CN115010184A - 一种基于免疫磁珠的金黄色葡萄球菌重组酶聚合酶扩增检测方法 - Google Patents
一种基于免疫磁珠的金黄色葡萄球菌重组酶聚合酶扩增检测方法 Download PDFInfo
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Abstract
本发明提供了一种基于免疫磁珠的金黄色葡萄球菌重组酶聚合酶扩增检测方法,首先利用溶剂热法制备超顺磁性羧基化四氧化三铁磁珠,然后将热灭活的金黄色葡萄球菌作为免疫原获得了兔抗金黄色葡萄球菌多克隆抗体,借助碳二亚胺法将二者相连,对偶联条件及磁珠抗体质量比进行优化以获得高性能免疫磁珠;其次,筛选重组酶聚合酶扩增技术最佳引物,检测其灵敏度、特异性,最终对免疫磁珠‑重组酶聚合酶扩增检测方法在人工污染样品及临床样品的应用效果进行评估。经充分验证,该检测方法具有高灵敏度、高特异性、简便快速的特点,是一种非常有效的畜产品中金黄色葡萄球菌的实时监测技术。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种基于免疫磁珠的金黄色葡萄球菌重组酶聚合酶扩增检测方法。
背景技术
金黄色葡萄球菌(Staphylococcus aureus,S.aureus)是一种常见的条件致病菌,危害人类和畜牧业的健康。其广泛分布于自然界中,如土壤内、空气中、皮肤表面、前鼻腔甚至胃肠道内,已发现几乎30%的人群是金黄色葡萄球菌的永久定植者[1]。金黄色葡萄球菌不仅是引起食物中毒的主要病原菌之一,还是牛羊乳腺炎的主要致病菌之一。近年来,耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)在牛奶、乳制品和肉中不断检出[2-5],并开始出现与食物链相关和畜牧业相关的跨宿主流行现象[6]。金黄色葡萄球菌对人和动物造成的危害愈发严重,对其预防、检测和控制等方面显得至关重要。目前常用的金黄色葡萄球菌检测方法有传统分离培养法、液相色谱法、ELISA、PCR方法,但存在耗时长、操作复杂、设备要求高、受样品基质影响大等缺点,这限制了对金葡菌进行现场快速检测的应用。
免疫磁分离技术的基本原理是借助偶联在磁珠表面的特异性抗体识别捕获目的抗原,在利用磁性纳米颗粒的超顺磁性从各种复杂的生物流体中分离富集出目的抗原。生物样品检测前的处理方式非常影响后续检测方法的灵敏度、特异性和时效性,典型的磁分离过程中,标记有抗体、适配体的磁珠能特异性结合到目标物(细胞、蛋白质、DNA、RNA、细菌、病毒)上,然后通过外部磁场的分离,使特定目标物从复杂基质中分离出来,达到富集浓缩的效果。现有的食品样品中微生物分离技术有传统分离培养法、低俗离心沉淀法、滤膜过滤法。传统分离培养仍为微生物检测的金标准,但该方法的最大缺点是过程繁琐,耗时长;低俗离心沉淀法则存在设备依赖性、灵敏度低的缺点;过滤法在用注射器推送食物样品、从滤器中抽取滤膜时较为费力。免疫磁珠(Immunomagnetic beads,IMBs)快速高效的富集分离作用在含少量目标物的待测样品预处理中具有很大的优势,即简便快速,灵敏度高,特异性强。
重组酶聚合酶扩增技术(Recombinase Polymerase Amplification,RPA)是一种能在等温下快速扩增目的片段的技术,最大的优势就是可在37-42℃下极快速扩增,仅10-20分钟即可观察结果,并且其扩增产物可用多种方式观察:琼脂糖凝胶电泳、实时定量荧光仪、侧流向试纸条。RPA既摆脱了传统PCR扩增变性、退火的复杂过程,又具有便捷、高灵敏度、特异性,是一种很有前途的快速检测病原菌的方法。与同为等温扩增技术的LAMP相比,RPA所需引物的设计更简单。经过近些年的快速发展,RPA已被广泛应用到各领域检测中,如病原菌检测、食品鉴定等。
[1]Kluytmans J,Van B A,Verbrugh H.Nasal carriage of Staphylococcusaureus:epidemiology,underlying mechanisms,and associated risks[J].ClinicalMicrobiology Reviews,1997,10(3):505-520.
