CN108445212B - 一种用于检测艰难梭菌的胶体金试纸条和试剂盒 - Google Patents
一种用于检测艰难梭菌的胶体金试纸条和试剂盒 Download PDFInfo
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Abstract
本公开提供了一种用于检测艰难梭菌的胶体金试纸条,该胶体金试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,其中,所述C线上包被有dCas9抗体,所述T线上包被有tcdB基因片段‑牛血清白蛋白复合物;所述金标垫中含有胶体金标记的dCas9‑gRNA蛋白核酸复合物。本公开提供了一种用于检测艰难梭菌的试剂盒,其中,该试剂盒包括如上所述的用于检测艰难梭菌的胶体金试纸条。通过上述技术方案,本公开以dCas9‑gRNA与DNA特异识别的方式取代了常用的通过抗原抗体结合所开发的胶体金检测,有效提高了检测特异性及反应灵敏度。
Description
技术领域
本公开涉及生物医药领域,具体地,涉及一种用于检测艰难梭菌的胶体金试纸条和试剂盒。
背景技术
艰难梭菌是引起住院病人肠道感染的主要致病菌之一,是一种在医院和护理机构可引起致命性传染的耐药性细菌。美国疾控中心将其列为“美国最紧急抗药性威胁榜单”的榜首,临床上,约30%的抗菌药物相关性腹泻(antibiotic associated diarrhea,AAD)、50%~75%的抗菌药物相关性结肠炎和95%~100%的伪膜性肠炎(pseudomembranouscolitis,PMC)是由艰难梭菌感染(Clostridium difficile infection,CDI)引起。CDI主要是由产毒素艰难梭菌过度繁殖导致肠道菌群失调并释放毒素所引起,主要临床症状为发热、腹痛、水样便腹泻。严重者引发伪膜性肠炎,且常伴有中毒性巨结肠、肠穿孔、感染性休克等并发症,甚至最终导致死亡。
艰难梭菌产毒株主要分泌毒素A和毒素B,分别由毒素基因tcdA和tcdB编码。目前,已发现毒素A阴性、毒素B阳性的菌株;而毒素A阳性,毒素B阴性的菌株尚未被发现,认为毒素B可单独致病。另外,毒素B的毒力较毒素A强10倍。毒素B主要通过解聚肌动蛋白,破坏细胞骨架,导致细胞坏死,直接损伤肠壁细胞。
目前,粪便中艰难梭菌检测主要通过三步法进行:首先使用谷氨酸脱氢酶(GDH)试验进行初筛,GDH阳性进行毒素酶免疫方法(EIAs)试验,二者结果不一致使用细胞毒性试验(CCTA)、产毒素培养(TC)或核酸扩增试验(NAATs)确证。CCTA和TC目前是检测CDI的“金标准”,但因培养条件苛刻、技术要求高、操作复杂及耗时,难以用于临床艰难梭菌的常规检测。EIAs检测则特异、快速,但灵敏性差;GDH较为快速,但该方法存在交叉反应、一些无毒素艰难梭菌也可分泌出GDH因此已出现假阳性,检测费用高,需要成批检测以降低人员操作及试剂成本,但同时使得检测周期延长。国内外广泛应用的PCR检测方法则因实验仪器要求高、操作繁琐、耗时,扩增完成后需要通过电泳、显影等步骤对产物进行判别,不适合基层医院及现场快速检测的要求。因此,开发一款能够适用于临床疾控快速筛查要求,且灵敏性高、特异性强且价格低廉的艰难梭菌检测试剂盒方法对于疾病感染的控制和临床诊治显得尤其重要。
发明内容
本公开的目的是提供一种灵敏性高、特异性强且价格低廉的艰难梭菌检测试剂盒。
本公开的发明人出乎意料地发现:利用CRISPR/Cas9基因编辑技术中失去DNA切割活性的dCas9蛋白,通过dCas9蛋白在特定gRNA介导下与艰难梭菌毒素B基因的特异结合,就能够提供一种灵敏性高、特异性强、价格低廉且可在数分钟内对大量待测DNA样本进行筛查的艰难梭菌检测试剂盒及其制备方法,由此得到了本公开的技术方案。
为了实现上述目的,本公开提供一种用于检测艰难梭菌的胶体金试纸条,该胶体金试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,其中,所述C线上包被有dCas9抗体,所述T线上包被有tcdB基因片段-牛血清白蛋白复合物;所述金标垫中含有胶体金标记的dCas9-gRNA蛋白核酸复合物。
另一方面,本公开提供了一种用于检测艰难梭菌的试剂盒,其中,该试剂盒包括样品提取液以及如上所述的用于检测艰难梭菌的胶体金试纸条。
通过上述技术方案,本公开利用了dCas9-gRNA精确识别目标DNA序列的能力,筛选到特异靶向tcdB保守基因序列片段且亲和力强的gRNA,开创性的以dCas9-gRNA与DNA特异识别的方式取代了常用的通过抗原抗体结合所开发的胶体金检测,从而避免了因抗体灵敏性和特异性差异所导致的检测结果差异,对找不到抗体的艰难梭菌变种同样有效。采用合理配方对玻璃纤维膜进行预处理保证了dCas9-gRNA胶体金纸片dCas9-gRNA蛋白核酸复合体的稳定性,有效提高产品稳定性及反应灵敏度。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本公开的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本公开,但并不构成对本公开的限制。在附图中:
图1是胶体金试纸的组装结构示意图。
