CN116064741A - 导向rna探针用于检测蠕形螨的应用和试剂盒及其使用 - Google Patents
导向rna探针用于检测蠕形螨的应用和试剂盒及其使用 Download PDFInfo
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Abstract
本发明公开了一种导向RNA探针在制备检测蠕形螨产品中的应用,所述导向RNA探针的编码DNA序列如SEQ ID No.1至SEQ ID No.10中至少一项所示。本发明公开了一种用于检测蠕形螨的CRISPR‑Cas13系统,包括:Cas13a核酸酶、上述应用中所用导向RNA探针、报告分子;所述导向RNA探针针对蠕形螨几丁质合成酶基因,Cas13a核酸酶通过在导向RNA探针识别靶基因后,激活酶活性,并对报告分子进行切割,释放检测信号。本发明公开了一种试剂盒,包括:等温扩增系统,上述用于检测蠕形螨的CRISPR‑Cas13系统,以及可视化系统。
Description
技术领域
本发明涉及分子生物技术领域,尤其涉及一种导向RNA探针在制备检测蠕形螨产品中的应用,以及一种用于检测蠕形螨的CRISPR-Cas13系统,以及一种试剂盒及其使用方法。
背景技术
蠕形螨(Demodicid mite)是一类在人体寄生的螨类,主要有毛囊蠕形螨(Demodexfolliculorum)和皮脂蠕形螨(Demodex brevis)两种。以往认为蠕形螨是不致病或低致病性的,因为绝大多数人体蠕形螨感染者无自觉症状,表现为无症状的带虫者,或仅有轻微痒感或烧灼感。蠕形螨感染所表现的临床症状因患者的免疫状态、营养状况、寄生的虫种及感染度等因素有关。但近年来研究发现,人体蠕形螨在皮肤内活动时对上皮细胞和腺细胞造成机械性破坏,使毛囊、皮脂腺失去正常的结构和功能,引起毛囊扩张,上皮变性。当寄生虫体较多时,可引起角化过度或角化不全,皮脂腺分泌阻塞及真皮层毛细血管增生并扩张等病变;虫体的机械刺激和其分泌物、代谢物的化学刺激可引起皮肤组织的炎症反应,导致宿主局部皮肤的非细菌性炎症反应。此外,虫体代谢物可引起变态反应,虫体的进出活动携带其他病原生物进入毛囊或皮脂腺可致继发感染,引起毛囊周围细胞浸润,纤维组织增生。临床上常见的症状有患处皮肤轻度潮红和异常油腻,继而出现弥漫性潮红、充血,继发性红斑湿疹或散在的针尖至粟粒大小不等的红色痤疮状丘疹、脓疮、结痂及脱屑,皮脂异常渗出、毛囊口扩大,表面粗糙,皮肤有瘙痒感及烧灼感等。此外,酒渣鼻、毛囊炎、痤疮、脂溢性皮炎和睑缘炎等皮肤病患者的蠕形螨感染率及感染度均显著高于健康人及一般皮肤病患者,表明这些现象可能与蠕形螨的感染有关。
对于蠕形螨病的检测,常用的方法有透明胶带法(cellophane tape method,CTP)、挤压法、睫毛镜检法、以及外耳道蠕形螨检测等。
其中,透明胶带法是目前用于面部蠕形螨检测的有效方法,具有检测面积大、检出率高、无痛无创的优点,适用于人群蠕形螨感染调查和获取成虫虫源,但需过夜,患者次日送检,依从性较差,不适合临床门诊快速检查。挤压法是我国临床门诊常用的检查方法,显微镜镜检可检查到各期螨虫,操作简单快捷,但受检面积有限,检出率较低。睫毛镜检法是眼蠕形螨常用的检测方法。裂隙灯下拔取眼睫毛,最好选择拔取带有圆柱状鳞屑的睫毛,可检出各期蠕形螨;若圆柱状鳞屑致密,则加入荧光素溶液以溶解致密的鳞屑并刺激蠕形螨向外迁徙,提高检出率。而外耳道蠕形螨检测则是通过收集耵聍等外耳道分泌物,镜检蠕形螨。
以上的方法都是基于形态学的方法,需要专业的设备和技术人类来识别蠕形螨,只能在专业的机构进行,不适合患者在家中自检。
发明内容
本发明的目的是为了解决现有技术中存在的缺点,而提出的一种导向RNA探针在制备检测蠕形螨产品中的应用,以及一种用于检测蠕形螨的CRISPR-Cas13系统,以及一种试剂盒及其使用方法。
本发明能够解决现有蠕形螨感染检测需要特殊设备和专业技术的问题,简化检测流程,患者在家中即可完成检测,适用于任何场景的蠕形螨感染的诊断和筛查,并且提高检测的灵敏度。
本发明目的之一在于:提供一种导向RNA探针在制备检测蠕形螨产品中的应用,所述导向RNA探针的编码DNA序列如SEQ ID No.1至SEQ ID No.10中至少一项所示。