[2]Cui M,Li J,Ali T,et al.Emergence of livestock-associated MRSAST398 from bulk tank milk,China[J].Journal of Antimicrobial Chemotherapy,2020,75(12):3471-3474.
[3]Soltan Dallal M M,Salehipour Z,Sharifi Yazdi M K,et al.Phenotypicand Genotypic Characteristics of Methicillin-Resistant Staphylococcus aureusIsolated from Dairy and Meat Products in Iran[J].Journal of Food Quality andHazards Control,2020,7:108-114.
[4]Tanomsridachchai W,Changkaew K,Changkwanyeun R,et al.AntimicrobialResistance and Molecular Characterization of Methicillin-ResistantStaphylococcus aureus Isolated from Slaughtered Pigs and Pork in the CentralRegion of Thailand[J].Antibiotics(Basel),2021,10(2),206.
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发明内容
针对现有技术的不足,本发明目的是提供一种基于免疫磁珠的金黄色葡萄球菌重组酶聚合酶扩增检测方法。将兔抗金黄色葡萄球菌多克隆抗体连接在自制羧基化四氧化三铁磁珠表面,获得针对于金黄色葡萄球菌的免疫磁珠,旨在捕获待检样品中的金黄色葡萄球菌,实现该菌的富集与纯化;结合重组酶聚合酶扩增技术对分离出的金黄色葡萄球菌进行检测,建立了畜产品中金黄色葡萄球菌IMBs-RPA快速检测方法,为食品中病原菌的现场快速检测提供技术支持。
为达到上述目的,本发明的解决方案是:
一种羧基化四氧化三铁磁珠的制备方法,其包括:
(1)、将六水三氯化铁和醋酸钠分别溶解在乙二醇中搅拌至溶解后混合,将混合液倒入反应釜中升温反应,待反应结束后降至室温,清洗,真空干燥,得到羧基化四氧化三铁磁珠。
优选地,六水三氯化铁的摩尔浓度0.1-0.15mol/L,醋酸钠的摩尔浓度0.5-1mol/L。
优选地,搅拌的转速为250-500rpm,优选为300rpm;时间为3-6h。
优选地,升温反应的温度为160-220℃,反应的时间为4-8h。
优选地,真空干燥的温度为50-70℃,时间为6-9h。
一种羧基化四氧化三铁磁珠,其由上述的制备方法得到。
一种制备金黄色葡萄球菌免疫磁珠的方法,其包括如下步骤:
用预冷的2-(N-吗啉)乙磺酸(MES)分别溶解1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺(EDC)、N-羟基琥珀酰亚胺(NHS),配制成EDC溶液、NHS溶液;将羧基化四氧化三铁磁珠离心,磁分离,弃上清,清洗数次;随之先加入EDC溶液混匀,再向溶液中加入NHS溶液混匀,活化;
(2)、使用MES溶液清洗步骤(1)中处理过的磁珠,然后加入MES溶液和兔抗金黄色葡萄球菌多克隆抗体,置于温箱孵育,磁分离,弃上清;
(3)、加入含牛血清白蛋白(BSA)的磷酸缓冲液(PBS),置于混匀仪上封闭;
(4)、用含叠氮化钠(NaN3)和牛血清白蛋白的溶液,磷酸缓冲液作为免疫磁珠保存液重悬免疫磁珠,保存备用。
优选地,步骤(1)中,活化的温度为25-37℃,时间为0.5-1h。
优选地,步骤(2)中,温箱的温度为37℃,时间为0.5-1.5h。
优选地,步骤(3)中,封闭的温度为37℃,时间为0.5-1.5h。
一种金黄色葡萄球菌免疫磁珠,其由上述的方法得到。