附图标记说明
1 样品垫 2 被衬底板 3 金标垫
4 T线 5 C线 6 基膜
7 吸水垫
具体实施方式
以下结合附图对本公开的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本公开,并不用于限制本公开。
本公开提供了一种用于检测艰难梭菌的胶体金试纸条,该胶体金试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,其中,所述C线上包被有dCas9抗体,所述T线上包被有tcdB基因片段-牛血清白蛋白偶联物;所述金标垫中含有胶体金标记的dCas9-gRNA蛋白核酸复合物。
其中,tcdB基因片段-牛血清白蛋白复合物的制备方法为将合成的tcdB基因片段400-600ng加入8-12wt%牛血清白蛋白中室温孵育30分钟,即得tcdB基因片段-牛血清白蛋白复合物。
其中,dCas9-gRNA蛋白核酸复合物的制备方法可以为将dCas9蛋白与体外转录获得的gRNA在含有3mol/L的NaAc且pH为5.2的缓冲液中37℃下孵育60分钟,即得dCas9-gRNA复合体。
可选地,其中,所述dCas9-gRNA蛋白核酸复合物中gRNA序列为CATATACAATTGAGACTGGA,如SEQ ID NO.1所示。
可选地,其中,所述dCas9-gRNA蛋白核酸复合物中dCas9的氨基酸序列为DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD,如SEQID NO.2所示。
可选地,其中,所述tcdB基因片段-牛血清白蛋白偶联物中,tcdB基因片段序列为GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGT ATATTATTTTGGAGAAACATATACAATTGAGACTGGATGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG,如SEQ IDNO.3所示。
可选地,其中,相对于1cm所述T线,tcdB基因片段的DNA量为400-600ng,牛血清白蛋白的量为400-600ng。
可选地,其中,dCas9抗体为购自Abcam的货号为Ab204448的抗体。
可选地,其中,所述金标垫中所含有的胶体金标记的dCas9-gRNA蛋白核酸复合物是由3-8μg/mL胶体金标记的胶体金标记的dCas9-gRNA蛋白核酸复合物溶液以0.5-5μL/cm的喷涂速度通过喷金点膜机喷涂在金标垫上干燥后得到的;所述胶体金标记的dCas9-gRNA蛋白核酸复合物溶液中,胶体金的含量为3-8μg/mL,dCas9蛋白的含量为10-50ng/mL,gRNA核酸的含量为10-50ng/mL。
可选地,其中,所述T线上包被的tcdB基因片段-牛血清白蛋白复合物是由20-50mg/mL的tcdB基因片段-牛血清白蛋白偶联物溶液以1-10μL/cm的喷涂速度通过喷金点膜机喷涂在T线上干燥后得到的;所述tcdB基因片段-牛血清白蛋白偶联物溶液中,tcdB基因片段的含量为20-50ng/mL,牛血清白蛋白的含量为0.1-0.3w/v%。
可选地,其中,所述C线上包被的dCas9抗体是由0.1-5mg/mL的dCas9抗体溶液以1-10μL/cm的喷涂速度通过喷金点膜机喷涂在C线上干燥后得到的。
本公开还提供了一种用于检测艰难梭菌的试剂盒,其中,该试剂盒包括样品提取液以及如上所述的用于检测艰难梭菌的胶体金试纸条。
可选地,所述样品提取液的成分可以为2.5-3.5mol/L的NaAc溶液。提取的操作可以包括:在沸水浴中煮沸30分钟。
本发明中,将单克隆抗体进行胶体金标记的方法可以为本领域常规的方法,例如可以包括:将0.01重量%的HAuCl4溶液加热煮沸后加入1重量%的柠檬酸三钠溶液直至溶液的颜色完全变为透明的红色时,继续回流10min后停止加热,冷却至室温,即得到胶体金;取1mL制取好的胶体金,用1重量%的K2CO3调节pH值到8.0,加入15μg的dCas9-gRNA蛋白核酸复合物,混合均匀,室温反应40min;加入5重量%的BSA至终浓度为0.1重量%,静置30min;先用低速(1500×g)离心15分钟,弃去由凝聚的金胶粒形成的沉淀;然后用10000×g离心30分钟;仔细吸出上清,沉淀物用0.1mL含1%BSA的0.1M PBS(pH7.4)复溶,加入5%叠氮钠至终浓度为0.05%,4℃保存。
本发明中,制备免疫胶体金试纸的方法可以为本领域常规的方法,例如可以包括:用喷金点膜机将第一单克隆抗体和羊抗鼠IgG喷于硝酸纤维素膜上,形成相互平行的检测T线和质控C线,然后烘干;将胶体金标记的第二单克隆抗体用喷金点膜机均匀的喷在金标垫上;然后将样品垫、金标垫、喷有T线和C线的基膜(硝酸纤维素膜)和吸水纸依次连接组装,而后裁切包装备用。
可选地,C线和T线的间距为2.0-7.0mm。
以下通过实施例进一步详细说明本发明。
以下实施例中所用的试剂均为商购得到,例如:dCas9蛋白购自上海近岸科技有限公司,货号为E368-01A;dCas9抗体购自Abcam,货号为Ab204448,tcdB基因片段和gRNA由生工生物工程(上海)股份有限公司合成,RNA酶抑制剂购自上海近岸科技有限公司,gRNA体外转录试剂盒购自北京英茂盛业生物科技有限公司。硝酸纤维素膜、玻璃纤维膜:购自德国赛多利斯公司(SARTORIUS)。
