一种核酸检测试剂盒、检测方法及应用与流程

本发明属于生物
技术领域：
，特别涉及一种包括cpf1检测体系的核酸检测试剂盒、检测方法。本发明还涉及所述核酸检测试剂盒的应用，特别是在严重急性呼吸综合征相关冠状病毒和中东呼吸综合征冠状病毒的核酸的快速检测中的应用。
背景技术：
：冠状病毒属于套式病毒目nidovirales，冠状病毒科coronaviridae，冠状病毒属coronavirus[6]。成熟的冠状病毒表面有囊膜，囊膜表面覆有纤突，纤突末端呈球形，故整个纤突呈花瓣状或梨状，纤突之间有较宽的间隙。电镜下，囊膜纤突规则地排列成王冠状，病毒颗粒因此而得名。目前尚无针对高度传染性的冠状病毒的有效疫苗和治疗药物，及时诊断和隔离是控制该病暴发流行的有效方法。快速、高效的核酸快速检测是临床上病毒性疾病防控的有效方法，针对病毒性疾病现有的检测方法多以核酸检测为主，who发布的严重急性呼吸系统综合症(severeacuterespiratorysyndrome，缩写为sars)和中东呼吸综合征冠状病毒(middleeastrespiratorysyndromecoronavirus，mers-cov)的实验室确诊程序指导文件中推荐使用实时反转录聚合酶链式反应(realtimereversetranscriptionpcr，rrt-pcr)方法对病毒进行检测确诊。rrt-pcr检测病毒核酸主要受制于：昂贵的pcr实验设备、能进行熟练稳定操作的工作人员人数、检测时间较长等。免疫学检测方法也是病毒检测的重要手段，如双抗体夹心elisa、胶体金免疫层析试纸等方法。免疫学检查容易受到病程的影响，早期病人体内抗体较少，需要等待一定的时间才可以产生相应的抗体，容易出现漏诊；患病后的康复病人体内可能携带该类抗体，容易导致免疫诊断出现假阳性。为了有效的控制病毒性传染疾病的传播，需要建立一种简便、快速、特异的新型检测方法。crispr-cas(clusteredregularlyinterspacedshortpalindromicrepeats，crisprs)是细菌中的一种适应性免疫系统，cas蛋白通过rna引导的核酸酶靶向降解外来核酸[1,2]。其中，crispr-cpf1(cpf1)属于cas酶第二家族，用来引导rna指导双链dna裂解单个ruvc催化结构域。cpf1酶识别富含胸腺嘧啶(thymine，t)核苷酸的间隔区相邻基序(pam)[3]，催化它们自身的指导crisprrna(crrna)成熟[4]，并特异性识别和切割互补配对的双链dna(dsdna)[3]。当crispr/cpf1蛋白以序列特异性方式识别切割靶双链dna时，能诱导强大的非特异性单链dna(ssdna)反式切割活性[5]。cpf1是具有核酸内切酶活性，核酸内切酶的配体离子通常为镁离子。现有以cpf1为基础的核酸检测，信号强度仍旧不高，从而导致在实际检测中的检测时间过长，低浓度样品检测效率低等缺陷。因此还需要进一步增强检测信号的强度以满足实际检测的需要。此外，在检测核酸时的灵敏度、特异性和稳定性也迫切需要提高。技术实现要素：本发明所要解决的技术问题是为了克服现有技术中检测核酸时成本过高、检测试剂较长、检测效率低、特异性和灵敏性不够高等缺陷，提供了一种核酸检测试剂盒、一种突变的cpf1蛋白、一种crrna、检测方法及应用。利用本发明所述的核酸检测试剂盒、突变的cpf1蛋白或crrna检测核酸(例如检测sars相关冠状病毒或mers冠状病毒的核酸)时，可以显著提高检测时的信号强度从而减少检测时间、提高检测效率，且灵敏度高、特异性高、准确性高、检测可视化、成本低、无需复杂大型实验设备、操作简便。cpf1是具有核酸内切酶活性，而在本领域中核酸内切酶的配体离子通常为镁离子。而本发明人首次发现锰离子能够促进cpf1对核酸探针(例如单链dna探针)的切割能力，进而提高cpf1检测的信号强度，从而可用于增强cpf1介导的体外检测的信号以减少检测时间、提高检测效率。此外，本发明还通过大量实验，意外发现当将某些cpf1蛋白进行特定突变后，也能够显著提高检测核酸时的信号强度。本发明人还首次采用cpf1荧光法检测sars相关冠状病毒和mers冠状病毒，经过大量实验摸索设计出能够用于检测这些病毒的crrna，将其用于检测病毒时灵敏度和特异性非常高，且准确性也很高。为了解决上述技术问题，本发明第一方面提供了一种核酸检测试剂盒，其包括cpf1检测体系，所述cpf1检测体系包括：向导rna、cpf1蛋白、核酸探针和含锰离子的溶液。较佳地，所述含锰离子的溶液为硫酸锰、氯化锰或醋酸锰溶液。较佳地，所述核酸探针为单链dna探针，所述单链dna探针优选包括荧光标记，更优选在其5’端和3’端分别具有荧光基团和荧光淬灭基团，进一步更优选为在其5’端具有荧光基团和3’端具有荧光淬灭基团。其中，所述荧光基团可以为本领域常规，例如为6-fam、tet、cy3、cy5或rox。所述荧光淬灭基团可以为本领域常规，例如为bhq1、bhq2或bhq3。所述单链dna探针的序列按照本领域常规方法进行制备，例如可以为tttattt。较佳地，所述cpf1蛋白选自ascpf1(acidaminococcussp.bv3l6)、bbcpf1(beauveriabassianaka00251)、bocpf1(bacteroidetesoral)、fncpf1(francisellanovicidau112)、hkcpf1(helcococcuskunzii)、lb4cpf1(lachnospiraceaebacteriummc2017)、lb5cpf1(lachnospiraceaebacteriumnc2008)、lbcpf1(lachnospiraceaebacteriumnd2006)、oscpf1(oribacteriumsp.)和tscpf1(thiomicrospirasp.xs5)中的一种或多种。较佳地，所述cpf1蛋白为经过密码子优化的cpf1蛋白，其核苷酸序列优选如seqidno.14-23中的一个或多个所示。较佳地，所述cpf1蛋白为突变的cpf1蛋白，所述突变的cpf1蛋白优选其氨基酸序列与天然cpf1蛋白具有98％优选99％以上的序列同源性。更佳地，所述突变的cpf1蛋白的序列包括将如seqidno.55所示的k180、e184、n607和k613中的一个或多个位点突变为r后的序列，或将如seqidno.54所示的t148、t152、g532和k538中的一个或多个位点突变为r后的序列。例如可以是将如seqidno.55所示的e184和n607突变为r后的序列，可以是将如seqidno.55所示的e184、n607和k613突变为r后的序列，可以是将如seqidno.55所示的k180、e184、n607和k613突变为r后的序列，可以是将如seqidno.54所示的t152和g532突变为r后的序列，可以是将如seqidno.54所示的t152、g532和k538突变为r后的序列，可以是将如seqidno.54所示的t148、t152、g532和k538突变为r后的序列。进一步更佳地，所述突变的cpf1蛋白的核苷酸序列如seqidno.30-41所示。较佳地，所述向导rna为引导cpf1蛋白特异性结合所述核酸的rna，优选为crrna；所述crrna优选为检测病毒核酸的crrna；更优选为检测sars相关冠状病毒或mers-cov病毒的crrna；所述sars相关冠状病毒优选为冠状病毒sars-cov(严重急性呼吸综合征冠状病毒)或sars-cov-2(严重急性呼吸综合征冠状病毒2型)。更佳地，所述crrna为检测sars-cov病毒orf1ab基因的crrna、检测mers-cov病毒orf1a基因的crrna、检测mers-cov病毒upe基因的crrna、检测sars-cov-2病毒e基因的crrna、检测sars-cov-2病毒s基因的crrna、检测sars-cov-2病毒m基因的crrna和/或检测sars-cov-2病毒n基因的crrna，所述orf1ab基因的核苷酸序列如seqidno.11所示，所述orf1a基因的核苷酸序列如seqidno.12所示，所述upe基因的核苷酸序列如seqidno.13所示，所述e基因的核苷酸序列如seqidno.46所示，所述sars-cov-2-s基因的核苷酸序列如seqidno.79所示；所述sars-cov-2-m基因的核苷酸序列如seqidno.80所示；所述sars-cov-2-n基因的核苷酸序列如seqidno.81所示。进一步更佳地，所述crrna的核苷酸序列优选如seqidno.1-10、seqidno.47、seqidno.48和seqidno.53、seqidno.56-72中的一个或多个所示。较佳地，所述含锰离子的溶液中锰离子的浓度为5-600mm，例如6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500mm等。所列点值仅为示例，并不是限制于此。并且应当理解的是，在这浓度范围内的任意点值均可以自由组合，从而任意组合所行成的浓度范围均应该在本发明保护的范围内。例如5-50、5-100、5-200、5-300、5-400、5-500、100-200、100-300、200-300等等这些均应该在本发明的保护范围内。在实验中发明人发现锰离子的工作浓度在0.5-60mm都有效果，试剂盒中一般是混合的10倍的储液，故储液浓度可以是5-600mm。在本发明某一较佳实施例中，所用的含锰离子的溶液中锰离子的浓度为100mm。较佳地，所述核酸探针的浓度为1-100pmol/μl；优选25pmol/μl。较佳地，所述cpf1蛋白的浓度为20-1000ng/μl；优选200ng/μl。较佳地，所述向导rna的浓度为0.1-50μm；优选1μm。较佳地，所述核酸检测试剂盒还包括rna酶抑制剂和缓冲液；所述rna酶抑制剂的浓度优选为10-200u/μl，优选40u/μl。较佳地，所述缓冲液优选包括nacl、tris和bsa，所述缓冲液的ph优选7.9。在本发明某一较佳实施例中，所用缓冲液为10×缓冲液，其组成为：1000mmnacl，500mmtris，1000ug/mlbsa，ph7.9。为了解决上述技术问题，本发明第二方面提供了一种突变的cpf1蛋白，其序列包括将seqidno.55所示的k180、e184、n607和k613中的一个或多个位点突变为r后的序列；或者，其序列包括将seqidno.54所示的t148、t152、g532和k538中的一个或多个位点突变为r后的序列。较佳地，所述突变的cpf1蛋白的序列包括将如seqidno.55所示的e184和n607突变为r后的序列。较佳地，所述突变的cpf1蛋白的序列包括将如seqidno.55所示的e184、n607和k613突变为r后的序列。较佳地，所述突变的cpf1蛋白的序列包括将如seqidno.55所示的k180、e184、n607和k613突变为r后的序列。较佳地，所述突变的cpf1蛋白的序列包括将如seqidno.54所示的t152和g532突变为r后的序列。较佳地，所述突变的cpf1蛋白的序列包括将如seqidno.54所示的t152、g532和k538突变为r后的序列。较佳地，所述突变的cpf1蛋白的序列包括将如seqidno.54所示的或t148、t152、g532和k538突变为r后的序列。更佳地，所述突变的cpf1蛋白的核苷酸序列如seqidno.30-41所示。为了解决上述技术问题，本发明第三方面提供了一种核酸检测试剂盒，其包括cpf1检测体系，所述cpf1检测体系包括：向导rna、核酸探针和如本发明第二方面所述的突变的cpf1蛋白。较佳地，所述向导rna为引导cpf1蛋白特异性结合所述核酸的rna，优选为crrna；所述crrna优选为检测病毒核酸的crrna；更优选为检测sars相关冠状病毒或mers-cov病毒的crrna；所述sars相关冠状病毒优选为冠状病毒sars-cov或sars-cov-2；所述crrna优选为检测sars-cov病毒orf1ab基因的crrna、检测mers-cov病毒orf1a基因的crrna、检测mers-cov病毒upe基因的crrna、检测sars-cov-2病毒e基因的crrna、检测sars-cov-2病毒s基因的crrna、检测sars-cov-2病毒m基因的crrna和/或检测sars-cov-2病毒n基因的crrna，所述orf1ab基因的核苷酸序列如seqidno.11所示，所述orf1a基因的核苷酸序列如seqidno.12所示，所述upe基因的核苷酸序列如seqidno.13所示，所述e基因的核苷酸序列如seqidno.46所示，所述sars-cov-2-s基因的核苷酸序列如seqidno.79所示；所述sars-cov-2-m基因的核苷酸序列如seqidno.80所示；所述sars-cov-2-n基因的核苷酸序列如seqidno.81所示。更佳地，所述crrna的核苷酸序列如seqidno.1-10、seqidno.47、seqidno.48和seqidno.53、seqidno.56-72中的一个或多个所示。较佳地，所述核酸探针为单链dna探针，所述单链dna探针优选包括荧光标记，更优选为在其两端分别具有荧光基团和荧光淬灭基团，进一步更优选为在5’端具有荧光基团和3’端具有荧光淬灭基团。