CN112410358A - 识别嗜水气单胞菌DNAzymes及筛选检测方法与用途 - Google Patents
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Abstract
本发明是用于特异性识别嗜水气单胞菌的DNAzymes,DNAzymes的核苷酸序列为单链DNA片段DAh1、DAh2、DAh3、DAh4或者DAh5,或者Dah1序列的优化序列DAh1‑T1、DAh1‑T2、DAh1‑T3或者DAh1‑T4。本发明得到5条嗜水气单胞菌特异性识别和切割的DNAzymes序列及其优化序列。本发明还提供了DNAzymes的筛选方法与检测方法。本发明提供了多条特异性强识别嗜水气单胞菌DNAzyme序列,用于传感器制作能够有效地对嗜水气单胞菌进行检测,可在食品,环境及水产养殖中应用。
Description
技术领域
本发明属病源性微生物检测技术领域,具体涉及一种用于嗜水气单胞菌特异性检测的DNAzymes的序列,本发明还涉及一种用于快速检测嗜水气单胞菌的方法与用途。
背景技术
DNAzyme是通过体外选择分离的催化性DNA序列,是具有核酸酶活性的单链寡聚核苷酸,可在一定条件下裂解特定的核苷酸底物,可以特异性识别靶标并发生裂解反应。1994年,Joyce和Breaker在体外从大量随机DNA序列中分离出可以被Pb2+激活DNAzyme。它们作为传感器领域中廉价,稳定和可放大的检测元件而引起人们的关注。自DNAzyme被发现以来,在传感领域作为廉价,稳定,耐受性强,可扩增的检测元件而受到人们的关注。这些独特的特征使其检测目标范围从金属离子到生物分子,并获得了良好的结果。例如用于检测Na+,K+,Ag+,Mg2+,Ca2+,Cu2+,Hg2+,Pb2+,Zn2+,Co2+,Ni2+,UO2+,脂多糖,胰岛素,组氨酸,葡萄糖,凝血酶等。
近年来,细菌和病毒的增长已日益污染食品。被污染的食物或饮料可以感染人类。嗜水气单胞菌是一种在食物中具有高致病性的细菌,分布在牲畜,生红肉,海鲜,乳制品,蔬菜和其他食物中人们会被嗜水气单胞菌感染各种疾病,包括菌血症,脑膜炎,肠胃炎和肺部感染。嗜水气单胞菌被认为是水生,陆地环境和人类中的主要病原体。它给食品行业造成巨大的经济损失并影响人们的健康。因此,快速、便捷地检测嗜水气单胞菌感染的方法对于预防和控制嗜水气单胞菌败血症至关重要。常规方法包括培养和生化鉴定,血清分型,ELISA分析,PCR技术,已被广泛使用和接受。大多数方法耗时且费力,不适合在现场应用。如,细胞培养需要很长时间。 pH和温度等环境因素的影响将直接影响血清反应的结果。PCR技术需要提取细菌DNA和许多类型的试剂,操作繁琐。
基于DNAzymes的生物传感器正逐步用于细菌检测,如:结合DNAzyme和脲酶的方法来检测幽门螺杆菌,结合DNAzyme和LAMP的方法检测沙门氏菌,并结合使用DNAzyme和PCR检测单核细胞增生李斯特菌等。简便,高效的嗜水气单胞菌检测方法已成为防止嗜水气单胞菌对食品,公共卫生和水环境污染的主要问题。因此,研究新的基于DNAzyme的传感器,用聚苯乙烯板检测嗜水气单胞菌具有重要的意义。
发明内容
本发明的目的在于针对现有技术的不足,提供一种新的特异性识别嗜水气单胞菌的DNAzyme序列,可用于特异性识别嗜水气单胞菌检测。
本发明的另一个目的是提供了一种利用上述特异性识别嗜水气单胞菌的DNAzyme序列快速检测嗜水气单胞菌的方法。
本发明的再一个目的是提供了一种上述特异性识别嗜水气单胞菌的DNAzyme序列的用途。
本发明的目的是通过以下的技术方案来实现的。本发明公开了一种用于特异性识别嗜水气单胞菌的DNAzymes,其特征在于:所述DNAzymes的核苷酸序列为单链DNA片段DAh1、DAh2、DAh3、DAh4或者DAh5,或者Dah1序列的优化序列DAh1-T1、DAh1-T2、DAh1-T3或者DAh1-T4,各单链DNA片段的核苷酸序列(5’-3’)为:
DAh1序列: GAAAAGCGGTCTGCTGCGCTTCTTCCTCTAGTCTGGGGCCTTCGGCG
CGATGAGCCCTATACAGCGAGTATTCACTTGTATC
DAh2序列: GAAAAGCGGTCTGCTGCGCTTCTCTCTCTAGTCTGGGGCCTTCGGCGC
GATGAGCCCTATA AGCGAGTATTCACTTGTATC
DAh3序列: GAAAAGCGGTCTGCTGACGTATGGTCTATCCGACCATAGAGGACCCTTT
TGCGAGGTGACCGTAGCGAGTATTCACTTGTATC
DAh4序列: GAAAAGCGGTCTACTGCGCTTCTTCCTCTAGTCTGGGGCCTTCGGCG
CGATGAGCCCTATACAGCGAGTATTCACTTGTATC
DAh5序列: GAAAAGCGGTCTACTGCGCTTCTCTCTCTAGTCTGGGGCCTTCGGCGC
GATGAGCCCTATACAGCGAGTATTCACTTGTATC
DAh1-T1序列: GAAAAGCGGTCTGCTGCGCTTCTTCCTCTAGTCTGTATACCTAT
GTTCACTTATGAGCGA
DAh1-T2序列: GAAAAGCGGTCTGCTGCGTCTGGCCTTGAGCTATACCTATGTT
CACTTATGAGCGA
DAh1-T3序列: GAAAAGCGGTCTGCTGCGTCTGTATACCTATGTTCACTT
ATGAGCGA
DAh1-T4: GAAAAGCGGTCTGCTGCGTCTGGGGCCTTATGAGCCCTATACC
TATGTTCACTTATGAGCGA。
本发明所述的一种用于特异性识别嗜水气单胞菌的DNAzymes序列,其进一步优选的技术方案是:可以对本发明DNAzymes序列的进行碱基的突变、改造而得到的DNAzymes序列,并用于嗜水气单胞菌的检测。DNAzymes 的序列的突变、改造包括:磷酸化、甲基化、氨基化、巯基化、同位素化;添加生物素、地高辛、荧光物质、淬灭集团、纳米材料、聚乙二醇、肽段、蛋白、叶酸或酶标记。
本发明还公开了一种用于特异性识别嗜水气单胞菌的DNAzymes序列的筛选方法,所述的DNAzymes序列为以上技术方案所述的序列,筛选步骤如下:
(1)使用primerpremier5软件设计文库引物,使用10%的变性聚丙烯酰胺凝胶10%dPAGE电泳纯化;
(2)用于实验过程的嗜水气单胞菌从甘油管移至LB液体培养基中进行培养,其他用于反筛的菌株根据各自的培养条件培养;
(3)将培养好的嗜水气单胞菌菌悬液进行梯度稀释并涂布在LB固体培养基上,于培养箱过夜培养以检测菌体数;
(4)将步骤(2)所得菌悬液分装于1.5 mL无菌EP管中离心,保留上清液,-20℃储存备用;
(5)正向引物使用生物素标记,DNA链中插入一个腺嘌呤核糖核苷酸,通过PCR过程可将中心区域含有50个随机碱基和两端分别有20个固定碱基数的文库与引物通过PCR反应进行连接,引入rA切割位点和生物素标签,醇沉法纯化回收DNA;
(6)将步骤(5)所得PCR产物使用结合缓冲液溶解,并与链霉亲和素包被的磁珠混合均匀,于37℃连接30 min;
(7)连接结束后磁性分离弃去上清,磁珠用结合缓冲液清洗三次;
(8)用0.2 M的NaOH洗涤磁珠两次,磁性分离弃上清,磁珠上连接有DNA单链;
(9)超纯水清洗磁珠,直至上清液pH值接近于中性;
(10)反向筛选: 使用2×SB混匀磁珠,并将等体积的用于反筛的细菌胞外产物混合液与磁珠混合,37℃孵育2小时,磁性分离,弃上清,使用2×SB清洗磁珠三次,并将其转移至干净无菌的1.5 mL EP管。
(11)正向筛选:与步骤(10)同样的方法,将混合磁珠的2×SB与等体积CEM-AH混合,37℃孵育2小时。磁性分离,保留上清液,醇沉法回收DNA.