实施例1
1.两步还原法制备胶体金
a)氯金酸溶液第一次还原:将6ml 0.0164mol/L的HAuCL4水溶液加入200ml双蒸水中,煮沸30分钟,缓慢搅拌并加入50ml 0.016mol/L柠檬酸三钠溶液。以30kHZ的频率超声振荡2分钟,冷却至室温,得到15nm粒径的胶体金原核溶液。
b)氯金酸溶液第二次还原:取第一次还原后得到的胶体金原核溶液26ml在4℃条件下,加入4℃预冷后的0.035mol/L的HAuCL4溶液,缓慢搅拌并以每秒1-2滴的速度滴加4℃预冷后的0.018mol/L的抗坏血酸与0.138g/L PVP混合液,反应1小时至溶液呈现透明酒红色,即得到40nm粒径的胶体金溶液。
2.制备胶体金包被的dCas9-gRNA
a)gRNA体外转录
合成体外转录所需引物,序列如下:5’-TTAATACGACTCACTATAGGG CATATACAATTGA GACTGGAGTTTTAGAGCTAGAAATAG-3’(SEQ ID NO.4),按照gRNA体外转录试剂盒中的操作步骤对gRNA进行体外转录。
b)制备dCas9-gRNA复合体
将dCas9蛋白与体外转录获得的gRNA在3M NaAc pH5.2缓冲液中37℃孵育60分钟,即得dCas9-gRNA复合体,孵育过程中务必采用无RNA酶的耗材进行实验,注意防止RNA酶污染。
3.dCas9-gRNA胶体金预处理
a)将获得的dCas9-gRNA复合体用0.1M pH7.8的磷酸盐缓冲液稀释至浓度1mg/ml。
b)将1000ml胶体金溶液与含有500u/ml RNA酶抑制剂的100ml0.1M pH7.8的磷酸盐缓冲液混合,快速搅拌3分钟。之后,以每秒1-2滴的速度滴加稀释后的dCas9-gRNA复合体溶液8ml,缓慢搅拌室温反应5分钟。
c)在上述反应液中快速加入20ml 10wt%的牛血清白蛋白溶液,缓慢搅拌室温反应5分钟。
d)将获得的溶液8000rpm/min离心20分钟,取沉淀,并收集上清,将上清12500rpm/min离心30分钟,取沉淀。将两次沉淀合并用含有0.1wt%BSA的硼酸缓冲液复溶到OD540值为14。
4.dCas9-gRNA胶体金纸制备
a)喷金缓冲液制备:在800ml双蒸水中加入100ml 1.0M Tris溶液,调pH至8.5。在溶液中加入3g聚乙二醇20000、2g牛血清白蛋白,2g脱脂牛奶,3g酪蛋白和0.5g叠氮钠,充分溶解,至总体积1000ml。
b)用喷金缓冲液稀释dCas9-gRNA胶体金,至溶液OD540值为2。
c)取7ml Tween-20,160g蔗糖,用双蒸水定容至1L,配置成玻璃纤维膜预处理液。用每30ml预处理液浸泡玻璃纤维膜261mm*220mm 30分钟后,至37℃干燥;再用OD540值为2的dCas9-gRNA胶体金溶液喷涂玻璃纤维膜,每261mm*220mm的玻璃纤维膜上喷涂20ml,干燥,制得dCas9-gRNA胶体金纸。
5.含检测线及质控线硝酸纤维素膜的制备
将dCAS9抗体用磷酸盐缓冲液稀释成0.5mg/ml,制得质控线(C线)溶液。
tcdB基因片段-牛血清白蛋白偶联物的制备方法为将合成的tcdB基因片段400-600ng加入10wt%牛血清白蛋白中室温孵育30分钟,即得tcdB基因片段-牛血清白蛋白复合物;将tcdB基因片段-牛血清白蛋白偶联物用磷酸盐缓冲液稀释成0.5mg/ml,tcdB基因片段DNA含量为500ng/ml,制得检测线(T线)溶液。
a)用点膜机喷点C、T线溶液,每1m长硝酸纤维素膜分别包被有1ml的C线和T线溶液,C线和T线间距6mm。
将滤样纸、dCas9-gRNA胶体金纸片、硝酸纤维素膜、吸水纸依次黏贴在胶板上,切割成宽度为4mm的试剂条。
实施例2
按照实施例1的方法制备胶体金试纸条,不同的是,gRNA序列更改为TATAGTGGTTTAGTTAGAGT(SEQ ID NO.5)。
测试实施例1
(1)胶体金试纸条特异性实验:
交叉实验检测样品为分别含有下列常见肠道细菌DNA浓度为200ng/ml的磷酸盐溶液:布氏瘤胃球菌、双歧杆菌、黄化瘤胃球菌、史密斯甲烷短杆菌、沙门氏菌、志贺氏菌感染、弯曲杆菌。
检测方法:将检测试剂条置于待测交叉反应实验的样品中吸取样品约150ul,5-8分钟后判读结果。每种菌检测3次。
表1特异性实验结果
菌种 | 实施例1制备的试纸条 | 实施例2制备的试纸条 |
布氏瘤胃球菌 | 阴性 | 阴性 |
双歧杆菌 | 阴性 | 阴性 |
黄化瘤胃球菌 | 阴性 | 阴性 |
史密斯甲烷短杆菌 | 阴性 | 阴性/阳性 |
沙门氏菌 | 阴性 | 阴性 |
志贺氏菌感染 | 阴性 | 阴性 |
弯曲杆菌 | 阴性 | 阴性/阳性 |
实验结果如表1所示,采用实施例1方法制备的试纸条在上述肠道细菌DNA浓度等于或小于200ng/ml时不会被检出,采用实施例1方法制备的试纸条的特异性显著大于采用实施例2方法制备的试纸条。
(2)试剂盒灵敏度检测:
检测样品:配制含有艰难梭菌基因组DNA浓度为50ng/ml、100ng/ml、120ng/ml、150ng/ml、180ng/ml、200ng/ml样品。