所述荧光基团可为本领域常规，例如为6-fam、tet、cy3、cy5或rox。所述荧光淬灭基团可为本领域常规，例如为bhq1、bhq2或bhq3。所述单链dna探针可按本领域常规方法进行制备，其序列例如可以为tttattt。较佳地，所述向导rna的浓度为0.1-50μm；优选1μm。较佳地，所述突变的cpf1蛋白的浓度为20-1000ng/μl；优选200ng/μl。较佳地，所述核酸探针的浓度为1-100pmol/μl；优选25pmol/μl。较佳地，所述核酸检测试剂盒还包括含金属离子的溶液，例如含镁离子的溶液、含锰离子的溶液等；所述溶液中金属离子的浓度优选为5-600mm，例如6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500mm等。所列点值仅为示例，并不是限制于此。并且应当理解的是，在这浓度范围内的任意点值均可以自由组合，从而任意组合所行成的浓度范围均应该在本发明保护的范围内。例如5-50、5-100、5-200、5-300、5-400、5-500、100-200、100-300、200-300等等这些均应该在本发明的保护范围内。在实验中发明人发现金属离子的工作浓度在0.5-60mm都有效果，试剂盒中一般是混合的10倍的储液，故储液浓度可以是5-600mm。在本发明某一较佳实施例中，所用的含金属离子的溶液中金属离子的浓度为100mm。较佳地，所述核酸检测试剂盒还包括rna酶抑制剂和缓冲液；所述rna酶抑制剂的浓度优选为10-200u/μl，优选40u/μl。较佳地，所述缓冲液优选包括nacl、tris和bsa，所述缓冲液的ph优选7.9。在本发明某一较佳实施例中，所用缓冲液为10×缓冲液，其组成为：1000mmnacl，500mmtris，1000ug/mlbsa，ph7.9。为了解决上述技术问题，本发明第四方面提供了一种检测冠状病毒核酸的crrna，所述crrna为检测sars-cov病毒orf1ab基因的crrna、检测mers-cov病毒orf1a基因的crrna、检测mers-cov病毒upe基因的crrna、检测sars-cov-2病毒e基因的crrna、检测sars-cov-2病毒s基因的crrna、检测sars-cov-2病毒m基因的crrna和/或检测sars-cov-2病毒n基因的crrna，所述orf1ab基因的核苷酸序列如seqidno.11所示，所述orf1a基因的核苷酸序列如seqidno.12所示，所述upe基因的核苷酸序列如seqidno.13所示，所述e基因的核苷酸序列如seqidno.46所示，所述sars-cov-2-s基因的核苷酸序列如seqidno.79所示；所述sars-cov-2-m基因的核苷酸序列如seqidno.80所示；所述sars-cov-2-n基因的核苷酸序列如seqidno.81所示。较佳地，所述crrna为检测sars-cov病毒orf1ab基因的crrna的核苷酸序列如seqidno.1-3中的一个或多个所示。较佳地，所述检测mers-cov病毒orf1a基因的crrna的核苷酸序列如seqidno.4-7中的一个或多个所示。较佳地，所述检测mers-cov病毒upe基因的crrna的核苷酸序列如seqidno.8-10中的一个或多个所示。较佳地，所述检测sars-cov-2病毒e基因的crrna的核苷酸序列如seqidno.47、seqidno.48和seqidno.53中的一个或多个所示。较佳地，所述检测sars-cov-2病毒s基因的crrna的核苷酸序列如seqidno.56-63中的一个或多个所示。较佳地，所述检测sars-cov-2病毒m基因的crrna的核苷酸序列如seqidno.64-68中的一个或多个所示。较佳地，所述检测sars-cov-2病毒n基因的crrna的核苷酸序列如seqidno.69-72中的一个或多个所示。在本发明某一较佳实施例中，所述crrna的制备方法包括：针对sars病毒核酸orf1ab基因，其序列为seqidno.11，寻找包含cpf1识别序列(pam)tttn的靶向序列，设计44bp长度的crrna。针对mers病毒核酸upe基因，其序列为seqidno.12，寻找包含cpf1识别序列(pam)tttn的靶向序列，设计44bp长度的crrna。针对mers病毒核酸orf1基因，其序列为seqidno.13，寻找包含cpf1识别序列(pam)tttn的靶向序列，设计44bp长度的crrna。针对sars-cov-2病毒e基因，其序列为seqidno.46，寻找包含cpf1识别序列(pam)tttn的靶向序列，设计44bp长度的crrna。针对sars-cov-2病毒s基因，其序列为seqidno.79，寻找包含cpf1识别序列(pam)tttn的靶向序列，设计44bp长度的crrna。针对sars-cov-2病毒m基因，其序列为seqidno.80，寻找包含cpf1识别序列(pam)tttn的靶向序列，设计44bp长度的crrna。针对sars-cov-2病毒n基因，其序列为seqidno.81，寻找包含cpf1识别序列(pam)tttn的靶向序列，设计44bp长度的crrna。设计完成后，制备crrna。crrna的制备，可构建到载体puc57-t7-crrna，通过体外转录获得目的crrna；或可公司合成rna序列。为了解决上述技术问题，本发明第五方面提供了一种核酸检测试剂盒，其包括cpf1检测体系，所述cpf1检测体系包括：如本发明第四方面所述的crrna、cpf1蛋白和核酸探针。较佳地，所述cpf1蛋白选自ascpf1(acidaminococcussp.bv3l6)、bbcpf1(beauveriabassianaka00251)、bocpf1(bacteroidetesoral)、fncpf1(francisellanovicidau112)、hkcpf1(helcococcuskunzii)、lb4cpf1(lachnospiraceaebacteriummc2017)、lb5cpf1(lachnospiraceaebacteriumnc2008)、lbcpf1(lachnospiraceaebacteriumnd2006)、oscpf1(oribacteriumsp.)和tscpf1(thiomicrospirasp.xs5)、或与其氨基酸序列具有98％优选99％以上序列同源性的一种或多种。较佳地，所述cpf1蛋白为经过密码子优化的cpf1蛋白，其核苷酸序列优选如seqidno.14-23中的一个或多个所示。较佳地，所述cpf1蛋白为突变的cpf1蛋白，所述突变的cpf1蛋白优选其氨基酸序列与天然cpf1蛋白具有98％优选99％以上的序列同源性。更佳地，所述突变的cpf1蛋白的序列包括将如seqidno.55所示的k180、e184、n607和k613中的一个或多个位点突变为r后的序列，或将如seqidno.54所示的t148、t152、g532和k538中的一个或多个位点突变为r后的序列。例如可以是将如seqidno.55所示的e184和n607突变为r后的序列，可以是将如seqidno.55所示的e184、n607和k613突变为r后的序列，可以是将如seqidno.55所示的k180、e184、n607和k613突变为r后的序列，可以是将如seqidno.54所示的t152和g532突变为r后的序列，可以是将如seqidno.54所示的t152、g532和k538突变为r后的序列，可以是将如seqidno.54所示的t148、t152、g532和k538突变为r后的序列。进一步更佳地，所述突变的cpf1蛋白的核苷酸序列如seqidno.30-41所示。较佳地，所述核酸探针为单链dna探针，所述单链dna探针优选包括荧光标记，更优选为在其两端分别具有荧光基团和荧光淬灭基团，进一步更优选为在5’端具有荧光基团和3’端具有荧光淬灭基团。所述荧光基团可以为本领域常规，例如为6-fam、tet、cy3、cy5或rox。所述荧光淬灭基团可以为本领域常规，例如为bhq1、bhq2或bhq3。所述单链dna探针可按本领域常规方法制备，其序列例如可以为tttattt。较佳地，所述crrna的浓度为0.1-50μm；优选1μm。较佳地，所述cpf1蛋白的浓度为20-1000ng/μl；优选200ng/μl。较佳地，所述核酸探针的浓度为1-100pmol/μl；优选25pmol/μl。较佳地，所述核酸检测试剂盒还包括含金属离子的溶液，例如含镁离子的溶液、含锰离子的溶液等；所述溶液中金属离子的浓度优选为5-600mm，例如6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500mm等。所列点值仅为示例，并不是限制于此。并且应当理解的是，在这浓度范围内的任意点值均可以自由组合，从而任意组合所行成的浓度范围均应该在本发明保护的范围内。例如5-50、5-100、5-200、5-300、5-400、5-500、100-200、100-300、200-300等等这些均应该在本发明的保护范围内。在实验中发明人发现金属离子的工作浓度在0.5-60mm都有效果，试剂盒中一般是混合的10倍的储液，故储液浓度可以是5-600mm。在本发明某一较佳实施例中，所用的含金属离子的溶液中金属离子的浓度为100mm。较佳地，所述核酸检测试剂盒还包括rna酶抑制剂和缓冲液；所述rna酶抑制剂的浓度优选为10-200u/μl，优选40u/μl。较佳地，所述缓冲液优选包括nacl、tris和bsa，所述缓冲液的ph优选7.9。在本发明某一较佳实施例中，所用缓冲液为10×缓冲液，其组成为：1000mmnacl，500mmtris，1000ug/mlbsa，ph7.9。为了解决上述技术问题，本发明第六方面提供了一种核酸的检测方法，利用如本发明第一、三、五方面所述的核酸检测试剂盒中所述的cpf1检测体系检测所述核酸。较佳地，利用核酸快速释放试剂释放待测样品中的核酸。较佳地，所述核酸通过扩增得到，优选利用rt-rpa扩增待测样品中的核酸。其中，所述扩增时的温度优选为25±5min，和/或，所述扩增的时间优选为39±5℃，和/或，所述扩增时的引物优选如seqidno.24-29、seqidno.44-45、seqidno.73-78所示。较佳地，所述cpf1检测体系的反应温度为37±5℃。较佳地，所述cpf1检测体系的反应时间25±5min。较佳地，所述方法还包括读取结果的步骤，优选利用酶标仪或荧光灯下肉眼进行读取。在本发明某一较佳实施例中，所述检测方法包括以下步骤：步骤a：利用核酸快速释放试剂对样品病毒灭活和释放待测样品中核酸；步骤b：利用等温扩增引物扩增待测样品中核酸：将步骤a获得产物，利用sars病毒核酸orf1ab基因片段的特异性引物seqidno.24和seqidno.25，或者mers病毒核酸upe基因片段的特异性引物seqidno.26和seqidno.27，或者mers病毒核酸upe基因片段的特异性引物，seqidno.28和seqidno.29，或者sars-cov-2病毒e基因片段的特异性引物，seqidno.44和seqidno.45加入rt-rpa等温扩增体系，或者sars-cov-2病毒s基因片段的特异性引物，seqidno.73和seqidno.74加入rt-rpa等温扩增体系，或者sars-cov-2病毒m基因片段的特异性引物，seqidno.75和seqidno.76加入rt-rpa等温扩增体系，或者sars-cov-2病毒n基因片段的特异性引物，seqidno.77和seqidno.78加入rt-rpa等温扩增体系，在37℃反应30min扩增获得特异性产物；步骤c：利用cpf1检测体系识别切割sars病毒核酸orf1ab或者mers病毒核酸upe基因片段或者mers病毒核酸orf1基因片段或者sars-cov-2病毒e基因片段或者sars-cov-2病毒s基因片段或者sars-cov-2病毒m基因片段或者sars-cov-2病毒n基因片段时：将步骤b中产物加入cpf1检测体系，于37℃反应25min；步骤d：利用荧光酶标仪或荧光灯肉眼直接检测判读待检测样品中是否存在病毒核酸。为了解决上述技术问题，本发明第七方面提供了如本发明第一、三、五方面所述的核酸检测试剂盒、如本发明第二方面所述的突变的cpf1蛋白或如本发明第四方面所述的crrna在检测核酸优选病毒核酸，或制备检测核酸优选病毒核酸的试剂中的应用。较佳地，所述病毒为sars相关冠状病毒或mers-cov病毒；所述sars相关冠状病毒优选为sars-cov或sars-cov-2。本发明提供的用于sars和mers病毒核酸快速检测的核酸检测试剂盒既可利用酶标仪荧光检测也可以利用荧光肉眼检测。本发明中核酸探针(例如ssdnafqreporter)，当使用酶标仪荧光检测时，设定检测激发光为485nm-520nm；当使用肉眼直接检测时，选用可产生485nm波长光源的发光器进行检测。本发明中，通常可以对临床样品进行病毒灭活处理，释放待检样品中核酸。在等温条件下，对病毒rna进行反转录(rt)获得dna，并进行重组酶聚合酶扩增(rpa)[9]。