(12)以步骤(10)所得DNA为模板,加入引物进行PCR扩增,启动下一轮筛选;
(13)将第九轮所得的PCR产物送往高通量测序并进行DNA序列分析;通过高通量测序,共得到156700条DNA原始序列,根据序列长度和所占百分比由高到低选择了前5条序列进行检测;所述的5条DNA序列即权利要求1所述的DAh1、DAh2、DAh3、DAh4和DAh5;
(14)候选DNAzyme活性及性质检测,并进行截短;检测DAh1的截短序列,得到切割活性高的4条截短序列即权利要求1所述的DAh1T1、DAh1T2、DAh1T3和DAh1T4。
以上技术方案中,优选的技术特征是:
在DNA序列的5’端标记生物素,并插入腺嘌呤核糖核苷酸;
所使用的嗜水气单胞菌菌体浓度为7×108CFU/mL;
DNA与磁珠结合,正反筛反应,醇沉回收均在1.5mL无菌EP管中进行;
使用超微量紫外分光光度计检测DNA浓度;使用全波长多功能微孔板检测仪检测DNAzyme反应荧光强度;DNAzyme筛选过程中使用的磁珠直径为0.5μm。
本发明还公开了 一种用DNAzymes序列进行特异性识别嗜水气单胞菌的检测方法,其特点是:采用所述的用于特异性识别嗜水气单胞菌的DNAzymes序列进行检测,在海藻糖和普鲁兰多糖条件下,将DNAzymes序列固定在聚苯乙烯板。检测时优选将样品加在固定了DNAzyme的检测板上,混合后,快速检测,与空白比较并做出判断。
与现有技术相比,本发明的有益效果是:
本发明提供了多条特异性强识别嗜水气单胞菌DNAzyme序列,用于传感器制作能够有效的对嗜水气单胞菌进行检测,可在食品,环境及水产养殖中应用。
本发明提供的基于DNAzyme的传感器,可使用聚苯乙烯板检测嗜水气单胞菌。添加嗜水气单胞菌后,传感器将释放荧光信号,并且可以使用简单的设备进行荧光成像。该方法简便,快速,可在10 min达到检测目的,可用于嗜水气单胞菌的快速检测。
附图说明
图1为本发明中所设计的DNAzyme裂解反应原理图;
图2为本发明在进行体外筛选时的筛选步骤图及传感器制作图;
图3为本发明中对5条候选DNA序列的切割活性检测图(15%变性聚丙烯酰胺凝胶电泳以及荧光检测方法);
图4为本发明中截短序列活性比较图,截短序列二级结构,截短序列特异性检测图;
图5为本发明中传感器可行性检测图;
图6为本发明中pH优化,传感器检测时间优化,传感器中DNAzyme浓度优化,以及荧光方法检测图;
图7为本发明中传感器敏感性检测图(荧光检测和15%变性聚丙烯酰胺凝胶电泳图);
图8为本发明中传感器特异性检测图;
图9为本发明中传感器稳定性检测图;
图10 为本发明的传感器实际样品检测。
具体实施方式
下面结合附图和具体实施方式,对本发明的特异性识别嗜水气单胞菌的DNAzyme序列的筛选及性质鉴定进行详细说明。
实施例:
一、筛选前准备
(1)含有50个随机碱基的DNA筛选文库及标记有生物素的引物均由上海生工合成:
Lib2: 5'- Phosp-GGAGACCGCTTTTCCGGTCT-N50-CTATGTTC ACTTAT
GAGCGA-3'
FP: 5'- Biotin-GTGAACATAGTrAGGAGACCGCTTTTC-3'
RP: 5'- TCGCTCATAAGTGAACATAG-3'
FS: 5'- FAM-TCGCTCATAAGTGAACATAGTrAGGAGACCGCTTTTC-3'
DF: 5'- GTGAACATAGTAGGAGACCGCTTTTC-3'
DR: 5'- TCGCTCATAAGTGAACATAG-3'
(2) 采用LB液体培养基在30℃,180转对嗜水气单胞菌进行培养,待其OD600nm约为1时停止培养,利用梯度稀释涂布法测定每毫升细菌数量。
(3) 将嗜水气单胞菌菌悬液按1 mL的量平均分装到1.5 mL的无菌EP管中,室温11000转离心5 min,将上清液移置新的无菌EP管,标记并-20℃储存备用。
(4) DNA文库,引物经过10%变性聚丙烯酰胺凝胶电泳(dPAGE)进行纯化。
(5)将随机文库、引物按照以下方案进行PCR扩增以制备初始DNA文库:
50 μL的PCR体系包括:2μL随机文库(终浓度为20~50 ng/μL),各2μL10 μM的引物,8 μL10×PCR反应缓冲液(含有MgCl2),1.5 μL10 mM的dNTP混合液,1 μL5 U/μL TaqDNA聚合酶,33.5 μL超纯水。
PCR循环数优化:选择11,13,15,17,19,21,23,25,27轮PCR过程进行循环数优化。
