CN110734990A - Detection reagents, kits and their applications - Google Patents
Detection reagents, kits and their applications Download PDFInfo
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- CN110734990A CN110734990A CN201911086292.4A CN201911086292A CN110734990A CN 110734990 A CN110734990 A CN 110734990A CN 201911086292 A CN201911086292 A CN 201911086292A CN 110734990 A CN110734990 A CN 110734990A
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Abstract
本发明公开了一种检测试剂、试剂盒及其应用。所述检测试剂包括如下四个引物对中的任意一种或多种的组合,所述四个引物对包括:第一引物对,包括序列分别如SEQ ID NO:1、SEQ ID NO:2所示的第一引物、第二引物;第二引物对,包括序列分别如SEQ ID NO:3、SEQ ID NO:4所示的第三引物、第四引物;第三引物对,包括序列分别如SEQ ID NO:5、SEQ ID NO:6所示的第五引物、第六引物;第四引物对,包括序列分别如SEQ ID NO:7、SEQ ID NO:8所示的第七引物、第八引物。本发明提供的检测方法是以标记的特异性引物扩增适当长度的基因片段,能够保证检测的高度特异性,提高了检测结果的准确性。
The invention discloses a detection reagent, a kit and an application thereof. The detection reagent includes any one or a combination of any one or more of the following four primer pairs, the four primer pairs include: a first primer pair, including sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The first primer and the second primer shown; the second primer pair, including the third primer and the fourth primer whose sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively; the third primer pair, including the sequence as shown in The fifth primer and sixth primer shown in SEQ ID NO: 5 and SEQ ID NO: 6; the fourth primer pair, including the seventh primer and the sixth primer whose sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Eight primers. The detection method provided by the present invention is to amplify gene fragments of appropriate length with labeled specific primers, which can ensure high specificity of detection and improve the accuracy of detection results.
Description
技术领域technical field
本发明涉及一种检测试剂、试剂盒,特别涉及一种用于检测大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌的检测试剂、试剂盒及其应用,属于分子生物学和免疫学技术领域。The invention relates to a detection reagent and a kit, in particular to a detection reagent, a kit and application thereof for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae and applications thereof, belonging to molecular biology and The field of immunology technology.
背景技术Background technique
大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌等致病菌在自然界中广泛存在,其可以对食品、水体等造成污染,进而对人类与动物的生命安全造成威胁。Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae and other pathogenic bacteria widely exist in nature, which can cause pollution to food and water bodies, thereby threatening the life safety of humans and animals.
为防止此类致病菌的危害,人们发展出了多种致病菌检测技术。例如对于大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌,目前常规的检测方法包括样品处理、增菌培养、分离培养和染色观察等步骤,该检测方法是临床检测“金标准”,但是该工作量大,检测周期长需要约一周时间。In order to prevent the harm of such pathogenic bacteria, a variety of pathogenic bacteria detection technologies have been developed. For example, for Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae, the current conventional detection methods include sample processing, enrichment culture, isolation and culture, and staining observation. This detection method is the "gold standard for clinical detection". ”, but the workload is large, and the detection cycle is long and takes about a week.
其它诸如生理生化鉴定虽然比较准确,缺点是操作复杂耗时费力但也比较费时。显色平板检测能有效的检测大肠杆菌,但缺点是费时,需要专业人员的操作,且试剂昂贵。膜过滤技术属于定量试验,具有许多和试管发酵技术相同的优点,膜过滤技术比试管发酵技术最明显的优越性是非常便于检测较大体积的水样,这样就能增加检出的敏感性和可靠性,缺点是特异性不高,结果容易受水样中其他细菌的影响而出现误判,一般作为推测性实验,还需用其它方法进一步证实;酶活性检测法(酶底物法)比传统方法省时省力(18-24h),而且特异性高,缺点是试剂花费较高;免疫学方法的最大问题就是商品化单克隆抗体或多克隆抗体与多种肠道菌的交叉反应性,容易造成假阳性,需要制备更高特异性的单克隆抗体或多克隆抗体。Others such as physiological and biochemical identification are more accurate, but the disadvantage is that the operation is complicated and time-consuming, but it is also time-consuming. The chromogenic plate detection can effectively detect E. coli, but the disadvantage is that it is time-consuming, requires professional operation, and the reagents are expensive. Membrane filtration technology is a quantitative test and has many of the same advantages as test-tube fermentation technology. The most obvious advantage of membrane filtration technology over test-tube fermentation technology is that it is very convenient to detect larger volumes of water samples, which can increase the detection sensitivity and Reliability, the disadvantage is that the specificity is not high, and the results are easily affected by other bacteria in the water sample and cause misjudgment. Generally, it is used as a speculative experiment and needs to be further confirmed by other methods; the enzyme activity detection method (enzyme substrate method) is better than The traditional method saves time and labor (18-24h), and has high specificity, but the disadvantage is that the cost of reagents is high; the biggest problem of immunological methods is the cross-reactivity of commercial monoclonal antibodies or polyclonal antibodies with various intestinal bacteria. It is easy to cause false positives, and it is necessary to prepare monoclonal or polyclonal antibodies with higher specificity.