在使用荧光检测时，当cpf1检测体系中存在病毒核酸，会由在特异性crrna介导下特异性激活cpf1蛋白的核酸内切酶活性。激活后的cpf1蛋白会切割由荧光基团和淬灭基团标记的ssdnafqreporter，从而释放激活荧光基团，使用酶标仪可以检测到荧光读数或肉眼可见绿色反应。相对应的，待检测样品中不存在病毒核酸时，不会有荧光读数或肉眼不可见绿色反应。本发明中，所述的核酸探针一般也可称为荧光-淬灭单链dna(ssdna)报告系统，其一般含有荧光基团和淬灭基团，在完整状态荧光被淬灭基团淬灭，在反应过程中被切割之后，从而发出荧光。在本发明某一较佳实施例中，所述核酸探针为5’端-6-fam/tttattt/bhq1-3’端。在本发明某一较佳实施例中，所述cpf1蛋白的制备方法可以包括：针对多种不同来源的cpf1蛋白核酸序列进行原核密码子优化获得序列seqidno.14-23，构建到pet28a表达载体，进行低温诱导可溶蛋白表达，通过亲和纯化和分子筛纯化获得目的蛋白。为了解决上述技术问题，本发明还提供了一种检测受试者中是否含有某种病毒(例如被某种病毒感染)的方法，其包括使用如本发明第一、三、五方面所述的核酸检测试剂盒、如本发明第二方面所述的突变的cpf1蛋白或如本发明第四方面所述的crrna进行检测。检测的目的是判断受试者中是否含有该病毒的核酸。术语解释术语crrna是指crisprrna，是短的引导cas12a(cpf1)到结合到靶标dna序列的rna。术语crispr是指成簇的、规律间隔的短回文重复序列(clusteredregularlyinterspacedshortpalindromicrepeats)，该序列是许多原核生物的免疫系统。术语cas蛋白是指crispr-associated蛋白，它是crispr系统中的相关蛋白。术语cpf1(又称cas12a)是指crrna依赖的内切酶，它是crispr系统分类中v型(typev)的酶。术语pam是指前间区序列邻近基序(protospacer-adjacentmotif)，是cas12a切割所必须，fncas12a的pam为ttn序列，lbcas12a的pam为tttn序列。术语rt-rpa(reversetranscription-recombinasepolymeraseamplification)为本领域常规含义，通常是指反转录等温扩增，又称逆转录重组酶聚合酶扩增。本发明中，所提供的核苷酸序列，一般默认是包含了终止子，这对于本领域技术人员来说都是可被理解的。在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。本发明所用试剂和原料均市售可得。本发明的积极进步效果在于：利用本发明所述的核酸检测试剂盒、突变的cpf1蛋白或crrna检测核酸(例如检测sars相关冠状病毒或mers冠状病毒的核酸)时，可以显著提高检测时的信号强度从而减少检测时间、提高检测效率，且灵敏度高、特异性高、准确性高、检测可视化(可在荧光灯下肉眼直接可视检测)、成本低、无需复杂大型实验设备、操作简便。这些优势使得本发明的试剂盒和检测方法更加适用于基层实验和临床一线对病毒例如sars相关冠状病毒和mers冠状病毒的基层快速检测和鉴定诊断。在本发明某一较佳实施例中，检测病毒时的信号强度较现有技术提高了高达3-5倍不等。在本发明某一较佳实施例中，检测病毒的dna样品时的检测灵敏度为1e8个拷贝病毒，检测病毒的rna样品时的检测灵敏度为5个拷贝病毒。附图说明图1为增强型cpf1快速检测病毒核酸方法示意图。图2为不同金属离子对于不同cpf1信号的影响。图3为锰离子对于不同cpf1蛋白荧光检测效果的影响。图4为不同锰盐对于不同cpf1蛋白荧光检测效果的影响。图5为不同crrna检测sars和mers病毒不同基因荧光检测效果。图6为增强型cpf1荧光检测sars和mers病毒dna灵敏度。图7为增强型cpf1荧光检测sars和mers病毒dna灵敏度。图8为荧光检测cpf1快速检测sars和mers病毒dna特异性。图9为荧光检测cpf1快速检测sars和mers病毒rna特异性。图10为模拟检测临床核酸样品荧光检测结果。图11为锰离子对于不同cpf1突变蛋白荧光检测效果的影响。图12为不同crrna检测sars-cov-2病毒基因的荧光检测效果。图13为模拟检测临床核酸样品荧光检测结果。图14为针对sars-cov-2的s基因、m基因和n基因的检测结果。图15为cpf1的不同突变体通过酶标仪检测sars-cov2的e基因的结果图。图16为cpf1的不同突变体通过酶标仪检测sars-cov2的s基因的结果图。图17为cpf1的不同突变体通过酶标仪检测sars-cov2的m基因的结果图。图18为cpf1的不同突变体通过酶标仪检测sars-cov2的n基因的结果图。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。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。本发明中，rpa扩增试剂盒basickit购自twistamp公司；crrna体外转录盒子megashortscriptt7transcriptionkit和纯化盒子megaclearkit购自ambion公司；常规试剂如tris-base,bsa,nacl,tris-hcl，edta,egta,cacl2,cocl2,cuso4,mgcl2,niso4,feso4,mgso4,mncl2,mnso4,znso4,bsa和甘油等购自thermofisher；检测用核酸片段、ssdna探针和rna合成由南京金斯瑞公司完成；本发明使用购自诺唯赞公司的快速核酸释放剂获得预处理的核酸。本发明的总体技术示意图如图1所示，包括以下3大部分：待检测核酸样品制备、cpf1检测成分设计制备和体系构建和荧光检测。实施例1：灵敏度增强型cpf1检测方法1.不同金属离子对于cpf1检测的荧光强度的影响本实施例中的不同物种的cpf1基因经过密码子优化后，序列分别如seqidno.14-23(密码子优化后的核苷酸序列分别对应ascpf1、bbcpf1、bocpf1、fncpf1(氨基酸序列如seqidno.55所示)、hkcpf1、lb4cpf1、lb5cpf1、lbcpf1(氨基酸序列如seqidno.54所示)、oscpf1、tscpf1)所示，将其克隆到pet28a质粒中(由南京金斯瑞公司完成该步骤)，在大肠杆菌中表达，经过纯化后用于检测实验。增强型cpf1检测采用20μl体系如表1所示，但不限于此，包含对相应成分的比例调整，金属离子包括cacl2、cocl2、cuso4、niso4、feso4、mgso4、mnso4、znso4。表1病毒cpf1检测体系其中，10×缓冲液的组成为：1000mmnacl，500mmtris，1000ug/mlbsa，ph7.9。ssdnafqreporter为6fam/tttattt/3bhq1。依次检测不同的金属离子针对mers病毒核酸的crrna3(具体信息如下表2-1中的seqidno.6所示)检测效率。在cpf1检测体系中，各加入1μl100mm金属离子母液，其它各组分维持一致，混合均匀，37℃反应30min，对上述反应产物进行后续结果检测判定。利用荧光检测判定cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，读取检测30min荧光数值为反应值。其反应检测结果如图2。结果表明，本发明检测体系中，针对ascpf1、fncpf1和lbcpf1，锰离子均能提高信号强度，其荧光强度较其他金属离子显著提高，较镁离子、铁离子提高3-5倍不等(图2中显示了ascpf1、bbcpf1、bocpf1的结果图，其他物种的cpf1所得结果类似)。2.锰离子对于不同种属的cpf1检测的荧光强度的影响依次检测锰离子和镁离子对于不同种属的cpf1的检测的荧光强度的影响。在cpf1的检测体系中，各加入1μl上述不同种属的cpf1，包括ascpf1、bbcpf1、bocpf1、fncpf1、hkcpf1、lb4cpf1、lb5cpf1、lbcpf1、oscpf1、tscpf1，其它各组分维持一致(同第1部分)，混合均匀，37℃反应30min，对上述反应产物进行后续荧光强度检测判定，用锰离子的荧光信号除以镁离子的荧光信号，其结果如图3所示。结果表明，本发明的不同种属的cpf1检测体系中，锰离子均能提高荧光信号强度，锰离子相对于镁离子的应该强度增加比例为3.2-8倍不等。3.锰离子对于不同种属的cpf1突变蛋白检测的荧光强度的影响依次检测锰离子和镁离子对于不同种属的cpf1(lbcpf1和fncpf1)的突变蛋白检测的荧光强度的影响。在cpf1的检测体系中，各加入1μl不同种属的cpf1突变蛋白，包括lbcpf1(氨基酸序列如seqidno.54所示)、lbcpf1-t148r(seqidno.36)、lbcpf1-t152r(seqidno.37)、lbcpf1-g532r(seqidno.38)、lbcpf1-k536r(seqidno.42)、lbcpf1-k538r(seqidno.39)、lbcpf1-t152r/g532r(seqidno.40)、lbcpf1-t152r/g532r/k538r(seqidno.41)、fncpf1(氨基酸序列如seqidno.55所示)、fncpf1-k180r(seqidno.30)、fncpf1-e184r(seqidno.31)、fncpf1-n607r(seqidno.32)、fncpf1-k611r(seqidno.43)、fncpf1-k613r(seqidno.33)、fncpf1-e184r/n607r(seqidno.34)、fncpf1-e184r/n607r/k613r(seqidno.35)，其它各组分维持一致(同第1部分)，混合均匀，37℃反应30min，对上述反应产物进行后续荧光强度检测，检测结果如图11所示。结果表明，本发明的不同种属的cpf1突变检测体系中，部分突变能够增强信号强度，但是lbcpf1-k536r和fncpf1-k611r不能增加信号强度。锰离子能提高具有活性的突变蛋白的荧光信号强度，锰离子相对于镁离子的应该强度增加比例为3-5倍不等。此外，相对于未突变的cpf1蛋白而言，cpf1的突变蛋白也相应提高了荧光信号强度，其中突变体fncpf1-e184r、fncpf1-n607r、fncpf1-k613r、fncpf1-e184r/n607r、fncpf1-e184r/n607r/k613r和lbcpf1-t152r、lbcpf1-g532r、lbcpf1-k538r、lbcpf1-t152r/g532r、lbcpf1-t152r/g532r/k538r相对于其未突变的蛋白而言，荧光信号强度提高了1/2-1倍不等。4.不同形式的锰盐对于信号的增强作用依次检测mnso4、mncl2和mn(ch3coo)2对于不同种属的cpf1的检测的荧光强度的影响。在cpf1的检测体系中，各加入1μl不同种属的cpf1，包括ascpf1、bbcpf1、bocpf1、fncpf1、hkcpf1、lb4cpf1、lb5cpf1、lbcpf1、oscpf1、tscpf1，其它各组分维持一致(同第1部分)，混合均匀，37℃反应30min，对上述反应产物进行后续荧光强度检测判定，其结果如图4所示。结果表明，本发明的检测体系中，不同形式的锰盐均能提高荧光信号强度。实施例2：sars和mers病毒基因片段快速灵敏检测2.1核酸制备本实施例中，sars-cov病毒orf1ab基因参考ncbi上的ay278487基因，mers病毒upe基因片段参考ncbi上的jx869059基因，mers病毒orf1a基因片段参考ncbi上的jx869059基因，sars-cov-2病毒e基因片段、s基因片段、m基因片段和n基因片段参考ncbi上的nc045512基因组，均由南京金斯瑞公司合成，分别命名为sars-orf1ab、mers-upe、mers-orf1a、sars-cov-2-e、sars-cov-2-s、sars-cov-2-m、sars-cov-2-n对应序列分别为seqidno.11-13、seqidno.46、seqidno.79-81。通过体外转录制备sars-cov病毒orf1ab基因，mers病毒upe基因片段、orf1a、sars-cov-2病毒e基因、sars-cov-2病毒s基因、sars-cov-2病毒m基因、sars-cov-2病毒n基因对应的rna核酸样品。具体操作如下：使用megashortscriptt7转录试剂盒(thermofisherscientific)，分别以上述sars-orf1ab、mers-upe和orf1a、sars-cov-2-e、sars-cov-2-s、sars-cov-2-m、sars-cov-2-n作为模板转录对应的基因rna样品pcrna-sars-orf1ab、pcrna-mers-upe和pcrna-mers-orf1a、pcrna-sars-cov-2-e、pcrna-sars-cov-2-s、pcrna-sars-cov-2-m、pcrna-sars-cov-2-n。转录后的rna用megaclear试剂盒(thermofisherscientific)纯化，并通过乙醇沉淀法回收。