PCR扩增条件如下:95°C预变性5 min,95°C变性30 s,57.5°C退火30 s,72°C延伸1min,以最优循环数为扩增轮数扩增,最终72°C延伸2 min。
(6)使用2%的琼脂糖凝胶在1×TBE中进行PCR产物检测。
TBE:0.5 M EDTA, pH8. Tris,硼酸。
(7)通过醇沉法纯化回收PCR产物并作为初始筛选文库启动体外筛选过程。
二、体外筛选
(1) 500 μL结合缓冲液洗涤50 μL浓度为50 mg/mL的链霉素包被的磁珠三次,磁性分离,移去上清液,在1.5 mL无菌EP管中进行。
结合缓冲液:10 mM Tris-HCl,1 mM EDTA,1M NaCl,0.01%~0.1% Tween-20,pH7.5
(2) 500 μL结合反应缓冲液溶解上述PCR产物,并与(1)中的磁珠均匀混合,37 ℃轻荡结合30 min,DNA与磁珠相连接。
(3)磁性分离,弃上清,用结合缓冲液清洗磁珠两次。
(4)分两次加入500 μL的0.2M NaOH,每次室温静置2 min后进行磁性分离弃上清。
(5)超纯水清洗磁珠,直至上清液pH值为7左右。
(6) 反筛过程,加入150 μL事先准备好的用于反筛的细菌的胞外产物混合液与等体积的2×筛选缓冲液混匀,置于DNA混合仪上37°C切割反应2 h。
(7) 磁性分离,弃上清,使用2×筛选缓冲液清洗磁珠三次,并转移至干净的1.5mL无菌EP管中。
(8)加入150 μL事先准备好的嗜水气单胞菌胞外产物(CEM-AH),与等体积的2×筛选缓冲液混合均匀后置于DNA混合仪上37°C切割反应1 h。
2×筛选缓冲液(2×SB):100 mM HEPES,300 mM NaCl,30 mMMgCl2,pH7.5
(9)磁性分离,醇沉法回收上清液,干燥,重溶,Q5000紫外分光光度计检测DNA浓度。
(10)PCR产物作为模板启动下一轮筛选。体外筛选过程如图2所示。
(11)传感器制作。整个设备是一种96孔聚苯乙烯板的盖子,它具有许多微区域天然疏水区域,荧光背景信号低,很适合作为DNAzyme的承载区,DNAzyme可以在0.25 M海藻糖和8%普鲁兰多糖中保留所有活性。所以,我们将DAh1T1与2×SB,8%普鲁兰多糖和0.25 M海藻糖在干净的避光管中混合。将悬浮液分配到微区域,根据水滴液封的原理,在微小区域会形成微小的液滴。在黑暗条件下,将样品在50°C的烘箱中干燥1小时,可以形成无色透明固体膜,并将其在室温下放置在干燥的地方直到使用。
高通量测序分析:
高通量测序由上海生工采用IIIumina公司的Miseq技术完成,共得到156700条DNA原始序列,根据序列长度和所占百分比由高到低选择了前5条序列进行检测。
表1为经高通量测序获得的前5条DNA序列以及其所占比例;
表1
Top5 | Sequences of random region (N50, 5'~3') |
DAh1 | GCTGCGCTTCTTCCTCTAGTCTGGGGCCTTCGGCGCGATGAGCCCTATAC |
DAh2 | GCTGCGCTTCTCTCTCTAGTCTGGGGCCTTCGGCGCGATGAGCCCTATAC |
DAh3 | GCTGACGTATGGTCTATCCGACCATAGAGGACCCTTTTGCGAGGTGACCGT |
DAh4 | ACTGCGCTTCTTCCTCTAGTCTGGGGCCTTCGGCGCGATGAGCCCTATAC |
DAh5 | ACTGCGCTTCTCTCTCTAGTCTGGGGCCTTCGGCGCGATGAGCCCTATAC |
候选DNA序列活性检测:
变性聚丙烯酰胺凝胶电泳法(15%dPAGE)。
15%变形聚丙烯酰胺凝胶由15%dPAGE储存液、10%过硫酸铵(现配现用)、TEMED组成。
添加20 μL 2×SB、20μL超纯水、10μLCEM-AH和5 μL 5 μM的DAh-FS,混合均匀后37℃避光切割反应30 min。
向上述反应液中加入8 μL 2×Gel Loading Dye Blue(8M尿素稀释)。
150V电泳50 min。
凝胶成像,拍照分析。