一种金黄色葡萄球菌免疫磁珠-重组酶聚合酶扩增的检测方法,其包括:结合重组酶聚合酶扩增技术,利用设计的引物对使用金黄色葡萄球菌免疫磁珠分离出的金黄色葡萄球菌进行扩增与检测;
其中,金黄色葡萄球菌免疫磁珠为上述的金黄色葡萄球菌免疫磁珠。
优选地,引物的序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16所示。
由于采用上述方案,本发明的有益效果是:
本发明用于金黄色葡萄球菌分离富集的免疫磁珠,是基于自制的四氧化三铁磁性微粒获得的,对金黄色葡萄球菌具有最高可达92.75%的高捕获率、2.5CFU/mL的高灵敏度以及强特异性。该免疫磁珠可以将金黄色葡萄球菌高效快速、简便、准确的从牛奶和猪肉匀浆中分离浓缩出来,以便进一步用于RPA快速检测,具有浓缩待测样品中金黄色葡萄球菌浓度、提高检测灵敏度、排除复杂基质影响、提高检测准确性、减少样品处理时间、非高昂设备依赖性的优点。IMBs-RPA的联合进一步缩短了畜产品样品中金黄色葡萄球菌的检测时间,免疫磁分离、DNA提取、RPA检测完成全过程可控制在2.5h以内。此外,IMBs-RPA具有较高灵敏度和特异性,其灵敏度约为单纯使用PCR方法的1000倍。
附图说明
图1为本发明的基于IMBs的金黄色葡萄球菌RPA快速检测过程示意图。
图2为本发明的实施例1中羧基化Fe3O4磁珠的形态结构示意图(A:Fe3O4磁珠的扫描电子显微镜图,B:Fe3O4磁珠的透射电子显微镜图)。
图3为本发明的实施例1中羧基化Fe3O4磁珠的傅里叶红外光谱图。
图4为本发明的实施例1中羧基化Fe3O4磁珠的磁滞回线图。
图5为本发明的实施例2中金黄色葡萄球菌免疫磁珠的灵敏度示意图。
图6为本发明的实施例2中免疫磁珠捕获金黄色葡萄球菌在透射电子显微镜下的形态图(扫描电子显微镜下金黄色葡萄球菌(A)、免疫磁珠与S.aureus免疫复合物(B)的形态图)。
图7为本发明的实施例2中金黄色葡萄球菌免疫磁珠的特异性示意图。
图8为本发明的实施例3中RPA引物的筛选示意图。
图9为本发明的实施例3中PCR检测的灵敏度示意图。
图10为本发明的实施例3中RPA检测的灵敏度(A)和特异性(B)示意图。
图11为本发明的实施例3中IMBs-RPA及IMBs-PCR在加标样品中的检测示意图(IMBs-RPA对牛奶(A)、IMBs-PCR对牛奶(B)、IMBs-RPA对猪肉(C)、IMBs-PCR对猪肉(D)人工污染样品的检测)。
图12为本发明的实施例3中IMBs-RPA在临床样品中的检测结果示意图(IMBs-RPA对牛奶(A)和猪肉(B)临床样品的检测)。
图13为本发明的实施例3中IMBs-PCR在临床样品中的检测结果示意图(IMBs-PCR对牛奶(A)和猪肉(B)临床样品的检测)。
具体实施方式
本发明提供了一种基于IMBs的金黄色葡萄球菌RPA快速检测方法。
如图1所示,本发明首先利用溶剂热法制备羧基化Fe3O4磁珠,借助碳二亚胺法将磁珠与兔抗金黄色葡萄球菌多克隆抗体偶联,获得金黄色葡萄球菌特异性免疫磁珠;然后将金黄色葡萄球菌免疫磁珠加入待检的牛奶和猪肉均质液中捕获金黄色葡萄球菌,施加外部磁场实现金黄色葡萄球菌的分离与富集,再结合RPA扩增技术实现实际样品中金黄色葡萄球菌的快速、高灵敏度、特异性的检测。
实施例1:
1.溶剂热法制备羧基化Fe3O4磁珠
采用溶剂热一锅法制备Fe3O4纳米粒子,过程如下:将3.51g的FeCl3·6H2O、7.46g的NaAc分别溶解在40mL乙二醇中高速搅拌30min至充分溶解;将两溶液混合后用乙二醇补足100mL继续高速搅拌3h,终浓度分别为0.13mol/L的Fecl3·6H2O、0.91mol/L的NaAc;最后将混合物倒入密封高温反应釜中温度200℃、反应时间5h;待反应温度降至室温后,从反应釜中倒出黑色目的产物,使用无水乙醇和水交替清洗产物各三次,将产物置于真空干燥箱,60℃干燥8h。
2.磁珠理化特性的检测
(1)表面形态检测