检测转录后所得单链rna(ssrna)的质量和浓度，并保存在-80℃直至使用。2.2针对特异性crrna的设计制备针对特异性crrna制备按照如下方案进行，针对sars-orf1ab、mers-upe基因、mers-orf1a基因、sars-cov-2-e基因、sars-cov-2-s基因、sars-cov-2-m基因、sars-cov-2-n基因，寻找包含cpf1识别序列(pam，protospaceradjacentmotif)tttn的靶向序列，设计44bp长度的crrna。分别命名为针对sars-orf1ab基因的sars-orf1ab-crrna1到sars-orf1a-crrna3、针对mers-upe基因的mers-upe-crrna1到mers-upe-crrna4、针对mers-orf1a基因的mers-orf1a-crrna1到mers-orf1a-crrna3、针对sars-cov2-e基因的cov2-e-crrna1到cov2-e-crrna7、针对sars-cov2-s基因的cov2-s-cr3到cov2-s-cr10、针对sars-cov2-m基因的cov2-m-cr1到cov2-m-cr5、针对sars-cov2-n基因的cov2-n-cr1到cov2-n-cr4。设计完成后，制备crrna。crrna的制备，可交与南京金斯瑞公司合成oligo，构建到载体puc57-t7-crrna，通过体外转录获得目的crrna，或可交由南京金斯瑞公司直接合成crrna序列对应的rna。本发明提供的sars-orf1ab、mers-upe基因、mers-orf1a基因和sars-cov-2-e基因、sars-cov-2-s基因、sars-cov-2-m基因、sars-cov-2-n基因的crrna包括seqidno.1到seqidno.10、seqidno.47到seqidno.53、seqidno.56到seqidno.72，具体信息如表2-1和表2-2所示：表2-1针对sars和mers病毒核酸特异性crrna表2-2针对sars和mers病毒核酸特异性crrna2.3反转录等温扩增反应本实施例中，利用反转录等温扩增(rt-rpa)对上述2.1中所制备的单链rna(ssrna)病毒核酸进行预扩增，以便进行cpf1检测反应。按照等温扩增反应要求，设计并合成rt-rpa扩增引物rt-rpa-f(正向引物)和rt-rpa-r(反向引物)(表3)。参考rt-rpa等温扩增操作步骤，扩增获得待检测样品。具体操作如下：等温扩增在50μl反应体系进行。将xμlrna样品，(18.5-x)μlddh2o,2μl表3中各自的rpa-f，2μlrpa-r和25μl反应缓冲液混匀，加入反应管溶解混匀。最后，加入2.5μl乙酸镁，混匀后在39℃下孵育30分钟。rt-rpa产物样品进行下一步检测。表3针对冠状病毒cov核酸特异性rt-rpa引物序列2.4本实施例中，增强型cpf1检测采用20μl体系如表4所示，但不限于此，包含对相应成分的比例调整：表4病毒cpf1检测体系成分用量/样品10×缓冲液2μl金属离子溶液1μlrnaseinhibitors(40u/μl)1μlcrispr-cpf1(200ng/μl)1μlssdnafqreporter(25pmol/μl)1μllcrrna(1μm)1μl样品xμlh2o(rnafree)upto20μl2.5全波长酶标仪荧光检测酶标仪荧光检测中，cpf1对目的基因检测体系依次加入2μl10×缓冲液、1μlrnaseinhibitors、1μlcpf1、1μlssdnafqreporter、5μl上述2.3中所得rt-rpa产物样品、1μlcrrna和9μlh2o。各组分混合均匀后于37℃反应30min。其中，上述反应体系中rnaseinhibitors浓度为40u/μl，cpf1为200ng/μl，ssdnafqreporter浓度为25pm/μl，crrna浓度为1nm/μl。首先，依次检测针对sars-cov病毒orf1ab基因、mers病毒upe基因、mers病毒orf1a基因、sars-cov-2病毒e基因、sars-cov-2病毒s基因、sars-cov-2病毒m基因、sars-cov-2病毒n基因的crrna检测效率。在cpf1检测体系中，各加入1μlcrrna，其它各组分维持一致，混合均匀，37℃反应30min，对上述反应产物进行后续结果检测判定。利用荧光检测判定cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，读取检测30min荧光数值为反应值。针对sars-cov病毒和mers病毒的不同crrna，其反应检测结果如图5。结果表明，本发明检测体系中，针对不同基因片段的crrna均能高效特异的检测对应的基因片段。针对sars-cov-2的e基因的检测结果如图12所示，其中，cov2-e-cr4和cov2-e-cr5无法检出，cov2-e-cr3和cov2-e-cr6检测效率较低，cov2-e-cr1、cov2-e-cr2和cov2-e-cr7检测效率较高。后续实验将cov2-e-cr1、cov2-e-cr2和cov2-e-cr7混合在一起制备sars-cov2-crmix用于后续检测sars-cov-2-e基因。针对sars-cov-2的s基因、m基因和n基因的检测结果如图14所示，针对不同基因片段的crrna均能高效特异的检测对应的基因片段。其中，cov2-s-cr3、cov2-s-cr5和cov2-s-cr6对sars-cov-2-s基因的检测效率较高，后续实验将这三个crrna混合，制备成cov2-s-crmix用于后续监测sars-cov-2-s基因；其中cov2-m-cr1、cov2-m-cr2和cov2-m-cr4对sars-cov-2-m基因的检测效率较高，后续实验将这三个crrna混合，制备成cov2-m-crmix用于后续监测sars-cov-2-m基因；其中cov2-n-cr1、cov2-n-cr2和cov2-n-cr6对sars-cov-2-n的检测效率较高，后续实验将这三个crrna混合，制备成cov2-n-crmix用于后续监测sars-cov-2-n基因。鉴于本实施例中针对sars-orf1ab和mers-upe基因的crrna有一致的检测效果，将针对sars-orf1ab基因的sars-o-crrna1到sars-o-crrna3等比例混合获得sars-o-crmix；将针对mers-upe基因的mers-e-crrna1到mers-e-crrna4等比例混合获得mers-e-crmix；将针对mers-orf基因的mers-o-crrna1到mers-o-crrna3等比例混合获得mers-o-crmix。后续检测中，sars-o-crmix检测sars-orf1ab基因，mers-e-crmix检测mers-upe基因，mers-o-crmix检测mers-orf基因。实施例3：基于cpf1荧光法高灵敏度检测病毒核酸本实施例中，为测定cpf1荧光法对检测灵敏度的影响，进行以下检测。(1)首先，根据实施例2中2.1所述的sars-orf1ab、mers-upe和mers-orf1基因片段对应的dna样品，计算检测片段分子量，并进行梯度稀释，获得每微升含有5e9、1e9、5e8、1e8、5e7、1e7拷贝数(copy/μl)的测试样品。利用荧光法，对上述稀释样品进行cpf1特异性反应，参照实施例2中检测步骤进行检测和荧光结果判读。操作简述如下：1μl梯度稀释样品，加入到20μlcpf1荧光核酸检测体系(其中所添加的crrna即为上述检测sars-orf1ab基因的sars-o-crmix、检测mers-upe基因的mers-e-crmix或检测mers-orf基因的mers-o-crmix)，混合均匀，37℃反应30min进行荧光结果判读。本实施例中，应用增强型cpf1荧光法检测sars和mers病毒，分别采用酶标仪检测(结果见图6)，可实现对1e8个拷贝病毒的高灵敏度检出，灵敏度高。(2)其次，根据实施例2中2.1所述的sars-orf1ab、mers-upe和mers-orf1基因片段对应的rna样品，计算检测片段分子量，并进行梯度稀释，获得每微升含有1000、100、10、5拷贝数(copy/μl)的测试样品。利用荧光法，对上述稀释样品进行cpf1特异性反应，参照实施例2中检测步骤进行检测和荧光结果判读。操作简述如下：1μl梯度稀释样品，加入到50μlrt-rpa等温扩增反应体系进行扩增。取10μl等温扩增产物，加入到20μlcpf1荧光核酸检测体系(其中所添加的crrna即为上述检测sars-orf1ab基因的sars-o-crmix、检测mers-upe基因的mers-e-crmix或检测mers-orf基因的mers-o-crmix)，混合均匀，37℃反应25min进行荧光结果判读。本实施例中，应用cpf1荧光法检测sars和mers病毒rna，分别采用酶标仪检测(结果见图7)，可实现对5个拷贝病毒的高灵敏度检出，灵敏度高。实施例4：基于cpf1荧光法高特异性检测病毒核酸本实施例中，为检测cpf1荧光法能否进行高特异反应，能否有效区分sars-cov病毒和mers病毒，进行以下检测。(1)首先，针对病毒dna的特异性进行测试。根据本发明中针对sars-cov病毒和mers病毒核酸检测区段sars-orf1ab和mers-upe，参照实施例2中的方法，制备mers病毒对应基因序列dna样品：sars-orf1ab，mers-upe和mers-orf1。参照实施例2，将上述dna样品混合，在增强型cpf1检测体系中，依次加入2μlbuffer、1μlrnaseinhibitors、1μlcpf1、1μlssdnafqreporter、1μlcrrna(其中所添加的crrna即为上述检测sars-orf1ab基因的sars-o-crmix、检测mers-upe基因的mers-e-crmix或检测mers-orf基因的mers-o-crmix)和1μl检测样品。各组分混合均匀，在37℃反应30min。本检测体系中，rnaseinhibitors浓度为40u/μl，cpf1浓度为200ng/μl，ssdnadbreporter浓度为25pm/μl，crrna浓度为1nm/μl。本实施例中，利用荧光检测判定cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，取检测30min时荧光数值为反应值。检测结果如图8a(图8中的t1、t2、t3、t4分别为配制样品，其中，t1：已知是没有sars或者mers-upe基因的dna，t2：已知是含有sars基因的dna；t3：已知是含有mers-upe基因的dna；t4：已知是含有sars和mers-upe基因的dna。图8中的mers-crmix是指上述mers-e-crmix，sars-crmix是指上述sars-o-crmix)。同时，将上述cpf1检测反应30min后产物，置于在485nm激光灯下，可由肉眼直接进行结果判读。当crrna特异性识别靶核酸片段后，反应产物颜色由无色变为荧光绿；对应的，如果待检测无对应靶核酸，反应产物颜色仍为无色。在cpf1检测反应30min后，在荧光下进行肉眼判读，并拍照记录。如图8b所示，针对不同基因片段的crrna均能高效特异的检测对应的基因片段，特异性高且检测结果准确。(2)其次，针对病毒rna的特异性进行测试。根据本发明中针对sars-cov病毒和mers病毒核酸检测区段sars-orf1ab，mers-upe和mers-orf1，参照实施例2中方法，制备mers病毒对应基因序列rna样品：sars-orf1ab，mers-upe和mers-orf1。参照实施例2，通过rt-rpa反应，分别扩增制备sars-cov病毒和mers病毒检测样品。将rna样品混合，在增强型cpf1检测体系中，依次加入2μlbuffer、1μlrnaseinhibitors、1μlcpf1、1μlssdnafqreporter、1μlcrrna和10μl检测样品。各组分混合均匀，在37℃反应30min。本检测体系中，rnaseinhibitors浓度为40u/μl，cpf1浓度为200ng/μl，ssdnadbreporter浓度为25pm/μl，crrna浓度为1nm/μl。本实施例中，利用荧光检测判定cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，取检测30min时荧光数值为反应值。检测结果如图9a(图9中的t5、t6、t7、t8分别为配制样品，其中，t5：已知是没有sars或者mers-upe基因的rna，t6：已知是含有sars基因的rna；t7：已知是含有mers-upe基因的rna；t8：已知是含有sars和mers-upe基因的rna。图9中的mers-crmix是指mers-e-crmix，sars-crmix是指上述sars-o-crmix)。同时，将上述cpf1检测反应30min后产物，置于在485nm激光灯下，可由肉眼直接进行结果判读。当crrna特异性识别靶核酸片段后，反应产物颜色由无色变为荧光绿；对应的，如果待检测无对应靶核酸，反应产物颜色仍为无色。