这样一来,以分子生物学为基础的方法就发挥着独特的优势,分子生物学鉴定可以克服以上缺点,病原菌能在数小时内检测出来,操作简单、便捷;有关于运用PCR检测技术检测微生物的研究越来越多,一方面PCR检测技术相比于传统的微生物分离、培养、鉴定方法而言,减少了检测的周期长度,节省了检测所需成本;它适合于临床上进行大肠杆菌的流行病学调查和奶牛群中个体奶牛和群牛感染情况的调查,对于大肠杆菌的早期预防控制和准确的确定病原,及早快速进行临床治疗,减少经济损失,是一种有效的好方法,另一方面PCR检测技术具有较高的特异性和敏感性,缺点是该方法需要结合电泳槽、琼脂糖凝胶等仪器试剂,使用核酸染料等有毒物质且后期分析需要核算凝胶成像仪,对实验安全防护要求高且有毒。In this way, the methods based on molecular biology have unique advantages. Molecular biological identification can overcome the above shortcomings. Pathogens can be detected within a few hours, and the operation is simple and convenient. On the one hand, compared with the traditional methods of microbial isolation, culture and identification, PCR detection technology reduces the length of the detection cycle and saves the cost of detection; it is suitable for the clinical detection of Escherichia coli. Epidemiological investigations and investigations on the infection of individual cows and herds in dairy herds are an effective and good method for early prevention and control of Escherichia coli, accurate identification of pathogens, early and rapid clinical treatment, and reduction of economic losses. On the one hand, PCR detection technology has high specificity and sensitivity, but the disadvantage is that this method needs to combine electrophoresis tank, agarose gel and other instrument reagents, uses nucleic acid dyes and other toxic substances, and requires a gel imager for later analysis. Safety requirements are high and toxic.
然而,现有的检测方法存在操作复杂、易受环境影响、容易感染、不适于早期诊断且检测结果误差较大等缺点。因此,亟需一种快速、准确、便捷的诊断方法。However, the existing detection methods have disadvantages such as complicated operation, easy to be affected by the environment, easy to be infected, not suitable for early diagnosis, and have large errors in the detection results. Therefore, a rapid, accurate and convenient diagnostic method is urgently needed.
发明内容SUMMARY OF THE INVENTION
本发明的主要目的在于提供一种能够更加灵敏、快速便捷、安全廉价的用于检测大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌的检测试剂、试剂盒及其应用,以克服现有技术中的不足。The main purpose of the present invention is to provide a more sensitive, fast, convenient, safe and cheap detection reagent, kit and application for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and application thereof. Overcome the deficiencies in the prior art.