取少量干燥的磁性粉末固定一薄层于硅片的双面胶带上,再小心吹去表面未粘住的颗粒,将其直接固定在场发射扫描电子显微镜的镜托,送入机舱内部开始扫描观察磁性颗粒的表面形态和粒径分布。
(2)结构成分检测
取磁性粉末溶于无水乙醇中,使其浓度为1mg/mL,超声分散3次,每次20min,使用吸管将混匀的磁性氧化铁的醇溶液透滴在碳膜铜网上,待自然晾干后用高分辨率场透射电子显微镜观察磁性纳米颗粒的结构、聚集状态。
(3)表面官能团检测
称量1mg干燥后的磁性粉末样品与KBr在研钵中研磨均匀,借助制片机制备压片样品,使用傅里叶变换红外光谱仪在400-4000cm-1波数范围内对磁性粉末的元素成分进行检测。
(4)磁性检测
取30mg干燥的磁性粉末样品送入振动样品磁强计内,选择在25℃下,检测其在(-10 000)-10 000Oe的外部磁场作用下的磁滞回线,用于确定磁性颗粒的超顺磁性及饱和磁强度。
实施例2:
1.金黄色葡萄球菌免疫磁珠的制备:
使用EDC-NHS对羧基化Fe3O4磁珠表面的羧基基团进行活化,活化后的中间酯结构可与兔抗金黄色葡萄球菌抗体上的氨基反应,进而将二者连接在一起制备成能够特异性分离富集金黄色葡萄球菌的免疫磁珠。
步骤如下:
洗涤:取羧基化Fe3O4磁珠于离心管中,磁分离,弃上清;用无水乙醇洗一次,含0.05%Tween-20的pH=5.0、0.1mol/L的2-(N-吗啉)乙磺酸(MES)溶液洗2次。
活化:用预冷的0.1mol/L MES分别配置浓度均为100mg/mL的EDC溶液、NHS溶液;取100μL羧基化Fe3O4磁珠于1.5mL离心管中,磁分离,弃上清;用300μL无水乙醇洗一次、300μLMEST Buffer洗2次;随之先向管中加入200μL EDC溶液混匀并等待2min,再向溶液中加入200μL NHS溶液混匀,置于旋转混匀仪上37℃活化30min。
偶联:将活化后的磁珠分别重悬于pH=7.2、0.01mol/L PBS溶液、pH=5.0、0.1mol/L MES溶液,0.1mol/L、pH=7.2的PBS溶液;随后加入一定量的抗体,固定在旋转混匀仪上37℃孵育1h;磁分离、测定上清中游离免疫球蛋白的浓度,计算抗体的偶联率,确定抗体偶联时的最优缓冲液。
封闭:将质量为5mg、6mg、7mg、8mg的磁珠与终浓度为1mg/mL、1.5mg/mL、2mg/mL的抗体作正交试验以确定磁珠抗体的最佳用量;在旋转混匀仪上37℃孵育1h;借助磁力架,用PBS洗三次;再加入500μL免疫磁珠封闭液(含5%BSA的PBS),在旋转混匀仪上37℃封闭1h;重复洗涤步骤;取103CFU的菌液加入免疫磁珠中,用PBS补足1mL,置于混匀仪上在37℃温箱中孵育1h;磁分离,获得细菌磁珠免疫复合物,用PBS洗三次后,对磁珠菌液免疫复合物进行平板计数计算细菌捕获率,确定最佳的磁珠抗体质量比。
保存:用含0.02%NaN3、0.1%BSA的pH 7.2,0.01mol/L的PBS溶液作为免疫磁珠保存液重悬免疫磁珠浓度为10mg/mL,4℃保存备用。在用1%BSA封闭之前,将免疫磁珠点样至SDS-PAGE以检测抗体是否成功偶联到磁珠上。
2.免疫磁珠的性能检测:
(1)免疫磁珠的灵敏度检测
取6mg制备的免疫磁珠分别对2.5×100、2.5×101、2.5×102、2.5×103、2.5×104、2.5×105CFU/mL的金黄色葡萄球菌进行捕获,同样使用平板计数的方法确定免疫磁珠捕获率,确定免疫磁珠的灵敏度。
(2)免疫磁珠的特异性检测
取免疫磁珠分别对1mL浓度为104CFU/mL的鼠伤寒沙门菌、单增李斯特菌、大肠杆菌、金黄色葡萄球菌的新鲜菌液进行捕获,根据平板计数确定免疫磁珠对不同细菌的捕获性能,从而判定该免疫磁珠的特异性。
实施例3:
基于IMBs-RPA方法的建立
(1)RPA引物的设计与筛选
该技术的核心在于引物的设计与筛选。用于设计金黄色葡萄球菌RPA引物的Nuc基因序列从GenBank中下载,该基因表达金黄色葡萄球菌的耐热核酸酶,是高度保守且特异性的一段序列。根据TwisDx公司RPA设计指南的建议下设计8对RPA引物(如表1所示),并对所设计的引物扩增效果进行评估。RPA反应的具体操作步骤按照Twist@basic试剂盒说明书进行,其扩增产物经苯酚-氯仿萃取纯化,DNA凝胶电泳检测。最终筛选出最佳引物nuc-2.1。