检测方法:将检测试剂条置于待测样品中吸取样品约150ul,5-8分钟后判读结果,每个浓度检测3次。
表2灵敏度检测结果
实验结果如表2所示,采用实施例1方法制备的试纸条的检出灵敏度为100ng/ml显著大于采用实施例2方法制备的试纸条的检出灵敏度150ng/ml。
(3)试剂盒批间差异检测
检测样品:将含有艰难梭菌基因组DNA浓度为200ng/ml、500ng/ml、1000ng/ml的样品分别标号为1,2,3;将不含艰难梭菌基因组DNA的溶液4份分别标号为4,5,6,7。
检测方法:将不同标号的待测样品,用三个不同批次的检测试剂盒进行检测,每个样品检测3次,比较待测样品总体阳性率检测及重复性。
表3
实验结果如表3所示,采用实施例1方法制备的试纸条批间差异优于采用实施例2方法制备的试纸条。
以上结合附图详细描述了本公开的优选实施方式,但是,本公开并不限于上述实施方式中的具体细节,在本公开的技术构思范围内,可以对本公开的技术方案进行多种简单变型,这些简单变型均属于本公开的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本公开对各种可能的组合方式不再另行说明。
此外,本公开的各种不同的实施方式之间也可以进行任意组合,只要其不违背本公开的思想,其同样应当视为本公开所公开的内容。
序列表
<110> 杭州观梓健康科技有限公司
<120> 一种用于检测艰难梭菌的胶体金试纸条和试剂盒
<130> 9427-K-HZGZ
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
catatacaat tgagactgga 20
<210> 2
<211> 1367
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
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Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
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Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
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Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1010 1015 1020
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn
1025 1030 1035 1040
Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
1045 1050 1055
Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val
1060 1065 1070
Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met
1075 1080 1085
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1090 1095 1100
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala
1105 1110 1115 1120
Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1125 1130 1135
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys
1140 1145 1150
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1155 1160 1165
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys
1170 1175 1180
Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1185 1190 1195 1200
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1205 1210 1215
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1250 1255 1260
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
1265 1270 1275 1280
Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
1285 1290 1295
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