该导向RNA探针的序列已记录在本申请人在先申请(申请号为202210917814.6,申请日为2022年08月01日,发明名称为:导向RNA探针及用于检测尘螨变应原的应用和试剂盒)中。
优选地,所述产品为药物、试剂或试剂盒。
优选地,所述导向RNA探针的靶基因为蠕形螨几丁质合成酶基因。蠕形螨几丁质合成酶基因如SEQ ID No.13所示。
优选地,所述导向RNA探针的编码DNA序列包括:用于Cas13a核酸酶识别的核酸序列,以及与靶基因互补的核酸序列。
本发明目的之二在于:提供一种用于检测蠕形螨的CRISPR-Cas13系统,包括:Cas13a核酸酶、上述应用中所用导向RNA探针、报告分子;所述导向RNA探针针对蠕形螨几丁质合成酶基因,Cas13a核酸酶通过在导向RNA探针识别靶基因后,激活酶活性,并对报告分子进行切割,释放检测信号。
优选地,报告分子为非特异性报告分子,其序列为/56-FAM/mArArUrGrGrCmAmArArUrGrGrCmA/3Bio/,其中5’端用FAM标记,3’端用生物素标记。
优选地,还包括:检测缓冲液,检测缓冲液包括:T7 DNA聚合酶、rNTPs和氯化镁。
优选地,检测缓冲液C包括:2U T7 DNA聚合酶、10mM rNTPs和3mM氯化镁。
本发明目的之三在于:提供针对蠕形螨几丁质合成酶基因的引物对,包括:CHS-F和CHS-R,其核酸序列依次如SEQ ID No.11和SEQ ID No.12所示。
本发明目的之四在于:提供上述针对蠕形螨几丁质合成酶基因的引物对在等温扩增中的应用。
本发明目的之五在于:提供一种试剂盒,包括:等温扩增系统,上述用于检测蠕形螨的CRISPR-Cas13系统,以及可视化系统。
优选地,等温扩增系统包括:上述针对蠕形螨几丁质合成酶基因的引物对。
优选地,等温扩增系统还包括:RPA重组酶、反应缓冲液V、醋酸镁溶液、无核酸水。
优选地,RPA重组酶包括:T4噬菌体重组酶UvsX、辅助因子UvsY、DNA聚合酶、单链DNA结合蛋白和dNTPs。
优选地,反应缓冲液V包括:40mM Tris(pH=7.9)、64mM醋酸钾、8mM乙酸镁、0.8mMDTT、2.4mM ATP、16mM磷酸肌酸、80ng/μL肌酸激酶和质量浓度为5%的聚乙二醇。
优选地,醋酸镁溶液摩尔浓度为250mM。
优选地,可视化系统包括:横向流动试纸条、检测层析液。
优选地,横向流动试纸条前端包被有带FAM抗体的纳米金粒,检测线上包被有生物素抗体,质控线上包被有固定抗体。
本发明目的之五在于:提供上述试剂盒的使用方法,包括如下步骤:将样品DNA通过等温扩增系统进行体外扩增;随后经用于检测蠕形螨的CRISPR-Cas13系统对蠕形螨几丁质合成酶基因进行特异性识别;通过可视化系统观察检测信号以判断待检样品是否含有蠕形螨。
横向流动试纸条前端包被有带FAM抗体的荧光微球,其荧光微球采用(羧基)偶联抗体两步法工艺,通过微球的稀释、活化、去除残留EDC、偶联抗体、封闭、去除未结合抗体等步骤制成。控制线(C线)在后端,其上包被有羊抗鼠抗体;检测线(T线)位于控制线前,其上包被有固定抗体。完整的非特异性报告分子不受检测线上的固定抗体限制,会与羊抗鼠抗体结合而被保留在控制线上显色,而经过Cas13a水解的非特异性报告分子可以结合检测线上的固定抗体而显色。
本发明能够对蠕形螨进行检测,采用恒温扩增的方式并结合横向流动试纸条,不需要复杂的温控,具有操作简单,灵敏度高,特异性强,检测速度快等诸多技术优势。本发明通过挤压法获取毛囊分泌物,利用导向RNA探针技术对毛囊分泌物中的蠕形螨几丁质合成酶基因(Chitin Synthase gene)进行检测,具有操作简单,灵敏度高的特点,且不需要特殊设备,患者在家即可完成检测和结果的判读,了解是否有蠕形螨的感染。
附图说明
图1为采用本发明方法检测疑似患者毛囊分泌物提取物为阳性的照片。
图2为透明胶纸法镜检疑似患者为阳性的照片。
具体实施方式
下面结合具体实施例对本发明作进一步解说。
实施例1
1.1引物及探针设计
针对蠕形螨几丁质合成酶基因进行引物设计,并合成DNA序列,具体如下:
名称 | 序列(5'to 3') |
CHS-F | GACCCGGATTATTATGAGT |
CHS-R | TTAGCTTAATCTTACACTAA |
设计探针,导向RNA探针的DNA序列具体如下:
1.2探针制备
