claims (8)
1.一种H7N9禽流感患病概率检测试剂盒，其特征在于，包含对于CPT2-chr1：53676448&53676401、CPT2-chr1：53679229&53679028、FCGR2A-chr1：161479745、IFITM3-chr11：320772、RPAIN-chr17：5326145和TLR3-chr4：187004074&187004217这5个基因9个位点检测的6对PCR引物。
2.如权利要求1所述的H7N9禽流感患病概率检测试剂盒，其特征在于，所述的CPT2-chr1：53676448&53676401位点的PCR检测引物序列为：CCTGGTCAATGCGTATCCC和CTGGCTGGCTCTGTGGAGT；所述的CPT2-chr1：53679229&53679028位点的PCR检测引物序列为：ACCGACACTTGTTTGCTCTGC和ATTACAGGCATGAGCCACCAT；所述的FCGR2A-chr1：161479745位点的PCR检测引物序列为：ACCCTTGGAATCTATCCTTACAACT和CAACAGCCTGACTACCTATTACCTG；所述的IFITM3-chr11：320772位点的PCR检测引物序列为：CCTCTTTCCTCCCTCTCCTA和GTTCCCTTCTCACTTTCTGG；所述的RPAIN-chr17：5326145位点的PCR检测引物序列为：CAGAGGCCAAAACTGAGATTT和GGCAGGATGGTAGAAGAAACA；所述的TLR3-chr4：187004074&187004217位点的PCR检测引物序列为：ATTTGTTTTCTCACTCTTTGCACG和TATGTTGGCTATGTTGTTGTTGCT。
3.如权利要求1所述的H7N9禽流感患病概率检测试剂盒，其特征在于，所述的CPT2-chr1：53676448&53676401位点的PCR退火温度为58±2℃，PCR产物长度为596bp。
4.如权利要求1所述的H7N9禽流感患病概率检测试剂盒，其特征在于，所述的CPT2-chr1：53679229&53679028位点的PCR退火温度为60±2℃，PCR产物长度为400bp。
5.如权利要求1所述的H7N9禽流感患病概率检测试剂盒，其特征在于，所述的FCGR2A-chr1：161479745位点的PCR退火温度为60±2℃，PCR产物长度为525bp。
6.如权利要求1所述的H7N9禽流感患病概率检测试剂盒，其特征在于，所述的IFITM3-chr11：320772位点的PCR退火温度为58±2℃，PCR产物长度为795bp。
7.如权利要求1所述的H7N9禽流感患病概率检测试剂盒，其特征在于，所述的RPAIN-chr17：5326145位点的PCR退火温度为58±2℃，PCR产物长度为758bp。
8.如权利要求1所述的H7N9禽流感患病概率检测试剂盒，其特征在于，所述的TLR3-chr4：187004074&187004217位点的PCR退火温度为60±2℃，PCR产物长度为629bp。
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| CN117448446A (zh) * | 2023-12-25 | 2024-01-26 | 广州嘉检医学检测有限公司 | Fcgr2a基因检测引物探针组合、试剂盒及其应用 |
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| CN109536600B (zh) * | 2018-12-17 | 2021-11-05 | 上海市疾病预防控制中心 | 基于ubxn11基因多态性位点预测a(h7n9)易感人群的用途及试剂盒及方法 |
| CN113945723A (zh) * | 2021-10-28 | 2022-01-18 | 复旦大学附属中山医院 | 预测免疫检查点抑制剂治疗相关肺炎发生风险的试剂盒、系统、储存介质及其应用 |
| CN113945723B (zh) * | 2021-10-28 | 2024-03-12 | 复旦大学附属中山医院 | 预测免疫检查点抑制剂治疗相关肺炎发生风险的系统、储存介质及其应用 |
| CN117448446A (zh) * | 2023-12-25 | 2024-01-26 | 广州嘉检医学检测有限公司 | Fcgr2a基因检测引物探针组合、试剂盒及其应用 |
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