CN113267623A - 一种同时检测新冠病毒等多种病原体的试剂盒及制备方法 - Google Patents
一种同时检测新冠病毒等多种病原体的试剂盒及制备方法 Download PDFInfo
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Abstract
本发明属于医学检测技术领域,提供了一种能同时检测新冠病毒等多种呼吸道病原体的微阵列芯片试剂盒及制备方法与应用。该试剂盒配合新冠病毒核酸检测方法,大大地增加了检测的灵敏度和降低了漏检率。达到高效、节省时间、高通量同时诊断的目的。无论对于基层还是大型医疗机构,配以芯片阅读仪后可以达到定量诊断的目的,本方法商业化后对基层人员的要求不高,是核酸检测的有力补充。是目前比较理想的技术平台方法,在国内外均未见到类似的方法。目前已经应用于新冠病毒疫苗接种后人体保护性抗体的监测中。
Description
技术领域
本发明属于医学检测技术领域,涉及一种同时检测新冠病毒等多种病原体的试剂盒及制备方法,具体涉及一种能同时检测新冠病毒等8种或18种呼吸道病原体的微阵列芯片试剂盒及制备方法与应用。
背景技术
呼吸道感染是由多种微生物包括细菌、病毒、支原体、真菌、寄生虫等引起的感染性疾病。
上呼吸道感染:90%左右由病毒引起,细菌感染常继发于病毒感染之后。该病四季、任何年龄均可发病,通过含有病毒的飞沫、雾滴,或经污染的用具进行传播。常于机体抵抗力降低时,如受寒、劳累、淋雨等情况,原已存在或由外界侵入的病毒或/和细菌,迅速生长繁殖,导致感染。该病预后良好,有自限性,一般5~7天痊愈。常继发支气管炎、肺炎、副鼻窦炎,少数人可并发急性心肌炎、肾炎、风湿热等。
下呼吸道感染:临床上供选择的抗生素日益增多,耐药菌株亦明显增多,由于大剂量头孢菌素的应用,导致院内感染特别是假单孢铜绿杆菌和肠球菌感染日益增多。无论是下呼吸道感染还是上呼吸道感染,绝大部分是由病毒引起的,称病毒性呼吸道感染。
病毒性的感染是多种病毒引起的急性呼吸道感染。分病毒性上呼吸道感染和病毒性下呼吸道感染,前者表现为普通感冒、急性咽炎、扁桃体炎、喉炎,后者表现为气管-支气管炎、肺炎。本类疾病发病率高,人群普遍易染,小儿、老人、营养不良及患有慢性病者更易患病。以冬春季发病较多。
一种新型冠状病毒被WHO命名“SARS-CoV-2”,其所引起的肺炎被命名为“COVID-19”。人感染冠状病毒后常见的症状包括呼吸道症状,发热、乏力、干咳、呼吸急促和呼吸困难等,严重的情况下,感染会导致肺炎、急性呼吸道综合症甚至死亡。目前对于新型冠状病毒所致疾病没有特异治疗药物,早诊断、早隔离、早接种疫苗预防是主要的诊断和预防手段!