1300 1305 1310
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
1315 1320 1325
Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
1330 1335 1340
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
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Leu Ser Gln Leu Gly Gly Asp
1365
<210> 3
<211> 143
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcagttgaat atagtggttt agttagagtt ggtgaagatg tatattattt tggagaaaca 60
tatacaattg agactggatg gatatatgat atggaaaatg aaagtgataa atattatttc 120
aatccagaaa ctaaaaaagc atg 143
<210> 4
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttaatacgac tcactatagg gcatatacaa ttgagactgg agttttagag ctagaaatag 60
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tatagtggtt tagttagagt 20
Claims (7)
1.一种用于检测艰难梭菌的胶体金试纸条,该胶体金试纸条包括依次相连接的吸水垫、基膜、金标垫和样品垫;所述基膜上设置有C线和T线,其特征在于,所述C线上包被有dCas9抗体,所述T线上包被有tcdB基因片段-牛血清白蛋白复合物;所述金标垫中含有胶体金标记的dCas9-gRNA蛋白核酸复合物;所述dCas9-gRNA蛋白核酸复合物中gRNA序列为CATATACAATTGAGACTGGA;所述dCas9-gRNA蛋白核酸复合物中dCas9的氨基酸序列为SEQ IDNO.2;所述tcdB基因片段-牛血清白蛋白复合物中,tcdB基因片段序列为GCAGTTGAATATAGTGGTTTAGTTAGAGTTGGTGAAGATGTATATTATTTTGGAGAAACATATACAATTGAGACTGGATGGATATATGATATGGAAAATGAAAGTGATAAATATTATTTCAATCCAGAAACTAAAAAAGCATG。
2.根据权利要求1所述的胶体金试纸条,其中,相对于1cm所述T线,tcdB基因片段的DNA量为400-600ng,牛血清白蛋白的量为0.1-0.3w/v%。
3.根据权利要求1所述的胶体金试纸条,其中,dCas9抗体为购自Abcam的货号为Ab204448的抗体。
4.根据权利要求1所述的胶体金试纸条,其中,所述金标垫中所含有的胶体金标记的dCas9-gRNA蛋白核酸复合物是由胶体金标记的dCas9-gRNA蛋白核酸复合物溶液以0.5-5μL/cm的喷涂速度通过喷金点膜机喷涂在金标垫上干燥后得到的;所述胶体金标记的dCas9-gRNA蛋白核酸复合物溶液中,胶体金的含量为3-8μg/mL,dCas9蛋白的含量为10-50ng/mL,gRNA核酸的含量为10-50ng/mL。
5.根据权利要求4所述的胶体金试纸条,其中,所述T线上包被的tcdB基因片段-牛血清白蛋白复合物是由tcdB基因片段-牛血清白蛋白偶联物溶液以1-10μL/cm喷涂速度通过喷金点膜机喷涂在T线上干燥后得到的;所述tcdB基因片段-牛血清白蛋白偶联物溶液中,tcdB基因片段的含量为20-50ng/mL,牛血清白蛋白的含量为0.1-0.3w/v%。
6.根据权利要求5所述的胶体金试纸条,其中,所述C线上包被的dCas9抗体是由0.1-5mg/mL的dCas9抗体溶液以1-10μL/cm的喷涂速度通过喷金点膜机喷涂在C线上干燥后得到的。
7.一种用于检测艰难梭菌的试剂盒,其特征在于,该试剂盒包括样品提取液以及权利要求1-6中任意一项所述的用于检测艰难梭菌的胶体金试纸条。
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WO2013049214A1 (en) * | 2011-09-26 | 2013-04-04 | Techlab, Inc. | Cysteine protease cwp84 (cd2728) as a diagnostic marker for clostridium difficile |
EP2841593A1 (en) * | 2012-04-26 | 2015-03-04 | TechLab, Inc. | Clostridium difficile dehydrogenase and toxin as a biomarker |
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