荧光信号监测法。由上海生工合成DNAzyme序列,并将其连接至底物链FS(DAh-FS)。在3'端带有淬灭剂的10µM DNAzyme,连接至10 µM FS(5'带有荧光标签),2×SB,在混合器中均匀混合,在95°C加热3 min,自然冷却至室温。通常,对于每种DNAzyme,裂解反应均以100 µL的体积进行:分为对照(仅2×SB)和样品实验(使用CEM-AH)。4 µL DNA(FS)和DAhDNAzyme混合物(DAh-FS)与45 µL的2×SB和41 µL ddH2O混合。在对照中加入10 µL 2×SB,在测试中加入10µL CEM-AH,混合并进行裂解反应。在(DAh-FS)中添加CEM-AH后,观察到的荧光信号会随时间变化。当裂解反应开始时,将酶标仪(Infinite M1000 Pro,Tecan,瑞士)用于96孔板的实时监测。激发波长为490 nm,发射波长为520 nm。每个实验的最终体积为100 µL,每30 s进行1 h的测试。对照和样品实验进行了三个平行实验,以实现实时荧光监测RNA裂解DNAzyme的活性。裂解反应原理图参照图1。
活性分析:
选择过程总共进行了8轮,使用枯草芽孢杆菌(BS),大肠杆菌(EC),鳗弧菌(VA)和创伤弧菌(VV)的胞外产物混和物以及无菌LB培养基(LB)在2、4、6轮选择中进行了反向选择。在第8轮中添加具有index的测序引物用于PCR,然后送至上海生工(中国上海)进行深度测序。合成了前5条富集度较高的序列进行测试。在存在CEM-AH的情况下,检测切割活性发现,DAh1的DNAzyme的活性最高,DAH2次之,DAh3, DAh4,和DAh5 也具有活性。 参照图3。
截短序列特异性检测
随后,检测DAh1的截短序列,结果如图4A 显示,DAh1T1, DAh1T2,DAh1T3, DAh1T4切割活性都很高。其中,DAh1T1 DNAzyme切割活性最高。所以将截短后的DNAzyme序列用于后续实验制作传感器。可以通过软件https://sg.idtdna.com/UNAFold 对DNAzymes的二级结构进行预判。如图4B显示, DAh1T1该DNAzyme包括一个特殊的茎环结构,可能与CEM-AH的活性有关,可能是靶标结合催化酶裂解的部位。其次检测,DAh1T1, DAh1T2,DAh1T3,和 DAh1T4的选择性,使用2×SB作为对照,使用枯草芽孢杆菌,大肠杆菌,杀鲑气单胞菌,铜绿假单胞菌,迟钝爱德华菌,鳗弧菌和创伤弧菌的胞外产物进行DNAzyme的切割性能检测,结果如图4C,4D所示,使用动力学方法和凝胶电泳法均得到: DNAzyme 的对照实验和其他细菌胞外产物不会诱导DAh1T1、DAh1T2、DAh1T3和 DAh1T4发生裂解反应。仅在CEM-AH存在DNAzyme下才发生裂解反应, 并产生大量荧光信号。
传感器可行性试验
为了测试传感器的可行性,首先在微区中添加了8%的普鲁兰多糖和0.25M海藻糖,DAh1T1-FS, DAh1T2-FS,DAh1T3-FS和DAh1T4-FS和2×SB,使 DNAzyme固定在中心。然后将装有样品的装置移至50°C的烘箱中30 min,可以形成固体膜,加入CEM-AH, 反应几分钟后,固定的DNAzyme溶解,随着时间的延长,荧光信号逐渐增强。结果如图5所示:使用蓝光检测荧光(450 nm-500 nm),对照组没有荧光变化,而实验组可以观察到荧光。
实验条件的优化
适度的pH环境,裂解反应时间和浓度对于传感器的实现都起着至关重要的作用,在我们的研究中对不同的测定条件进行了优化。对反应中的缓冲液pH值进行优化,优化范围为pH(4.5-8.0),如图6A所示:当pH值为7.0时,DNAzyme的活性最高,所以,pH7的缓冲液用于后续实验。背景荧光强度对传感器的干扰比较大,所以对固定在聚苯乙烯板中心的DNAzyme浓度进行优化(DNAzyme的浓度为50nM、100nM、200nM、300nM),致力于在低背景荧光强度和明显的检测信号之间寻找平衡点。结果如图6B显示:当DNAzyme浓度为200nM时,传感器显示出较低背景信号和较高的检测信号,所以我们将200nM DAh1T1-FS, DAh1T2-FS,DAh1T3-FS,和 DAh1T4-FS固定于96孔板盖微型区域制作传感器,进行后续实验。同时,对传感器的检测时间进行检测。结果如图6C和6D,当加入CEM-AH时,10min就可以显示出肉眼可见的荧光信号,普鲁兰多糖固定的DNAzyme复合物先要溶解,才开始发生反应。使用动力学方法检测到随着时间的延长,传感器的信号强度不断增加。
传感器敏感性检测
检测范围是反映传感器可行性的重要指标之一。将不同浓度的细菌嗜水气单胞菌加入到传感器区域,并通过酶标仪和15%dPAGE检测其活性,以此来检测传感器对嗜水气单胞菌的检测范围。结果如图7所示,在蓝光下观察荧光,传感器装置和荧光检测方法可以检测到105CFU / mL的细菌,而15%dPAGE方法能够检测104 CFU/mL细胞,说明该传感器可以实现对嗜水气单胞菌的快速检测。