由于新冠病毒属于乙类呼吸道传染病,传播力非常强,致死率高于普通流感病毒,且疾病的初期以及后期,经常伴有其他呼吸道病原感染,较难与新冠病毒鉴别,因此,无论在疾病初期还是后期,鉴别诊断始终是很重要的环节。本发明进行了大约400份新冠病毒标本的检测,从早期、中期、晚期患者的血清标本中,检测出了其他常见病原体的伴随感染状况,总之来说,大约有75%的病例伴随其他常见呼吸道病毒如流感病毒A、B型和肺炎支原体及肺炎衣原体、呼吸道和博爱病毒等的混合感染现象。
基于上述实验数据及本发明现有技术情况,本发明提供了联合检测临床上最常见呼吸道病原体(包括新冠病毒)的微阵列芯片技术,一次实验可以检测出8 种或18种目前临床上最常见的呼吸道病原体,包括新型冠状病毒 (SARS-CoV-2),为鉴别新发传染病提供有力的辅助诊断手段。
众所周知,呼吸道病原体的检测主要有:
(1)病原学分离培养;
(2)血清学检测:监测免疫系统抗体反应(IgM/IgG/IgA);
(3)核酸检测:目前在新型冠状病毒临床诊断主要靠核酸检测,其具有灵敏度高、可缩短窗口期检测时间等优点,但令人担心的问题是2019-nCoV核酸检测的假阴性,就目前的数据来看,疑似病例的病毒核酸检测假阴性率颇高。抗体的检测对于进一步区分隐性或无症状感染者是极为有效的手段,对协助CT检查阳性而核酸检测阴性患者有重要辅助意义。另外对于普通流感及上下呼吸道感染与新型冠状病毒的即时鉴别诊断尤其意义重大,也对疫苗效果的监测具有无可替代的作用。
上述方法都各有优缺点,但都需要一次实验只能检测一种病原体,或者一个抗体,不能同时检测出8种或18种病原体抗体。在检测效率方面费时费力,尤其传统的分离培养需要3-5天时间。PCR方法固然敏感和特异,但也要几个小时,也需要昂贵仪器,一次实验只能作出一种病原体的诊断,在基层医疗机构无法实施,因为它需要有严格控制的操作空间和结净环境且假阴性高,经常需要CDC 进行复核,检测成本进一步提高。
发明内容
本发明涉及呼吸道病原体感染检测中一种用于同时检测新冠病毒 (SARS-CoV-2或2019nCoV)等18种常见呼吸道病原体的微阵列芯片方法。包括其他呼吸道病原体(腺病毒(ADV)、呼吸道合胞病毒(RSV)、A型流感病毒(IFV-A)、B型流感病毒(IFV-B)、副流感病毒(PIV-I、II、III)、肺炎支原体(MP)、肺炎衣原体(CP)、人偏肺病毒(HMPV)、细小病毒(HRV)、嗜血杆菌(HI)、军团杆菌(LP)、肺炎链球菌(SP)、金黄色葡萄球菌(SA)、铜绿假单胞菌(PA)、肺炎克雷白杆菌(KP)。具体地说是一种用微阵列蛋白质芯片方法同时高通量检测临床上常见相似症状的呼吸道病原体IgM/IgG/IgA 抗体的方法的建立。
本发明所要解决的技术问题在于提供一种微阵列蛋白质芯片点阵的方法来实现高通量同时检测临床上最常见的18种呼吸道病原体感染(包含新型冠状病毒),即一次检测可得知是否有新型冠状病毒感染,亦或是其他普通呼吸道病原体感染,具有鉴别感染类型和鉴别诊断的作用,尤其在新发传染病流行初期以及新冠病毒混合感染的患者的治疗方面,只有严格的分型和鉴别诊断才能达到有效的治疗及阻断传染病的目的。
为解决上述技术问题,本发明设计了一种能同时检测临床上常见18种呼吸道病原体(包含新冠病毒)感染的生物芯片方法,其采用微阵列蛋白芯片技术对上述型别进行同时检测。
首先,本发明提供了一种同时检测新冠病毒等多种病原体的试剂盒及制备方法,包括如下步骤:
A:准备好已活化处理的玻璃芯片片基;
B:准备待点样的呼吸道病原体纯化或基因工程抗原;
C:准备质控蛋白,经过多次试验确定,无论待检样品是阳性还是阴性,该蛋白均能在反应结束后显示质控点,在阅读仪或扫描仪下能清晰出现,以监测实验的有效性;
D:进行位点图案排布设计,图案中的各位点分别代表质控点、病原体抗原位点,采用双位点设计;