在cpf1检测反应30min后，在荧光下进行肉眼判读，并拍照记录。如图9b所示，针对不同基因片段的crrna均能高效特异的检测对应的基因片段，特异性高且检测结果准确。实施例5：模拟cpf1荧光法快速检测临床病毒核酸本实施例模拟临床组织样品的核酸进行快速检测，本检测不涉及任何临床sars和mers病毒样品，且所有操作合格按照有关法律法规和相关规定进行。本实施例中，首先，将实施例2中sars-cov病毒核酸对应rna样品(sars-orf1ab)和mers病毒核酸对应rna样品(mers-orf1ab和mers-upe)，点涂到咽拭子，模拟临床样品进行检测。(1)首先，对病毒灭活和病毒核酸释放。本实施例使用购自诺唯赞公司的快速核酸释放剂获得预处理的核酸。步骤如下：将咽拭子加入50μlpbs，模拟溶解病毒到液体。取3μl待检测样品加入20μl核酸裂解液，加入rna酶抑制剂，常温静置3分钟，加入20μl中和液，混匀后进行下一步检测。在cpf1检测中，每个待检测样品取5μl进行rt-rpa预扩增，步骤与实施例1中相同，获得cpf1检测样品。在cpf1检测体系中，依次加入2μlbuffer、1μlrnaseinhibitors、1μlcpf1、1μlssdnafqreporter、10μlrpasample、1μlcrrna。各组分混合均匀，在37℃反应25min。本实施例中，可通过荧灯裸眼观察或者酶标仪直接判定cpf1检测结果。如图10所示，本发明中cpf1荧光法可通过酶标仪检测可有效对模拟临床阴阳性样品判定。同样的，在荧光灯下裸眼可有效对模拟临床阴阳性样品判定(图10，图中的cs1-cs6分别为配制样品，其中，cs1、cs2和cs6为已知是sars-cov病毒阳性的样品，其余均为已知是sars阴性和mers阴性的样品。图中的mers-crmix是mers-e-crmix，sars-crmix是指上述sars-o-crmix)。可见本发明的检测方法快速、可视、成本低、操作简便的同时，所得结果准确性还很高。(2)类似的，模拟临床制备cs7到cs12的样品，其中cs8、cs10和cs11已知为sars-cov-2阳性的样品，cs9已知为mers-cov阳性的样品，其余均为已知是sars-cov-2阴性和mers-cov阴性的样品。通过与上述(1)中类似的方法进行检测，然后通过荧灯裸眼观察或者酶标仪直接判定cpf1检测结果。如图13所示(图中mers-crmix是mers-e-crmix)，本发明中cpf1荧光法可通过酶标仪检测可有效对模拟临床阴阳性样品判定。可见本发明的检测方法准确性很高。此外，综合上述实施例的结果进一步可知，将反应体系中cpf1的配体离子替换为锰离子时，在检测不同的病毒(sars-cov、mers-cov、sars-cov-2等)核酸时都能够显著提高检测的信号强度，可见该锰离子体系具有普适性。实施例6：模拟cpf1荧光法快速检测临床sars-cov-2病毒核酸的不同基因本实施例模拟临床组织样品的核酸进行快速检测，本检测不涉及任何临床sars-cov-2病毒样品，且所有操作合格按照有关法律法规和相关规定进行。本实施例中，首先，将sars-cov-2病毒核酸对应rna样品(cov2-e-基因，cov2-s-基因,cov2-m-基因,cov2-n-基因)，点涂到咽拭子，模拟临床样品进行检测。(1)首先，对病毒灭活和病毒核酸释放。本实施例使用购自诺唯赞公司的快速核酸释放剂获得预处理的核酸。步骤如下：将咽拭子加入50μlpbs，模拟溶解病毒到液体。取3μl待检测样品加入20μl核酸裂解液，加入rna酶抑制剂，常温静置3分钟，加入20μl中和液，混匀后进行下一步检测。在cpf1检测中，每个待检测样品取5μl进行rt-rpa预扩增，步骤与实施例1中相同，获得cpf1检测样品。在cpf1检测体系中，依次加入2μlbuffer、1μlrnaseinhibitors、1μlcpf1或cpf1突变蛋白、1μlssdnafqreporter、10μlrpasample、1μlcrrna、加入1μlmn2+或者mg2+，各组分混合均匀，在37℃反应25min。本实施例中，可通过酶标仪直接判定cpf1及其突变的检测结果。如图15、16、17、18所示，本发明中cpf1的不同突变体可以通过酶标仪检测sars-cov2的s基因、m基因、n基因和e基因。在加入mg2+和mn2+离子均可以有效的检测，但是mn2+的加入信号更强，锰离子的体系具有普适性。参考文献1.r,b.,etal.,crisprprovidesacquiredresistanceagainstvirusesinprokaryotes.science(newyork,n.y.),2007.315(5819):p.1709-12.2.la,m.ands.ej,crisprinterferencelimitshorizontalgenetransferinstaphylococcibytargetingdna.science(newyork,n.y.),2008.322(5909):p.1843-5.3.b,z.,etal.,cpf1isasinglerna-guidedendonucleaseofaclass2crispr-cassystem.cell,2015.163(3):p.759-71.4.i,f.,etal.,thecrispr-associateddna-cleavingenzymecpf1alsoprocessesprecursorcrisprrna.nature,2016.532(7600):p.517-21.5.js,c.,etal.,crispr-cpf1targetbindingunleashesindiscriminatesingle-strandeddnaseactivity.science(newyork,n.y.),2018.360(6387):p.436-439.6.gonzález,jm.etal.acomparativesequenceanalysistorevisethecurrenttaxonomyofthefamilycoronaviridae.,gomez-puertasp,cavanaghd,gorbalenyaae,enjuanesl.archvirol.2003；148(11):p.2207-35.7.ksiazek,tg.etal.,anovelcoronavirusassociatedwithsevereacuterespiratorysyndrome.nengljmed,2003,348(20):p.1953-19668.zaki,am.etal.,isolationofanovelcoronavirusfromamanwithpneumoniainsaudiarabia.nengljmed,2012,367(19):p.1814-20.9o,p.,etal.,dnadetectionusingrecombinationproteins.plosbiology,2006.4(7):p.e204.sequencelisting<110>上海科技大学<120>一种核酸检测试剂盒、检测方法及应用<130>p20012584c<150>202010251367.6<151>2020-04-01<160>81<170>patentinversion3.5<210>1<211>44<212>rna<213>artificialsequence<220><223>sars-o-cr1<400>1uaauuucuacuaaguguagauuacagguuagcuaacgagugugc44<210>2<211>44<212>rna<213>artificialsequence<220><223>sars-o-cr2<400>2uaauuucuacuaaguguagauucaagcuguuacagccaauguaa44<210>3<211>44<212>rna<213>artificialsequence<220><223>sars-o-cr3<400>3uaauuucuacuaaguguagauaacugaugguaauaagauagcug44<210>4<211>44<212>rna<213>artificialsequence<220><223>mc-e-crrna1<400>4uaauuucuacuaaguguagaugacauauggaaaacgaacuaugu44<210>5<211>44<212>rna<213>artificialsequence<220><223>mc-e-crrna2<400>5uaauuucuacuaaguguagauuccaagaacgaauaggguuguuc44<210>6<211>44<212>rna<213>artificialsequence<220><223>mc-e-crrna3<400>6uaauuucuacuaaguguagaucuaugaacaacccuauucguucu44<210>7<211>44<212>rna<213>artificialsequence<220><223>mc-e-crrna4<400>7uaauuucuacuaaguguagaucauauguccaaagagagacuaau44<210>8<211>44<212>rna<213>artificialsequence<220><223>mc-o-cr1<400>8uaauuucuacuaaguguagauacuuaugcaaacauagucuacga44<210>9<211>44<212>rna<213>artificialsequence<220><223>mc-o-cr2<400>9uaauuucuacuaaguguagaugucagcgcugauugcaguugcaa44<210>10<211>44<212>rna<213>artificialsequence<220><223>mc-o-cr3<400>10uaauuucuacuaaguguagaucaacugcaaucagcgcugacgaa44<210>11<211>444<212>dna<213>artificialsequence<220><223>sars-orf1ab<400>11taatacgactcactataggtctagaaataattttgtttaactttaagaaggagatatacc60atgctaacatgcttaggataatggcctctcttgttcttgctcgcaaacataacacttgct120gtaacttatcacaccgtttctacaggttagctaacgagtgtgcgcaagtattaagtgaga180tggtcatgtgtggcggctcactatatgttaaaccaggtggaacatcatccggtgatgcta240caactgcttatgctaatagtgtctttaacatttgtcaagctgttacagccaatgtaaatg300cacttctttcaactgatggtaataagatagctgacaagtatgtccgcaatctacaacaca360ggctctatgagtgtctctatagaaatagggatgttgatcatgaattcgtggatgagtttt420acgcttacctgtttttttgaattc444<210>12<211>345<212>dna<213>artificialsequence<220><223>mers-upe<400>12taatacgactcactataggtctagaaataattttgtttaactttaagaaggagatatacc60atgctgttttcgtgcctgcaacgcgcgattcagttcctcttcacataatcgccccgagct120cgcttatcgtttaagcagctctgcgctactatgggtcccgtgtagaggctaatccattag180tctctctttggacatatggaaaacgaactatgttaccctttgtccaagaacgaatagggt240tgttcatagtaaactttttcatttttaccgtagtatgtgctataacactcttggtgtgta300tggctttccttacggctactagattatgtgtgtttttttgaattc345<210>13<211>449<212>dna<213>artificialsequence<220><223>mers-orf1a<400>13taatacgactcactatagggagatctagaaataattttgtttaactttaagaaggagata60taccatgactattcccacacagttgttcccactcttatttgtgactatggccttcgttat120gttgttggttaaacacaaacacacctttttgacacttttcttgttgcctgtggctatttg180tttgacttatgcaaacatagtctacgagcccactactcccatttcgtcagcgctgattgc240agttgcaaattggcttgcccccactaatgcttatatgcgcactacacatactgatattgg300tgtctacattagtatgtcacttgtattagtcattgtagtgaagagattgtacaacccatc360actttctaactttgcgttagcattgtgcagtggtgtaatgtggttgtacacttatagcat420tggagaagcctcaagctttttttgaattc449<210>14<211>3924<212>dna<213>artificialsequence<220><223>ascpf1<400>14atgactcagttcgaggggttcaccaatttgtaccaggtctctaagacacttcgctttgag60cttatccctcagggcaagacgctgaagcatatccaggagcaaggatttatcgaggaggac120aaggcacgtaatgaccactataaagagctgaaaccgatcattgatcgcatttacaagacc180tatgcagatcagtgtctgcagcttgtgcaattagattgggaaaatctgagcgccgcaatc240gactcctatcgtaaagagaagactgaagaaacacgtaatgcattaattgaagaacaggcc300acgtatcgcaatgctatccacgattattttattgggcgcaccgacaatttaactgatgcc360atcaataagcgtcacgcagagatctacaagggactttttaaagccgaattatttaatggc420aaggttttaaaacagcttggtactgtaacaacaacagagcacgagaatgcccttctgcgc480tcgttcgataagtttaccacgtacttctcgggcttttacgaaaaccgcaaaaacgtgttc540tcggcggaggacatctctacagctatcccacaccgtattgtacaagataacttccccaaa600tttaaggagaattgtcacattttcacgcgtttgattactgcggtaccgtctttacgtgag660catttcgaaaacgttaagaaggcaattggcatttttgtcagcacgtctattgaagaagtg720ttctcgtttcccttctacaatcagctgttgactcaaacgcagatcgacttatataaccag780ctgttgggtggcatctcacgcgaggcaggcacagaaaaaattaaaggcctgaacgaggtc840ttgaatttggctatccagaagaacgatgagaccgcgcacatcattgcttccttacctcat900cgctttattccgttgtttaaacaaattcttagtgatcgtaatactttatccttcatctta960gaggaatttaaatccgacgaggaggttattcagtccttctgcaaatataaaactctgtta1020cgcaacgagaacgtgcttgaaaccgcggaggctttattcaacgagctgaactcgattgac1080ttgacacatattttcatctcacacaagaaactggaaacgatttcgtcagccctttgcgac1140cactgggatactctgcgtaacgcattatatgaacgtcgcatttccgaattgacaggaaaa1200atcactaagagcgcaaaggagaaggtacagcgttcgctgaaacatgaagacatcaacctt1260caggagattatctctgcggcgggcaaagaacttagtgaagcatttaagcaaaaaaccagt1320gagatccttagccatgcgcacgctgctcttgaccaaccacttccgacaacgctgaaaaaa1380caggaggaaaaggagattctgaagagccagctggattctttacttggactttatcatttg1440ttggattggtttgccgtggacgaaagt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tcggac3600aagaaattcttcgcaaaactgacgtctgttcttaataccatccttcaaatgcgtaattcc3660aagacgggtacggagctggattacctgatcagtcccgttgccgatgttaatggtaatttt3720ttcgactcgcgtcaggccccgaagaacatgcctcaggacgccgatgccaacggtgcgtac3780catattggccttaagggcttaatgctgttaggacgtattaagaacaaccaggagggtaaa3840aaactgaatctggtcattaaaaacgaagaatatttcgaatttgtacagaaccgcaataac3900tga3903<210>36<211>3687<212>dna<213>artificialsequence<220><223>lbcpf1-t148r<400>36atgtctaagctggaaaaattcaccaactgctacagtttaagcaagacgttacgcttcaag60gcgatcccggttgggaaaacacaagagaatattgacaacaaacgccttttagttgaagat120gagaagcgtgcagaggactataaaggagttaagaagcttttggaccgttactacttatcg180tttattaatgatgtgctgcattccatcaaactgaagaatctgaataattacattagtctt240ttccgtaagaaaacacgtactgagaaggaaaacaaggagctggaaaatcttgagatcaat300ttgcgcaaggagattgctaaggcattcaaaggtaacgagggatataagtcgttgtttaaa360aaagacattattgaaacgatccttccggaattccttgacgataaagacgagattgcactg420gtgaactccttcaacgggttccgtactgcctttacgggtttttttgacaaccgcgagaat480atgttcagtgaagaagccaaatctacctctatcgctttccgctgcattaatgaaaacctg540actcgttatatctctaacatggacatcttcgaaaaggtagacgcgatctttgataaacac600gaagtgcaggagatcaaggagaaaattcttaatagcgactatgatgttgaagatttcttt660gagggggaattctttaactttgtactgacgcaagaagggatcgacgtctataacgctatt720atcggaggattcgtaaccgagagtggagagaaaattaagggtcttaatgagtacattaat780ttgtacaatcaaaaaacgaaacagaaattgcccaagtttaaacctctgtataaacaagtg840ctgagtgatcgcgaaagtttgtcattctatggagaggggtatactagcgacgaggaggtt900cttgaggtgtttcgcaacaccttaaacaagaactctgagattttcagctcaatcaaaaaa960cttgagaaattatttaaaaatttcgacgaatactcgtcagccggcattttcgtgaaaaat1020ggtccggccatttccacaatctctaaggacatctttggagaatggaacgttatccgtgac1080aaatggaacgctgaatatgatgacatccatttgaaaaagaaggctgtcgttacagaaaaa1140tatgaagatgatcgccgcaagagcttcaaaaagatcggttcgttctcgcttgagcagttg1200caagaatacgcagatgccgatctgtcagtagtagagaaattaaaggaaatcattattcaa1260aaagtggacgagatttacaaagtttacggatcctctgagaagctttttgatgcagatttt1320gtgttggagaaatcgttaaaaaagaatgatgccgtcgtggcaattatgaaagacttatta1380gatagcgtgaagtccttcgagaattatattaaggcgttctttggagagggtaaggagaca1440aaccgcgacgaatcgttttacggcgatttcgtgttggcctacgacatcttgctgaaagtt1500gatcacatttacgacgccatccgtaactatgtaactcagaaaccctacagtaaggacaag1560tttaagttatactttcagaatccgcagttcatgggtggatgggataaggacaaagagaca1620gactaccgtgctactatcttacgctacgggtcgaagtattaccttgcaattatggataag1680aagtacgcaaaatgcttgcagaagatcgataaagacgacgtcaacggaaattatgaaaag1740atcaattataaattattacccgggcctaataaaatgttgcccaaagtatttttctctaag1800aaatggatggcctactacaatccatcagaagacattcagaagatctataaaaacggcaca1860ttcaaaaaaggcgatatgttcaacttgaacgattgccacaaactgattgatttctttaag1920gatagcattagccgttacccgaagtggtccaacgcttatgactttaacttcagcgaaaca1980gagaagtataaagacattgcagggttctaccgtgaggtagaagaacaaggatataaagtt2040tctttcgaaagtgcaagtaagaaggaagtagataaattggttgaggagggcaaactgtat2100atgtttcagatttacaataaggatttctctgacaagtcccacggcacacccaatttgcat2160actatgtacttcaaacttctgtttgacgagaataaccatgggcagatccgtcttagtggg2220ggagctgagttatttatgcgccgtgcgtctttgaaaaaggaggagttagttgttcacccg2280gcaaattcacccattgccaacaaaaatccagataaccccaagaagacaaccactctttca2340tacgatgtgtacaaagataagcgtttctctgaagatcaatacgaactgcatattccgatc2400gcaatcaacaagtgcccaaagaacatctttaaaatcaatacggaagttcgcgttcttctg2460aaacatgatgataacccgtatgttattggaattgatcgtggggagcgtaacttgttgtat2520attgtcgtagtagatggaaaaggtaacatcgtcgaacagtactcgctgaatgagattatt2580aacaacttcaacgggattcgcatcaagactgattaccactcacttcttgacaaaaaggag2640aaggagcgctttgaagcccgtcaaaattggacatcaatcgaaaatattaaggagctgaaa2700gccggatacatctctcaagtggtccataaaatttgtgaactggtcgagaaatatgatgcc2760gtgatcgcgcttgaggacctgaatagtggattcaaaaactcgcgtgtgaaagttgagaaa2820caggtgtatcagaaatttgagaaaatgttaattgacaaattgaactacatggtagataaa2880aagagtaacccatgtgcgacgggcggtgcattgaagggataccagatcaccaataagttt2940gagtctttcaaaagtatgtctacgcagaatggttttattttctacatcccggcttggctg3000acatctaaaattgacccttcgacgggttttgtcaatctgctgaaaacaaagtatacaagt3060attgcggattcaaaaaaattcatctcatctttcgatcgcatcatgtacgtcccagaagaa3120gacttatttgagtttgcactggattataagaatttttcccgtactgacgcagactatatc3180aagaaatggaaattatattcatacgggaaccgcattcgtatcttccgtaatcctaaaaag3240aacaacgttttcgactgggaagaagtatgcttaaccagtgcttacaaagaattgttcaac3300aaatacggcattaactaccagcagggcgacattcgcgcattattatgcgaacagtcagat3360aaagcgttctactccagctttatggcgttaatgtcgcttatgttacagatgcgcaacagc3420attacaggccgcaccgatgtcgattttctgatttcgccggtgaaaaacagcgatggcatt3480ttttacgattctcgcaattacgaggcgcaggaaaatgcaattcttccgaagaacgcagac3540gcaaacggcgcttacaacatcgcacgtaaagttctttgggcgatcggacaatttaaaaaa3600gccgaggatgaaaaattggacaaggttaaaattgctatctcgaataaagagtggcttgag3660tacgcgcaaacttctgttaagcactga3687<210>37<211>3687<212>dna<213>artificialsequence<220><223>lbcpf1-t152r<400>37atgtctaagctggaaaaattcaccaactgctacagtttaagcaagacgttacgcttcaag60gcgatcccggttgggaaaacacaagagaatattgacaacaaacgccttttagttgaagat120gagaagcgtgcagaggactataaaggagttaagaagcttttggaccgttactacttatcg180tttattaatgatgtgctgcattccatcaaactgaagaatctgaataattacattagtctt240ttccgtaagaaaacacgtactgagaaggaaaacaaggagctggaaaatcttgagatcaat300ttgcgcaaggagattgctaaggcattcaaaggtaacgagggatataagtcgttgtttaaa360aaagacattattgaaacgatccttccggaattccttgacgataaagacgagattgcactg420gtgaactccttcaacgggttcacgactgcctttcgtggtttttttgacaaccgcgagaat480atgttcagtgaagaagccaaatctacctctatcgctttccgctgcattaatgaaaacctg540actcgttatatctctaacatggacatcttcgaaaaggtagacgcgatctttgataaacac600gaagtgcaggagatcaaggagaaaattcttaatagcgactatgatgttgaagatttcttt660gagggggaattctttaactttgtactgacgcaagaagggatcgacgtctataacgctatt720atcggaggattcgtaaccgagagtggagagaaaattaagggtcttaatgagtacattaat780ttgtacaatcaaaaaacgaaacagaaattgcccaagtttaaacctctgtataaacaagtg840ctgagtgatcgcgaaagtttgtcattctatggagaggggtatactagcgacgaggaggtt900cttgaggtgtttcgcaacaccttaaacaagaactctgagattttcagctcaatcaaaaaa960cttgagaaattatttaaaaatttcgacgaatactcgtcagccggcattttcgtgaaaaat1020ggtccggccatttccacaatctctaaggacatctttggagaatggaacgttatccgtgac1080aaatggaacgctgaatatgatgacatccatttgaaaaagaaggctgtcgttacagaaaaa1140tatgaagatgatcgccgcaagagcttcaaaaagatcggttcgttctcgcttgagcagttg1200caagaatacgcagatgccgatctgtcagtagtagagaaattaaaggaaatcattattcaa1260aaagtggacgagatttacaaagtttacggatcctctgagaagctttttgatgcagatttt1320gtgttggagaaatcgttaaaaaagaatgatgccgtcgtggcaattatgaaagacttatta1380gatagcgtgaagtccttcgagaattatattaaggcgttctttggagagggtaaggagaca1440aaccgcgacgaatcgttttacggcgatttcgtgttggcctacgacatcttgctgaaagtt1500gatcacatttacgacgccatccgtaactatgtaactcagaaaccctacagtaaggacaag1560tttaagttatactttcagaatccgcagttcatgggtggatgggataaggacaaagagaca1620gactaccgtgctactatcttacgctacgggtcgaagtattaccttgcaattatggataag1680aagtacgcaaaatgcttgcagaagatcgataaagacgacgtcaacggaaattatgaaaag1740atcaattataaattattacccgggcctaataaaatgttgcccaaagtatttttctctaag1800aaatggatggcctactacaatccatcagaagacattcagaagatctataaaaacggcaca1860ttcaaaaaaggcgatatgttcaacttgaacgattgccacaaactgattgatttctttaag1920gatagcattagccgttacccgaagtggtccaacgcttatgactttaacttcagcgaaaca1980gagaagtataaagacattgcagggttctaccgtgaggtagaagaacaaggatataaagtt2040tctttcgaaagtgcaagtaagaaggaagtagataaattggttgaggagggcaaactgtat2100atgtttcagatttacaataaggatttctctgacaagtcccacggcacacccaatttgcat2160actatgtacttcaaacttctgtttgacgagaataaccatgggcagatccgtcttagtggg2220ggagctgagttatttatgcgccgtgcgtctttgaaaaaggaggagttagttgttcacccg2280gcaaattcacccattgccaacaaaaatccagataaccccaagaagacaaccactctttca2340tacgatgtgtacaaagataagcgtttctctgaagatcaatacgaactgcatattccgatc2400gcaatcaacaagtgcccaaagaacatctttaaaatcaatacggaagttcgcgttcttctg2460aaacatgatgataacccgtatgttattggaattgatcgtggggagcgtaacttgttgtat2520attgtcgtagtagatggaaaaggtaacatcgtcgaacagtactcgctgaatgagattatt2580aacaacttcaacgggattcgcatcaagactgattaccactcacttcttgacaaaaaggag2640aaggagcgctttgaagcccgtcaaaattggacatcaatcgaaaatattaaggagctgaaa2700gccggatacatctctcaagtggtccataaaatttgtgaactggtcgagaaatatgatgcc2760gtgatcgcgcttgaggacctgaatagtggattcaaaaactcgcgtgtgaaagttgagaaa2820caggtgtatcagaaatttgagaaaatgttaattgacaaattgaactacatggtagataaa2880aagagtaacccatgtgcgacgggcggtgcattgaagggataccagatcaccaataagttt2940gagtctttcaaaagtatgtctacgcagaatggttttattttctacatcccggcttggctg3000acatctaaaattgacccttcgacgggttttgtcaatctgctgaaaacaaagtatacaagt3060attgcggattcaaaaaaattcatctcatctttcgatcgcatcatgtacgtcccagaagaa3120gacttatttgagtttgcactggattataagaatttttcccgtactgacgcagactatatc3180aagaaatggaaattatattcatacgggaaccgcattcgtatcttccgtaatcctaaaaag3240aacaacgttttcgactgggaagaagtatgcttaaccagtgcttacaaagaattgttcaac3300aaatacggcattaactaccagcagggcgacattcgcgcattattatgcgaacagtcagat3360aaagcgttctactccagctttatggcgttaatgtcgcttatgttacagatgcgcaacagc3420attacaggccgcaccgatgtcgattttctgatttcgccggtgaaaaacagcgatggcatt3480ttttacgattctcgcaattacgaggcgcaggaaaatgcaattcttccgaagaacgcagac3540gcaaacggcgcttacaacatcgcacgtaaagttctttgggcgatcggacaatttaaaaaa3600gccgaggatgaaaaattggacaaggttaaaattgctatctcgaataaagagtggcttgag3660tacgcgcaaacttctgttaagcactga3687<210>38<211>3687<212>dna<213>artificialsequence<220><223>lbcpf1-g532r<400>38atgtctaagctggaaaaattcaccaactgctacagtttaagcaagacgttacgcttcaag60gcgatcccggttgggaaaacacaagagaatattgacaacaaacgccttttagttgaagat120gagaagcgtgcagaggactataaaggagttaagaagcttttggaccgttactacttatcg180tttattaatgatgtgctgcattccatcaaactgaagaatctgaataattacattagtctt240ttccgtaagaaaacacgtactgagaaggaaaacaaggagctggaaaatcttgagatcaat300ttgcgcaaggagattgctaaggcattcaaaggtaacgagggatataagtcgttgtttaaa360aaagacattattgaaacgatccttccggaattccttgacgataaagacgagattgcactg420gtgaactccttcaacgggttcacgactgcctttacgggtttttttgacaaccgcgagaat480atgttcagtgaagaagccaaatctacctctatcgctttccgctgcattaatgaaaacctg540actcgttatatctctaacatggacatcttcgaaaaggtagacgcgatctttgataaacac600gaagtgcaggagatcaaggagaaaattcttaatagcgactatgatgttgaagatttcttt660gagggggaattctttaactttgtactgacgcaagaagggatcgacgtctataacgctatt720atcggaggattcgtaaccgagagtggagagaaaattaagggtcttaatgagtacattaat780ttgtacaatcaaaaaacgaaacagaaattgcccaagtttaaacctctgtataaacaagtg840ctgagtgatcgcgaaagtttgtcattctatggagaggggtatactagcgacgaggaggtt900cttgaggtgtttcgcaacaccttaaacaagaactctgagattttcagctcaatcaaaaaa960cttgagaaattatttaaaaatttcgacgaatactcgtcagccggcattttcgtgaaaaat1020ggtccggccatttccacaatctctaaggacatctttggagaatggaacgttatccgtgac1080aaatggaacgctgaatatgatgacatccatttgaaaaagaaggctgtcgttacagaaaaa1140tatgaagatgatcgccgcaagagcttcaaaaagatcggttcgttctcgcttgagcagttg1200caagaatacgcagatgccgatctgtcagtagtagagaaattaaaggaaatcattattcaa1260aaagtggacgagatttacaaagtttacggatcctctgagaagctttttgatgcagatttt1320gtgttggagaaatcgttaaaaaagaatgatgccgtcgtggcaattatgaaagacttatta1380gatagcgtgaagtccttcgagaattatattaaggcgttctttggagagggtaaggagaca1440aaccgcgacgaatcgttttacggcgatttcgtgttggcctacgacatcttgctgaaagtt1500gatcacatttacgacg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60aaacatgatgataacccgtatgttattggaattgatcgtggggagcgtaacttgttgtat2520attgtcgtagtagatggaaaaggtaacatcgtcgaacagtactcgctgaatgagattatt2580aacaacttcaacgggattcgcatcaagactgattaccactcacttcttgacaaaaaggag2640aaggagcgctttgaagcccgtcaaaattggacatcaatcgaaaatattaaggagctgaaa2700gccggatacatctctcaagtggtccataaaatttgtgaactggtcgagaaatatgatgcc2760gtgatcgcgcttgaggacctgaatagtggattcaaaaactcgcgtgtgaaagttgagaaa2820caggtgtatcagaaatttgagaaaatgttaattgacaaattgaactacatggtagataaa2880aagagtaacccatgtgcgacgggcggtgcattgaagggataccagatcaccaataagttt2940gagtctttcaaaagtatgtctacgcagaatggttttattttctacatcccggcttggctg3000acatctaaaattgacccttcgacgggttttgtcaatctgctgaaaacaaagtatacaagt3060attgcggattcaaaaaaattcatctcatctttcgatcgcatcatgtacgtcccagaagaa3120gacttatttgagtttgcactggattataagaatttttcccgtactgacgcagactatatc3180aagaaatggaaattatattcatacgggaaccgcattcgtatcttccgtaatcctaaaaag3240aacaacgttttcgactgggaagaagtatgcttaaccagtgcttacaaagaattgttcaac3300aaatacggcattaactaccagcagggcgacattcgcgcattattatgcgaacagtcagat3360aaagcgttctactccagctttatggcgttaatgtcgcttatgttacagatgcgcaacagc3420attacaggccgcaccgatgtcgattttctgatttcgccggtgaaaaacagcgatggcatt3480ttttacgattctcgcaattacgaggcgcaggaaaatgcaattcttccgaagaacgcagac3540gcaaacggcgcttacaacatcgcacgtaaagttctttgggcgatcggacaatttaaaaaa3600gccgaggatgaaaaattggacaaggttaaaattgctatctcgaataaagagtggcttgag3660tacgcgcaaacttctgttaagcactga3687<210>40<211>3687<212>dna<213>artificialsequence<220><223>lbcpf1-t152r-g532r<400>40atgtctaagctggaaaaattcaccaactgctacagtttaagcaagacgttacgcttcaag60gcgatcccggttgggaaaacacaagagaatattgacaacaaacgccttttagttgaagat120gagaagcgtgcagaggactataaaggagttaagaagcttttggaccgttactacttatcg180tttattaatgatgtgctgcattccatcaaactgaagaatctgaataattacattagtctt240ttccgtaagaaaacacgtactgagaaggaaaacaaggagctggaaaatcttgagatcaat300ttgcgcaaggagattgctaaggcattcaaaggtaacgagggatataagtcgttgtttaaa360aaagacattattgaaacgatccttccggaattccttgacgataaagacgagattgcactg420gtgaactccttcaacgggttcacgactgcctttcgtggtttttttgacaaccgcgagaat480atgttcagtgaagaagccaaatctacctctatcgctttccgctgcattaatgaaaacctg540actcgttatatctctaacatggacatcttcgaaaaggtagacgcgatctttgataaacac600gaagtgcaggagatcaaggagaaaattcttaatagcgactatgatgttgaagatttcttt660gagggggaattctttaactttgtactgacgcaagaagggatcgacgtctataacgctatt720atcggaggattcgtaaccgagagtggagagaaaattaagggtcttaatgagtacattaat780ttgtacaatcaaaaaacgaaacagaaattgcccaagtttaaacctctgtataaacaagtg840ctgagtgatcgcgaaagtttgtcattctatggagaggggtatactagcgacgaggaggtt900cttgaggtgtttcgcaacaccttaaacaagaactctgagattttcagctcaatcaaaaaa960cttgagaaattatttaaaaatttcgacgaatactcgtcagccggcattttcgtgaaaaat1020ggtccggccatttccacaatctctaaggacatctttggagaatggaacgttatccgtgac1080aaatggaacgctgaatatgatgacatccatttgaaaaagaaggctgtcgttacagaaaaa1140tatgaagatgatcgccgcaagagcttcaaaaagatcggttcgttctcgcttgagcagttg1200caagaatacgcagatgccgatctgtcagtagtagagaaattaaaggaaatcattattcaa1260aaagtggacgagatttacaaagtttacggatcctctgagaagctttttgatgcagatttt1320gtgttggagaaatcgttaaaaaagaatgatgccgtcgtggcaattatgaaagacttatta1380gatagcgtgaagtccttcgagaattatattaaggcgttctttggagagggtaaggagaca1440aaccgcgacgaatcgttttacggcgatttcgtgttggcctacgacatcttgctgaaagtt1500gatcacatttacgacgccatccgtaactatgtaactcagaaaccctacagtaaggacaag1560tttaagttatactttcagaatccgcagttcatgcgtggatgggataaggacaaagagaca1620gactaccgtgctactatcttacgctacgggtcgaagtattaccttgcaattatggataag1680aagtacgcaaaatgcttgcagaagatcgataaagacgacgtcaacggaaattatgaaaag1740atcaattataaattattacccgggcctaataaaatgttgcccaaagtatttttctctaag1800aaatggatggcctactacaatccatcagaagacattcagaagatctataaaaacggcaca1860ttcaaaaaaggcgatatgttcaacttgaacgattgccacaaactgattgatttctttaag1920gatagcattagccgttacccgaagtggtccaacgcttatgactttaacttcagcgaaaca1980gagaagtataaagacattgcagggttctaccgtgaggtagaagaacaaggatataaagtt2040tctttcgaaagtgcaagtaagaaggaagtagataaattggttgaggagggcaaactgtat2100atgtttcagatttacaataaggatttctctgacaagtcccacggcacacccaatttgcat2160actatgtacttcaaacttctgtttgacgagaataaccatgggcagatccgtcttagtggg2220ggagctgagttatttatgcgccgtgcgtctttgaaaaaggaggagttagttgttcacccg2280gcaaattcacccattgccaacaaaaatccagataaccccaagaagacaaccactctttca2340tacgatgtgtacaaagataagcgtttctctgaagatcaatacgaactgcatattccgatc2400gcaatcaacaagtgcccaaagaacatctttaaaatcaatacggaagttcgcgttcttctg2460aaacatgatgataacccgtatgttattggaattgatcgtggggagcgtaacttgttgtat2520attgtcgtagtagatggaaaaggtaacatcgtcgaacagtactcgctgaatgagattatt2580aacaacttcaacgggattcgcatcaagactgattaccactcacttcttgacaaaaaggag2640aaggagcgctttgaagcccgtcaaaattggacatcaatcgaaaatattaaggagctgaaa2700gccggatacatctctcaagtggtccataaaatttgtgaactggtcgagaaatatgatgcc2760gtgatcgcgcttgaggacctgaatagtggattcaaaaactcgcgtgtgaaagttgagaaa2820caggtgtatcagaaatttgagaaaatgttaattgacaaattgaactacatggtagataaa2880aagagtaacccatgtgcgacgggcggtgcattgaagggataccagatcaccaataagttt2940gagtctttcaaaagtatgtctacgcagaatggttttattttctacatcccggcttggctg3000acatctaaaattgacccttcgacgggttttgtcaatctgctgaaaacaaagtatacaagt3060attgcggattcaaaaaaattcatctcatctttcgatcgcatcatgtacgtcccagaagaa3120gacttatttgagtttgcactggattataagaatttttcccgtactgacgcagactatatc3180aagaaatggaaattatattcatacgggaaccgcattcgtatcttccgtaatcctaaaaag3240aacaacgttttcgactgggaagaagtatgcttaaccagtgcttacaaagaattgttcaac3300aaatacggcattaactaccagcagggcgacattcgcgcattattatgcgaacagtcagat3360aaagcgttctactccagctttatggcgttaatgtcgct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