(2)RPA灵敏度和特异性检测
使用最优的引物nuc-2.1,对104、103、102和101CFU金黄色葡萄球菌细胞的DNA进行扩增,用于检测RPA灵敏度。同时,对105CFU的鼠伤寒沙门氏菌、大肠杆菌、单增李斯特菌的基因组进行扩增,研究RPA检测方法的特异性。扩增产物经纯化后在凝胶电泳中观察。
(3)IMBs-RPA在人工污染牛奶和猪肉中的检测性能
将从超市购买的巴氏杀菌牛奶和新鲜猪肉样品参照国标GB 4789.10-2016执行进行黄色葡萄球菌的检验,选择检验结果为金黄色葡萄球菌阴性的样品用于人工污染样品的制备。先用PBS按1:10的比例稀释牛奶和猪肉匀浆,该步骤在无菌操作台中进行,取10mL稀释样品,分别加入终浓度为104、103、102和101CFU/mL的金黄色葡萄球菌培养物。采用IMBs捕获人工污染样品中的金黄色葡萄球菌,并提取金黄色葡萄球菌培养物的基因组DNA。将提取DNA用作模板,采用建立的RPA法进行检测。并用PCR对IMBs-RPA的结果进行验证,扩增产物经纯化后在凝胶电泳中观察。
(4)IMBs-RPA在临床样品中的应用效果
对来自不同奶牛场的20份原料奶和来自不同菜市场的20份冰鲜猪肉,分别用PBS按1:9的比例稀释或均质。再使用已建立的IMBs-RPA方法对其进行检测,同时用PCR对IMBs-RPA的结果进行验证。扩增结果使用DNA凝胶电泳分析。
表1
结果:
(1)形态与结构分析
在SEM(图2中A)和TEM(图2中B)下观察可知,该羧基化Fe3O4磁珠颗粒的尺寸为200nm、形貌均一且单分散的黑色纳米颗粒。
(2)表面官能团分布
如图3,FT-IR图谱显示该磁珠在584cm-1处出现Fe-O键的特征吸收峰,在1536cm-1和1411cm-1处观察到与ν(COO-)非对称振动和ν(COO-)对称振动对应的峰;在3436cm-1、2961cm-1和1042cm-1分别观测到特征吸收带,这可分别归因于O-H键、C-H键和C-O键的伸缩振动。最后,1627cm-1处的吸收带对应于-OH基团的面外弯曲振动(γOH···O面外)。故上述方法成功制备出表面含有多种官能团的磁性颗粒。
(3)磁性测定
如图4所示,羧基化Fe3O4磁珠的磁滞回线从原点经过,说明该磁珠具有良好的顺磁性;饱和磁强度为74.37emu/g,矫顽力为80Oe,故可用于后续制备免疫磁珠。
(4)免疫磁珠的灵敏度
如图5所示,该免疫磁珠捕获率最高可达92.75%,灵敏度为2.5CFU/mL,说明该免疫磁珠的灵敏度高。图6为免疫磁珠捕获金黄色葡萄球菌在透射电子显微镜下的形态图片,可以看到免疫磁珠吸附在金黄色葡萄球菌表面上。
(5)免疫磁珠的特异性
如图7所示,本发明制备的免疫磁珠对金黄色葡萄球菌有极高的捕获率,对鼠伤寒沙门菌、单增李斯特菌、大肠杆菌的非特异性捕获率都在1%以下,这表明该免疫磁珠的特异性也较为良好。
(6)RPA最佳引物
如图8所示,泳道3的电泳条带最亮,且没有二聚体存在,故引物nuc-2.1具有最佳的扩增性能,使用该引物的扩增产物的大小为130bp。
(7)RPA灵敏度和特异性
如图9所示,PCR对金黄色葡萄球菌的敏感性为2.5×103CFU/mL;如图10所示,RPA为2.5CFU/mL。此外,该RPA方法对鼠伤寒杆菌、单核增生李斯特菌和大肠杆菌的特异性检测中,未发现条带。因此,RPA方法对金黄色葡萄球菌的检测具有较高的敏感性和特异性。
(8)IMBs-RPA在加标牛奶和猪肉中的检测性能
如图11所示,在牛奶和猪肉样品中,IMBs-RPA和IMBs-PCR方法对金黄色葡萄球菌的检测灵敏度均为2.5CFU/mL。这些结果表明,IMBs-RPA和IMBs-PCR方法的敏感性约为单纯使用PCR方法的1000倍。同时,使用IMBs-PCR对该结果进行检验,结果与RPA一致。
(9)IMBs-RPA在临床样品中的检测