传感器特异性检测
使用2×结合缓冲液作为对照,结果如图8所示。传感器仅在CEM-AH存在的情况下显示出强烈的荧光信号,除了嗜水气单胞菌的胞外产物能诱导DAh1T1, DAh1T2,DAh1T3,和DAh1T4发生裂解反应,来自其他细菌的胞外产物均不能发生切割反应,近一步说明该传感器可以用于食品,环境中嗜水气单胞菌的检测。
传感器稳定性检测
该检测传感器在室温下保存6个月时,结果显示在图9中,分别加入CEM-AH以及嗜水气单胞菌,该传感器均能保持正常的性能,并能观察到荧光信号变化。 普鲁兰多糖和海藻糖确实可以保证DNAzyme 长期的活性等,这一特性也证实了该传感器的可行性,其检测速度快,可操作性强,简单,稳定性高的性质。
样品检测
将嗜水气单胞菌(108 CFU / mL)分别与牛奶和饮用水混合。将10 µL混合样品添加到传感器的微小区域,反应10 min。使用2×SB(缓冲液)作为对照,实验一式三份。图10的结果表明该传感器确实可以用于现场检测嗜水气单胞菌。
序列表
<110> 江苏海洋大学
<120> 识别嗜水气单胞菌DNAzymes及筛选检测方法与用途
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 82
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
gaaaagcggt ctgctgcgct tcttcctcta gtctggggcc ttcggcgcga tgagccctat 60
acagcgagta ttcacttgta tc 82
<210> 2
<211> 81
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
gaaaagcggt ctgctgcgct tctctctcta gtctggggcc ttcggcgcga tgagccctat 60
aagcgagtat tcacttgtat c 81
<210> 3
<211> 83
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
gaaaagcggt ctgctgacgt atggtctatc cgaccataga ggaccctttt gcgaggtgac 60
cgtagcgagt attcacttgt atc 83
<210> 4
<211> 82
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
gaaaagcggt ctactgcgct tcttcctcta gtctggggcc ttcggcgcga tgagccctat 60
acagcgagta ttcacttgta tc 82
<210> 5
<211> 82
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
gaaaagcggt ctactgcgct tctctctcta gtctggggcc ttcggcgcga tgagccctat 60
acagcgagta ttcacttgta tc 82
<210> 6
<211> 60
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
gaaaagcggt ctgctgcgct tcttcctcta gtctgtatac ctatgttcac ttatgagcga 60
<210> 7
<211> 56
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
gaaaagcggt ctgctgcgtc tggccttgag ctatacctat gttcacttat gagcga 56
<210> 8
<211> 47
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
gaaaagcggt ctgctgcgtc tgtataccta tgttcactta tgagcga 47
<210> 9
<211> 62
<212> DNA
<213> 人工引物(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