合成探针的DNA序列。取5μL导向RNA探针对应的DNA序列(带T7)加入转录反应体系中,混匀;将反应管置于37℃反应16h。反应结束后,用磁珠纯化反应产物,得到导向RNA序列。
转录反应体系为:
试剂 | 用量 |
检测缓冲液C | 12μL |
导向RNA对应的DNA序列(带T7) | 5μL |
无核酸水 | 3μL |
1.3等温扩增
取5μL待检测样品和无核酸水分别加入等温扩增反应体系中,混匀;将反应板置于39℃反应15min,反应结束后,得到待检测样品的等温扩增反应产物。
试剂 | 用量 |
提供针对蠕形螨几丁质合成酶基因的引物对 | 2μL |
缓冲液V和RNA冻干酶 | 25μL |
醋酸镁 | 2.5μL |
无核酸水 | 15.5μL |
模板 | 5μL |
1.4靶向检测
取1μL等温扩增产物进行检测。其中检测反应体系如下:
试剂 | 含量 |
Cas13a核酸酶 | 82.58nM |
导向RNA探针 | 0.4μM |
非特异性报告分子 | 200nM |
检测缓冲液 | 8μL |
等温扩增产物 | 1μL |
非特异性报告分子如下:
反应结束后,向产物添加20μL试纸条检测层析液,混匀离心后将横向流动试纸条的载样区插入检测液中,2-5min后根据检测线条带的明亮度差异,可以通过肉眼直接观察并进行判读。
实施例2
通过门诊选取疑似蠕形螨感染的患者,经挤压法获取其毛囊分泌物,样本保存在Eppendorf管(EPs)中,保存温度为-20℃。本研究中无需伦理许可。将样本置于液氮中冻融1min,然后球磨10min,重复进行4次。使用DNA提取试剂盒(OMEGA,Georgia,USA)提取gDNA,并在-20℃下保存在EPs中。按实施例1的步骤对所得提取物进行检测,如图1所示。
同时对上述患者采用透明胶纸法,其结果如图2所示。
将本发明发明和透明胶纸法的检测结果进行对比,具体如下:
虽然,蠕形螨的检测往往采用透明胶纸法。该方法检测结果准确可靠,但是需要专门的设备和专业的技术知识,而且检测时间较长,需要过夜。
通过与传统的透明胶纸法对比,虽然本发明方法的特异度78.0%不如透明胶纸法的91.4%,但是本方法的灵敏度为85.2%,明显高于透明胶纸法的65.8%;而且检测时间短,能更快的缩小确诊范围。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种导向RNA探针在制备检测蠕形螨产品中的应用,其特征在于,所述导向RNA探针的编码DNA序列如SEQ ID No.1至SEQ ID No.10中至少一项所示。
2.根据权利要求1所述应用,其特征在于,所述产品为药物、试剂或试剂盒。
3.根据权利要求1所述应用,其特征在于,所述导向RNA探针的靶基因为蠕形螨几丁质合成酶基因。
4.根据权利要求1所述应用,其特征在于,所述导向RNA探针的编码DNA序列包括:用于Cas13a核酸酶识别的核酸序列,以及与靶基因互补的核酸序列。
5.一种用于检测蠕形螨的CRISPR-Cas13系统,其特征在于,包括:Cas13a核酸酶、根据权利要求1-4任一项所述应用中所用导向RNA探针、报告分子;
所述导向RNA探针针对蠕形螨几丁质合成酶基因,Cas13a核酸酶通过在导向RNA探针识别靶基因后,激活酶活性,并对报告分子进行切割,释放检测信号。
6.根据权利要求5所述用于检测蠕形螨的CRISPR-Cas13系统,其特征在于,还包括:检测缓冲液,检测缓冲液包括:T7 DNA聚合酶、rNTPs和氯化镁。
7.针对蠕形螨几丁质合成酶基因的引物对,其特征在于,包括:CHS-F和CHS-R,其核酸序列依次如SEQ ID No.11和SEQ ID No.12所示。
8.一种试剂盒,其特征在于,包括:等温扩增系统,如权利要求5或6所述用于检测蠕形螨的CRISPR-Cas13系统,以及可视化系统。
9.如权利要求8所述试剂盒,其特征在于,等温扩增系统包括:如权利要求7所述针对蠕形螨几丁质合成酶基因的引物对。
10.一种如权利要求8或9所述试剂盒的使用方法,其特征在于,包括如下步骤:将样品DNA通过等温扩增系统进行体外扩增;随后经用于检测蠕形螨的CRISPR-Cas13系统对蠕形螨几丁质合成酶基因进行特异性识别;通过可视化系统观察检测信号以判断待检样品是否含有蠕形螨。
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