E:点样,将质控蛋白和各型别抗原按步骤D中设计的图案用机械手或人工点样与芯片上,制作成微阵列检测芯片。用于同时检测不同种类呼吸道病原体及亚型所产生的抗体,包括IgM和IgG及IgA型抗体。
所述的呼吸道病原体包括:
腺病毒(ADV)、呼吸道合胞病毒(RSV)、A型流感病毒(IFV-A)、B 型流感病毒(IFV-B)、副流感病毒(PIV-I、II、III)、肺炎支原体(MP)、肺炎衣原体(CP)、人偏肺病毒(HMPV)、细小病毒(HRV)、嗜血杆菌(HI)、军团杆菌(LP)、肺炎链球菌(SP)、金黄色葡萄球菌(SA)、铜绿假单胞菌 (PA)、肺炎克雷白杆菌(KP)、新型冠状病毒(SARS-CoV-2)。
所述质控蛋白包括质控点和阴性对照点及阳性对照。
所述的呼吸道病原体微阵列芯片的一种规格为呼吸道病原体18联检微阵列芯片,其微阵列设计图形如下图1所示。
所述的位点排布图案为7×7的芯片点阵,为双位点设计,从上至下用A、B、 C、D、E、F、G分别代表行,从左至右用数字1、2、3、4、5、6、7分别代表列。其中,各位点A1、B1、C1、D1、E1、F1、G1均代表质控点(PC位点);
各位点:
A2 B2代表IFV-A位点;C2 D2代表RSV位点;E2F2代表SA位点
A3 B3代表IFV-B位点;C3D3代表HRV位点;E3F3代表LP位点
A4 B4代表PIV-I位点;C4D4代表HMPV位点;E4F4代表SP位点 A5 B5代表PIV-II位点;C5D5代表SARS-CoV-2位点;E5F5代表KP位点
A6 B6代表PIV-III位点;C6D6代表HI位点;E6F6代表MP位点
A7 B7代表ADV位点;C7D7代表PA位点;E7F7代表CP位点
G2G3代表空白对照位点;G4 G5代表阴性对照(NC)位点,G6G7代表阳性对照(PC)位点。
其中,阵列图中C5D5为2019-nCoV,即新冠病毒抗原位点。
所述的呼吸道病原体微阵列芯片的另一种规格为呼吸道病原体8联检微阵列芯片,其微阵列设计图形如下图2所示。
所述的位点排布图案为5×5的芯片点阵,为双位点设计,从上至下用A、B、 C、D、E分别代表行,从左至右用数字1、2、3、4、5分别代表列。其中,各位点A1、B1、C1、D1、E1均代表质控点(PC位点);
各位点:
A2B2代表ADV位点;A3 B3代表RSV位点;A4 B4代表IFV-A位点;
A5 B5代表IFV-B位点;B1C1代表阳性对照(PC)位点;
C2D2代表PIV位点;C3 D3代表MP位点;C4 D4代表CP位点;
C5D5代表SARS-CoV-2位点;
E2E3代表空白对照位点;E4 E5代表阴性对照(NC)位点。
其中,阵列图中C5D5为2019-nCoV。
本发明进一步提供了一种同时检测新冠病毒等多种病原体的试剂盒,通过所述的制备方法及阵列设计方案制备得到。
本发明进一步提供了所述的试剂盒用于同时检测不同种类呼吸道病原体及亚型所产生的抗体,包括IgM和IgG及IgA型抗体。
用上述微阵列芯片检测结果的意义如下:
1、可同时得知8种或18种不同类型的呼吸道病原体(含新冠病毒)感染的组合,混合感染情况一目了然,有利于针对性的选择治疗方法。
2、NC位点始终不出现反应,是阴性对照;用于质控目的;
3、BC位点也始终不出现反应,是空白对照;用于质控目的;
4、PC位点始终为阳性对照反应。用于质控目的;
5、各位点的反应呈现蓝色或红色斑点(与示踪物有关),弱反应可能呈现半圆型斑点或淡淡的斑点;
6、各位点用于不同标记系统时可出现不同的信号显示,用发光标记物时,各位点为黑色背景下呈白色或蓝色斑点;用荧光标记物时各位点呈各色荧光斑点 (以绿色或黄色为常见)。
本发明相对于现有技术的有益效果包括:
本发明的微阵列蛋白质芯片法应用于新冠病毒诊断,发挥了重大作用和社会效益。配合新冠病毒核酸检测方法,大大地增加了检测的灵敏度和降低了漏检率。达到高效、节省时间、高通量同时诊断的目的。无论对于基层还是大型医疗机构,配以芯片阅读仪后可以达到定量诊断的目的,本方法商业化后对基层人员的要求不高,是核酸检测的有力补充。是目前比较理想的技术平台方法,在国内外均未见到类似的方法。目前已经应用于新冠病毒疫苗接种后人体保护性抗体的监测中。
附图说明
图1:7×7阵列芯片设计图(用于18种呼吸道病原体芯片);
图2:5×5阵列设计图(用于8种呼吸道病原体芯片);
图3:PVC覆膜后已经处理好的玻璃片基;
图4:新冠病毒感染患者微阵列芯片结果检测实例判读(8种呼吸道病原体检测)。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,并参照附图,对本发明作进一步的详细说明。
实施例1
步骤(1)、微阵列芯片的制备方法
(一)微阵列芯片片基的制备
(1)准备好常用显微镜载玻片:
尺寸26mm×76mm,表面洁净,无划痕及油渍(必要时经过浓硫酸浸泡清洗过夜,第二天用自来水冲洗三遍,最后用蒸馏水冲洗三遍,在烤箱里烤干待用)
(2)载玻片的表面处理
将上述洁净载玻片浸泡于3%的KH550烷化剂中过夜,第二天用自来水及蒸馏水分别清洗三遍,烤箱中烤干。再将载玻片置于含5%6-氨基己酸的0.1M pH 值为7.2的PBS溶液中,震荡摇床上反应24h,然后加入碳二亚胺(EDC)至浓度为1.5%,室温下反应2h,再用H2O、1M NaCl及H2O反复清洗至表面不挂水珠为止,再用蒸馏水超声清洗三遍,每遍30秒钟。拿出载玻片,放置于烤箱烤干。待用。
(3)将上述处理好的载玻片,双面分别贴上设计并印刷好文字和标识的 PVC标贴,每个标贴的尺寸大小与载玻片一致,即26mm×76mm,标贴背面敷有3M胶,黏贴于载玻片的两面,如下图3所示。
如图所示,贴好标贴后,载玻片正反面如上述图形,每个载玻片由PVC标贴固定后形成两个方形的标本框,即可以加两份标本。反面的标贴没有印刷,是空白的,这样一个玻璃片基会有两个标本框,是透明的,印刷文字和标志的一面作为正面,两个标本框作为实验反应区。根据方框的大小,每个载玻片可以设计成最多10个小方框,即10个标本框。
(二)微阵列芯片的点样
(1)准备好图1所示的18种呼吸道病原体抗原,或图2所示的8种呼吸道病原体抗原(纯化抗原或/基因工程抗原);
(2)上述抗原均为蛋白质抗原,统一用0.05M pH为9.5的碳酸缓冲液(内含0.1%的碳二亚胺)混匀稀释,按照每种抗原的特定比例进行稀释于96孔微孔板种,标记好每种抗原的位置。
(3)将微孔板置于生物芯片点样仪的点样平台上,按照预先设置好参数,进行微阵列的点样操作,由自动化生物芯片点样仪自动点样,每个样点重复两各位点,点样于每个方框中。
点样设计按照图1或图2的设计位点进行。如果要制作8种呼吸道病原体芯片,就按照图2设计进行,制作18种呼吸道病原体芯片,就按照图1设计图进行。点样环境最好为2-8℃。
(三)微阵列芯片的封闭处理
(1)在0.01M pH 7.2的PBS溶液中,加入1%的BSA,0.4%的硼氢化钠, 2%甘油、0.5%TW-20,0.1%的NaN3,混匀后,每个反应框里加入350μl,在37℃孵育箱里放置两小时,取出微阵列芯片,甩掉液体后在吸水纸上吸干反应框周围液滴。置托盘种排列整齐后置于干燥室干燥4-6小时。
(2)封口:将干燥后的微阵列芯片取出,用封口膜黏贴封住两个反应框,置于2-8°环境下保存备用。
步骤(2)、微阵列芯片试剂盒的样品检测
微阵列芯片试剂盒的检测步骤如下
1、待检测标本:检测IgM、IgG抗体时用血清标本,检测IgA标本时,除过血清标本外,可以用人体分泌物如唾液、乳汁、尿道分泌物及粪便等,只需10-20μl的量。
2、标本稀释:上述标本经过0.01M PBS pH 7.2的缓冲液含5%的牛血清白蛋白(BSA)及防腐剂稀释,稀释比例根据具体标本而不同,如1:20、40、100、 200等。特别是在检测IgM抗体时,标本稀释液中需要另加葡萄球菌A蛋白(SPA) 或者抗人IgG抗体等中和因子,来消除IgG的干扰。
3、标本与微阵列芯片反应:上述稀释后的标本加入到微阵列芯片反应框里,一般根据反应框的大小,来确定加入的标本量大小,从100μl至400μl/框不等,以不溢出反应框为准。然后将芯片放入恒温孵育箱或水浴锅中,37℃反应,时间为15分钟至30分钟不等,可根据标本类型确定反应时间。
4、洗涤:将芯片取出,用洗涤液(0.01M PBS缓冲液含0.5%的TW-20)连续洗涤3次,用吸水纸吸干表面液滴。
5、加入二抗标记物:本步骤可以分别加入抗人IgG、IgM、IgA的酶标记物 (主要为辣根过氧化物酶或碱性磷酸酶)、化学发光标记物(如丫靛酯)或荧光标记物如FITC、CY3、CY5等常用荧光物质。同上37℃反应15-30分钟(依据标本类型不同而定)。同上法洗涤3次并吸干。
6、加底物:加入酶的底物或发光物的底物,如是荧光标记,则不加底物,省去此步骤。
7、结果判断:在含有酶标、发光及荧光阅读功能的生物芯片阅读仪中,读取酶标的显色值、发光值(光子数)或荧光强度值。可以在生物芯片阅读仪上根据标记物不同进行选择任意一种方法的阅读按键,由软件自动识别并判读芯片结果。
8、以酶标记为例,下面是8种呼吸道病原体微阵列芯片结果判读实例,如图4所示。
9、判读结果说明:从图4新冠病毒患者血清标本实测结果可以看出,本微阵列芯片除过检测出新冠病毒(SARS-CoV-2)感染位点外,还同时检测出呼吸道合胞病毒(RSV)、副流感病毒(FLU-B)、肺炎衣原体(CP)感染同时并存的混合感染。而正常阴性样本(图中最后一个反应框)中无任何位点出现反应,只有质控点反应。
本发明于2020年2月20日至3月18日,在广医附一钟南山院士实验室用此微阵列芯片方法共检测了约400份新冠病毒患者血清标本以及80份正常血清标本及其他非新冠患者标本,检测结果显示约有75%的新冠病毒患者合并有其他呼吸道病原感染,为进一步治疗提供了有力佐证。
上述实例测定只是举例了以碱性磷酸酶标记的一种检测方式,但本发明不限于碱性磷酸酶,还包括辣根过氧化物酶(HRP)、葡萄糖氧化酶等,呈现颜色斑点。
若用发光物质标记,可同样测定出与以上例证类似的结果,只不过不是颜色图片,而是发光斑点,在黑色背景下可见的白色或蓝色光斑,大小与上述颜色斑点类似,软件同样可以判断其数值。
用荧光标记物进行检测时,其结果则为黑色背景下出现荧光光点,大小同颜色斑点一致。出现苹果绿或黄色的荧光斑点,只是荧光阅读模式时,其采用激发光,不用加入底物。
实施步骤说明,另一种微阵列芯片试剂盒的样品检测,参照步骤(2)检测方法,采用18种呼吸道病原体微阵列芯片检测时,出现的结果与判断模式与上述图4结果类似,只是反应框比较大(约15×15mm),一次标本实验能够同时检测出18种呼吸道病原体的抗体,对于常见呼吸道感染的鉴别诊断有重要的诊断价值。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (11)
1.一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,包括如下步骤:
A:准备好已活化处理的玻璃芯片片基;
B:准备待点样的呼吸道病原体纯化或基因工程抗原;
C:准备质控蛋白,经过多次试验确定,无论待检样品是阳性还是阴性,该蛋白均能在反应结束后显示质控点,在阅读仪或扫描仪下能清晰出现,以监测实验的有效性;
D:进行位点图案排布设计,图案中的各位点分别代表质控点、病原体抗原位点,采用双位点设计;
E:点样,将质控蛋白和各型别抗原按步骤D中设计的图案用机械手或人工点样与芯片上,制作成微阵列检测芯片。
2.根据权利要求1所述的一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,所述的呼吸道病原体包括:
腺病毒(ADV)、呼吸道合胞病毒(RSV)、A型流感病毒(IFV-A)、B型流感病毒(IFV-B)、副流感病毒(PIV-I、II、III)、肺炎支原体(MP)、肺炎衣原体(CP)、人偏肺病毒(HMPV)、细小病毒(HRV)、嗜血杆菌(HI)、军团杆菌(LP)、肺炎链球菌(SP)、金黄色葡萄球菌(SA)、铜绿假单胞菌(PA)、肺炎克雷白杆菌(KP)、新型冠状病毒(SARS-CoV-2)。
3.根据权利要求1所述的一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,所述质控蛋白包括质控点和阴性对照点及阳性对照。
4.根据权利要求1所述的一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,所述的呼吸道病原体微阵列芯片为呼吸道病原体18联检微阵列芯片,所述的位点排布图案为7×7的芯片点阵,为双位点设计,从上至下用A、B、C、D、E、F、G分别代表行,从左至右用数字1、2、3、4、5、6、7分别代表列;其中,各位点A1、B1、C1、D1、E1、F1、G1均代表质控点(PC位点);
各位点:
A2 B2代表IFV-A位点;C2 D2代表RSV位点;E2F2代表SA位点
A3 B3代表IFV-B位点;C3D3代表HRV位点;E3F3代表LP位点
A4 B4代表PIV-I位点;C4D4代表HMPV位点;E4F4代表SP位点
A5 B5代表PIV-II位点;C5D5代表SARS-CoV-2位点;E5F5代表KP位点
A6 B6代表PIV-III位点;C6D6代表HI位点;E6F6代表MP位点
A7 B7代表ADV位点;C7D7代表PA位点;E7F7代表CP位点
G2G3代表空白对照位点;G4 G5代表阴性对照(NC)位点,G6G7代表阳性对照(PC)位点。
5.根据权利要求1所述的一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,所述的呼吸道病原体微阵列芯片为呼吸道病原体8联检微阵列芯片,
所述的位点排布图案为5×5的芯片点阵,为双位点设计,从上至下用A、B、C、D、E分别代表行,从左至右用数字1、2、3、4、5分别代表列;其中,各位点A1、B1、C1、D1、E1均代表质控点(PC位点);
各位点:
A2B2代表ADV位点;A3 B3代表RSV位点;A4 B4代表IFV-A位点;
A5 B5代表IFV-B位点;B1C1代表阳性对照(PC)位点;
C2D2代表PIV位点;C3 D3代表MP位点;C4 D4代表CP位点;
C5D5代表SARS-CoV-2位点;
E2E3代表空白对照位点;E4 E5代表阴性对照(NC)位点。
6.根据权利要求4或5所述的一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,阵列中C5D5为2019-nCoV,即新冠病毒抗原位点。
7.根据权利要求4或5所述的一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,
步骤A包括:
(1)准备好常用显微镜载玻片:尺寸26mm×76mm,表面洁净,无划痕及油渍;
(2)载玻片的表面处理
将上述洁净载玻片浸泡于3%的KH550烷化剂中过夜,第二天用自来水及蒸馏水分别清洗三遍,烤箱中烤干;再将载玻片置于含5%6-氨基己酸的0.1M pH值为7.2的PBS溶液中,震荡摇床上反应24h,然后加入碳二亚胺(EDC)至浓度为1.5%,室温下反应2h,再用H2O、1MNaCl及H2O反复清洗至表面不挂水珠为止,再用蒸馏水超声清洗三遍,每遍30秒钟;拿出载玻片,放置于烤箱烤干,待用;