gaaaagcggt ctgctgcgtc tggggcctta tgagccctat acctatgttc acttatgagc 60
ga 62
Claims (8)
1.一种用于特异性识别嗜水气单胞菌的DNAzymesy,其特征在于:所述DNAzymes的核苷酸序列为单链DNA片段DAh1、DAh2、DAh3、DAh4或者DAh5,或者Dah1序列的优化序列DAh1-T1、DAh1-T2、DAh1-T3或者DAh1-T4,各单链DNA片段的核苷酸序列(5’-3’)为:
DAh1序列: GAAAAGCGGTCTGCTGCGCTTCTTCCTCTAGTCTGGGGCCTTCGGCG
CGATGAGCCCTATACAGCGAGTATTCACTTGTATC
DAh2序列: GAAAAGCGGTCTGCTGCGCTTCTCTCTCTAGTCTGGGGCCTTCGGCGC
GATGAGCCCTATA AGCGAGTATTCACTTGTATC
DAh3序列: GAAAAGCGGTCTGCTGACGTATGGTCTATCCGACCATAGAGGACCCTTT
TGCGAGGTGACCGTAGCGAGTATTCACTTGTATC
DAh4序列: GAAAAGCGGTCTACTGCGCTTCTTCCTCTAGTCTGGGGCCTTCGGCG
CGATGAGCCCTATACAGCGAGTATTCACTTGTATC
DAh5序列: GAAAAGCGGTCTACTGCGCTTCTCTCTCTAGTCTGGGGCCTTCGGCGC
GATGAGCCCTATACAGCGAGTATTCACTTGTATC
DAh1-T1序列: GAAAAGCGGTCTGCTGCGCTTCTTCCTCTAGTCTGTATACCTAT
GTTCACTTATGAGCGA
DAh1-T2序列: GAAAAGCGGTCTGCTGCGTCTGGCCTTGAGCTATACCTATGTT
CACTTATGAGCGA
DAh1-T3序列: GAAAAGCGGTCTGCTGCGTCTGTATACCTATGTTCACTT
ATGAGCGA
DAh1-T4: GAAAAGCGGTCTGCTGCGTCTGGGGCCTTATGAGCCCTATACC
TATGTTCACTTATGAGCGA 。
2.根据权利要求1中所述的一种用于特异性识别嗜水气单胞菌的DNAzymes,其特征在于:对DNAzymes序列的进行碱基的突变、改造得到的DNAzymes序列,并用于嗜水气单胞菌的检测。
3. 根据权利要求2中所述的一种用于特异性识别嗜水气单胞菌的DNAzymes,其特征在于:DNAzymes 的序列的突变、改造包括:磷酸化、甲基化、氨基化、巯基化、同位素化;添加生物素、地高辛、荧光物质、淬灭集团、纳米材料、聚乙二醇、肽段、蛋白、叶酸或酶标记。
4.一种用于特异性识别嗜水气单胞菌的DNAzymes序列的筛选方法,其特征在于:DNAzymes序列为权利要求1或2或3所述的序列,筛选步骤如下:
(1)使用primerpremier5软件设计文库引物,使用10%的变性聚丙烯酰胺凝胶10%dPAGE电泳纯化;
(2)用于实验过程的嗜水气单胞菌从甘油管移至LB液体培养基中进行培养,其他用于反筛的菌株根据各自的培养条件培养;
(3)将培养好的嗜水气单胞菌菌悬液进行梯度稀释并涂布在LB固体培养基上,于培养箱过夜培养以检测菌体数;
(4)将步骤(2)所得菌悬液分装于1.5 mL无菌EP管中离心,保留上清液,-20℃储存备用;
(5)正向引物使用生物素标记,DNA链中插入一个腺嘌呤核糖核苷酸,通过PCR过程可将中心区域含有50个随机碱基和两端分别有20个固定碱基数的文库与引物通过PCR反应进行连接,引入rA切割位点和生物素标签,醇沉法纯化回收DNA;
(6)将步骤(5)所得PCR产物使用结合缓冲液溶解,并与链霉亲和素包被的磁珠混合均匀,于37℃连接30 min;
(7)连接结束后磁性分离弃去上清,磁珠用结合缓冲液清洗三次;
(8)用0.2 M的NaOH洗涤磁珠两次,磁性分离弃上清,磁珠上连接有DNA单链;
(9)超纯水清洗磁珠,直至上清液pH值接近于中性;
(10)反向筛选: 使用2×SB混匀磁珠,并将等体积的用于反筛的细菌胞外产物混合液与磁珠混合,37℃孵育2小时,磁性分离,弃上清,使用2×SB清洗磁珠三次,并将其转移至干净无菌的1.5 mL EP管;
(11)正向筛选:与步骤(10)同样的方法,将混合磁珠的2×SB与等体积CEM-AH混合,37℃孵育2小时;磁性分离,保留上清液,醇沉法回收DNA.