通过IMBs-RPA方法检测了来自奶牛场和超市的原料奶和冰鲜猪肉各20份样品中金黄色葡萄球菌的存在情况。结果如图12所示,1份牛奶样本金黄色葡萄球菌检测呈阳性;阳性率为5%;2份冷鲜猪肉样品金黄色葡萄球菌检测呈阳性,相应的阳性率为10%。这些结果与PCR得到的结果吻合良好(图13)。值得注意的是,IMBs-RPA法的检测时间比IMB-PCR法短了近1h。
由上述可知,本发明成功制备出粒径200nm的羧基化Fe3O4磁珠,与购买的羧基磁珠相比,自制磁珠具有更高的捕获率。使用5mg IMBs在1h内对2.5-2.5×104(CFU)/mL梯度稀释的金黄色葡萄球菌进行捕获,其平均捕获效率(CE)在62.74-92.75%之间,且具有较高灵敏度和特异性。IMBs-RPA方法对人工污染样品的检测灵敏度达到2.5CFU/mL,该方法在2.5h内可完成细菌捕获、DNA提取、扩增、电泳等全过程;利用IMBs-RPA法对20份生牛奶和冷鲜猪肉的临床样品进行检测,检出率分别为5%(1/20)和10%(2/20),与PCR结果一致。综上所述,本发明制备的200nm羧基化Fe3O4磁珠,可用于临床样品中细菌的分离和富集,建立的金黄色葡萄球菌IMBs-RPA方法具有高灵敏度、高特异性、简便快速的特点,经过充分验证,不仅缩短了检测时间,而且具有较高的灵敏度和特异性,是一种非常有效的畜产品中金黄色葡萄球菌监测技术。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
序列表
<110> 北京农学院
<120> 一种基于免疫磁珠的金黄色葡萄球菌的重组酶聚合酶扩增检测方法
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<213> 人工序列(Artificial Sequence)
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tgtttcaggt gtatcaacca ataatagtct 30
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<213> 人工序列(Artificial Sequence)
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tcacaaacag gtaacggcgt aaatagaagt 30
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<213> 人工序列(Artificial Sequence)
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aggtgtatca accaataata gtctgaatgt 30
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<213> 人工序列(Artificial Sequence)
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caaacagata acggcgtaaa tagaagtggt 30
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<212> DNA
<213> 人工序列(Artificial Sequence)
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taatttaacc gtatcaccat caatcgcttt a 31
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<213> 人工序列(Artificial Sequence)
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acaaacagat aacggcgtaa atagaagtgg t 31