(3)将上述处理好的载玻片,双面分别贴上设计并印刷好文字和标识的PVC标贴,每个标贴的尺寸大小与载玻片一致,即26mm×76mm,标贴背面敷有3M胶,黏贴于载玻片的两面;
贴好标贴后,每个载玻片由PVC标贴固定后形成两个方形的标本框,即可以加两份标本;反面的标贴没有印刷,是空白的,这样一个玻璃片基会有两个标本框,是透明的,印刷文字和标志的一面作为正面,两个标本框作为实验反应区;根据方框的大小,每个载玻片可以设计成最多10个小方框,即10个标本框。
8.根据权利要求4或5所述的一种同时检测新冠病毒等多种病原体的试剂盒的制备方法,其特征在于,
步骤B-E包括:
(1)准备好18种呼吸道病原体抗原,或8种呼吸道病原体抗原;
(2)上述抗原均为蛋白质抗原,统一用0.05M pH为9.5的碳酸缓冲液(内含0.1%的碳二亚胺)混匀稀释,按照每种抗原的特定比例进行稀释于96孔微孔板种,标记好每种抗原的位置;
(3)将微孔板置于生物芯片点样仪的点样平台上,按照预先设置好参数,进行微阵列的点样操作,由自动化生物芯片点样仪自动点样,每个样点重复两各位点,点样于每个方框中;
点样设计采用所述的位点排布图案设计,点样环境为2-8℃;
进一步地,微阵列芯片的封闭处理包括:
(1)在0.01M pH 7.2的PBS溶液中,加入1%的BSA,0.4%的硼氢化钠,2%甘油、0.5%TW-20,0.1%的NaN3,混匀后,每个反应框里加入350μl,在37℃孵育箱里放置两小时,取出微阵列芯片,甩掉液体后在吸水纸上吸干反应框周围液滴,置托盘种排列整齐后置于干燥室干燥4-6小时;
(2)封口:将干燥后的微阵列芯片取出,用封口膜黏贴封住两个反应框,置于2-8°环境下保存备用。
9.一种同时检测新冠病毒等多种病原体的试剂盒,其特征在于,所述试剂盒通过权利要求1-8任一项所述的制备方法及阵列设计方案制备得到。
10.根据权利要求9所述的试剂盒用于同时检测不同种类呼吸道病原体及亚型所产生的抗体的应用,其特征在于,包括IgM和IgG及IgA型抗体。
11.根据权利要求10的应用,其特征在于,可以以碱性磷酸酶、辣根过氧化物酶(HRP)、葡萄糖氧化酶等标记的方式检测,呈现颜色斑点;也可以用发光物质标记的方式检测,呈现发光斑点,在黑色背景下可见的白色或蓝色光斑,软件可以判断其数值,用荧光标记物进行检测时,其结果则为黑色背景下出现荧光光点,大小同颜色斑点一致,出现苹果绿或黄色的荧光斑点,只是荧光阅读模式时,其采用激发光,不用加入底物。
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CN114657286A (zh) * | 2022-04-06 | 2022-06-24 | 中国人民解放军军事科学院军事医学研究院 | 一种同时检测12种呼吸道病原体的引物探针组合、试剂盒及检测方法 |
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CN111679083A (zh) * | 2020-02-17 | 2020-09-18 | 浙江大学医学院附属第一医院 | 用于检测新型冠状病毒抗体的碱性磷酸酶蛋白芯片试剂盒及其制备方法 |
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CN114657286A (zh) * | 2022-04-06 | 2022-06-24 | 中国人民解放军军事科学院军事医学研究院 | 一种同时检测12种呼吸道病原体的引物探针组合、试剂盒及检测方法 |
CN114657286B (zh) * | 2022-04-06 | 2022-10-14 | 中国人民解放军军事科学院军事医学研究院 | 一种同时检测12种呼吸道病原体的引物探针组合、试剂盒及检测方法 |
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