(12)以步骤(10)所得DNA为模板,加入引物进行PCR扩增,启动下一轮筛选;
(13)将第九轮所得的PCR产物送往高通量测序并进行DNA序列分析;通过高通量测序,共得到156700条DNA原始序列,根据序列长度和所占百分比由高到低选择了前5条序列进行检测;所述的5条DNA序列即权利要求1所述的DAh1、DAh2、DAh3、DAh4和DAh5;
(14)候选DNAzyme活性及性质检测,并进行截短;检测DAh1的截短序列,得到切割活性高的4条截短序列即权利要求1所述的DAh1T1、DAh1T2、DAh1T3和DAh1T4。
5.根据权利要求4所述的筛选方法,其特征在于:所使用的嗜水气单胞菌菌体浓度为7×108CFU/mL;在DNA序列的5’端标记生物素,并插入腺嘌呤核糖核苷酸;DNA与磁珠结合,正反筛反应,醇沉回收均在1.5mL无菌EP管中进行;使用超微量紫外分光光度计检测DNA浓度;使用全波长多功能微孔板检测仪检测DNAzyme反应荧光强度;DNAzyme筛选过程中使用的磁珠直径为0.5μm。
6.一种用DNAzymes序列进行特异性识别嗜水气单胞菌的检测方法,其特征在于:采用权利要求1或2或3所述的用于特异性识别嗜水气单胞菌的DNAzymes序列进行检测,在海藻糖和普鲁兰多糖条件下,将DNAzymes序列固定在聚苯乙烯板。
7.根据权利要求6所述的检测方法,其特征在于,其步骤如下:样品加在固定了DNAzyme的检测板上,混合后,快速检测,与空白比较并做出判断。
8.一种如权利要求1或2或3所述的DNAzymesy的用途,其特征在于:该用途为于将DNAzymes用于嗜水气单胞菌的特异性识别或检测。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103468696A (zh) * | 2012-01-05 | 2013-12-25 | 集美大学 | 嗜水气单胞菌适体及其筛选方法与应用 |
CN107164348A (zh) * | 2017-04-11 | 2017-09-15 | 大连民族大学 | 嗜水气单胞菌表面特异性抗原AhsA及其抗体和应用 |
CN110106178A (zh) * | 2019-05-17 | 2019-08-09 | 淮海工学院 | 用于鳗弧菌识别和切割的DNAzyme及筛选方法与用途 |
-
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- 2020-09-27 CN CN202011035972.6A patent/CN112410358A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103468696A (zh) * | 2012-01-05 | 2013-12-25 | 集美大学 | 嗜水气单胞菌适体及其筛选方法与应用 |
CN107164348A (zh) * | 2017-04-11 | 2017-09-15 | 大连民族大学 | 嗜水气单胞菌表面特异性抗原AhsA及其抗体和应用 |
CN110106178A (zh) * | 2019-05-17 | 2019-08-09 | 淮海工学院 | 用于鳗弧菌识别和切割的DNAzyme及筛选方法与用途 |
Non-Patent Citations (1)
Title |
---|
XIAOYI MA 等: "Rapid detection of Aeromonas hydrophila with a DNAzyme-based sensor", 《FOOD CONTROL 》, pages 1 - 8 * |
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