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<213> 人工序列(Artificial Sequence)
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catcacaaac agataacggc gtaaatagaa g 31
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<213> 人工序列(Artificial Sequence)
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Claims (8)
1.一种羧基化四氧化三铁磁珠的制备方法,其特征在于:其包括:
将六水三氯化铁和醋酸钠分别溶解在乙二醇中搅拌至溶解后混合,将混合液倒入反应釜中进行升温反应,待反应结束后降至室温,清洗,真空干燥,获得羧基化四氧化三铁磁珠。
2.根据权利要求1所述的羧基化四氧化三铁磁珠的制备方法,其特征在于:所述六水三氯化铁的摩尔浓度0.1-0.15mol/L,醋酸钠的摩尔浓度0.5-1mol/L;和/或,
所述搅拌的转速为250-500rpm,时间为3-6h;和/或,
所述升温反应的温度为160-220℃,反应的时间为4-8h;和/或,
所述真空干燥的温度为50-70℃,时间为6-9h。
3.一种羧基化四氧化三铁磁珠,其特征在于:其由权利要求1或2所述的制备方法得到。
4.一种利用权利要求3所述的羧基化四氧化三铁磁珠来制备金黄色葡萄球菌免疫磁珠的方法,其特征在于:其包括如下步骤:
(1)、用预冷的2-(N-吗啉)乙磺酸分别溶解1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺、N-羟基琥珀酰亚胺,配制成1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺溶液、N-羟基琥珀酰亚胺溶液;将羧基化四氧化三铁磁珠离心,磁分离,弃上清,清洗数次;然后先加入1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺溶液混匀,再向溶液中加入N-羟基琥珀酰亚胺溶液混匀,活化;
(2)、使用2-(N-吗啉)乙磺酸溶液清洗步骤(1)中处理过的磁珠,然后加入2-(N-吗啉)乙磺酸溶液和兔抗金黄色葡萄球菌多克隆抗体,置于温箱孵育,磁分离,弃上清;
(3)、加入含牛血清白蛋白的磷酸缓冲液,置于混匀仪上封闭;
(4)、用含叠氮化钠和牛血清白蛋白的溶液,磷酸缓冲液作为免疫磁珠保存液重悬免疫磁珠,保存备用。
5.根据权利要求4所述的制备金黄色葡萄球菌免疫磁珠的方法,其特征在于:步骤(1)中,所述活化的温度为25-37℃,时间为0.5-1h;和/或,
步骤(2)中,所述温箱的温度为37℃,时间为0.5-1.5h;和/或,
步骤(3)中,所述封闭的温度为37℃,时间为0.5-1.5h。
6.一种金黄色葡萄球菌免疫磁珠,其特征在于:其由权利要求4所述的方法得到。
7.一种金黄色葡萄球菌免疫磁珠-重组酶聚合酶扩增的检测方法,其特征在于:其包括:结合重组酶聚合酶扩增技术,利用设计的引物对使用金黄色葡萄球菌免疫磁珠分离出的金黄色葡萄球菌进行扩增与检测;
所述金黄色葡萄球菌免疫磁珠为权利要求6所述的金黄色葡萄球菌免疫磁珠。
8.根据权利要求7所述的检测方法,其特征在于:所述引物的序列如SEQ ID NO.1、SEQID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ IDNO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ IDNO.14、SEQ ID NO.15、SEQ ID NO.16所示。
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