为实现前述发明目的,本发明采用的技术方案包括:In order to realize the foregoing invention purpose, the technical scheme adopted in the present invention includes:
本发明实施例提供了一种检测试剂,其包括如下四个引物对中的任意一种或多种的组合,所述四个引物对包括:An embodiment of the present invention provides a detection reagent, which includes any one or a combination of the following four primer pairs, wherein the four primer pairs include:
第一引物对,包括序列分别如SEQ ID NO:1、SEQ ID NO:2所示的第一引物、第二引物;The first primer pair, including the first primer and the second primer whose sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;
第二引物对,包括序列分别如SEQ ID NO:3、SEQ ID NO:4所示的第三引物、第四引物;The second primer pair includes the third primer and the fourth primer whose sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;
第三引物对,包括序列分别如SEQ ID NO:5、SEQ ID NO:6所示的第五引物、第六引物;The third primer pair includes the fifth primer and the sixth primer whose sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively;
第四引物对,包括序列分别如SEQ ID NO:7、SEQ ID NO:8所示的第七引物、第八引物。The fourth primer pair includes the seventh primer and the eighth primer whose sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
进一步的,至少一个引物对所含的至少一个引物的5’端还具有核酸标记物,所述核酸标记物均包括FITC、FAM、HEX、TET、TAMRA、ROX、VIC、NED、Alexa Flour、生物素、地高辛、cy3、cy5中的任意一种。Further, the 5' end of at least one primer contained in at least one primer pair also has a nucleic acid label, and the nucleic acid label includes FITC, FAM, HEX, TET, TAMRA, ROX, VIC, NED, Alexa Flour, biological Any one of prime, digoxin, cy3, and cy5.
更进一步的,每一引物对所含的两个引物的5’端均具有所述核酸标记物。Further, the 5' ends of the two primers contained in each primer pair have the nucleic acid label.
进一步的,所述的检测试剂还包括DNA聚合酶和PCR常规组件。Further, the detection reagent also includes DNA polymerase and conventional PCR components.
本发明实施例还提供了一种试剂盒,其包括所述的检测试剂以及核酸免疫层析试纸条,所述核酸免疫层析试纸条包括底板、样品垫、金标垫、硝酸纤维素膜以及吸水垫,所述硝酸纤维素膜上设置有检测带(喷涂地高辛抗体)和质控带(喷涂羊抗鼠二抗)。The embodiment of the present invention also provides a kit, which includes the detection reagent and a nucleic acid immunochromatography test strip, wherein the nucleic acid immunochromatography test strip includes a bottom plate, a sample pad, a gold label pad, a nitrocellulose Membrane and absorbent pad, the nitrocellulose membrane is provided with a detection strip (sprayed with digoxigenin antibody) and a quality control strip (sprayed with goat anti-mouse secondary antibody).
进一步的,所述金标垫上附有由第一抗体和有色颗粒形成的复合物,所述检测带上设置有第二抗体,所述质控带上设置有第三抗体;其中,所述第一抗体为所述核酸标记物的抗体,所述第二抗体为所述酸标记物的抗体或配体;所述第三抗体为能够结合指定种属来源抗体的抗体。Further, a complex formed by a first antibody and colored particles is attached to the gold label pad, a second antibody is arranged on the detection band, and a third antibody is arranged on the quality control band; The first antibody is the antibody of the nucleic acid label, the second antibody is the antibody or ligand of the acid label; the third antibody is the antibody capable of binding to the antibody of the specified species.
进一步的,所述第三抗体为羊抗鼠多抗,所述有色颗粒为经异硫氰酸荧光素抗体标记的球形红色胶体金颗粒。Further, the third antibody is goat anti-mouse polyclonal antibody, and the colored particles are spherical red colloidal gold particles labeled with fluorescein isothiocyanate antibody.
在一些较为具体的实施方案中,所述样品垫、金标垫、硝酸纤维素膜和吸水垫均放置在底板上,所述样品垫位于底板一端,所述吸水垫位于底板的另一端,所述金标垫位于靠近样品垫一端并且一部分压置于样品垫底部,所述硝酸纤维素膜设置在金标垫和吸水垫中间并与金标垫、吸水垫有部分重合,所述硝酸纤维素膜上有喷涂的检测带和质控带,所述检测带位于靠近金标垫一侧,所述质控带位于靠近吸水垫一侧,所述吸水滤纸垫、吸水垫分别设置在侧向层析基质膜的两端,所述结合物垫设置在吸水滤纸垫上,金标垫和吸水垫之间有一定间隔。In some specific embodiments, the sample pad, the gold standard pad, the nitrocellulose membrane and the absorbent pad are all placed on the bottom plate, the sample pad is located at one end of the bottom plate, and the absorbent pad is located at the other end of the bottom plate, so The gold label pad is located near one end of the sample pad and a part of it is placed at the bottom of the sample pad. The nitrocellulose membrane is arranged in the middle of the gold label pad and the absorbent pad and overlaps with the gold label pad and the absorbent pad. There are sprayed detection strips and quality control strips on the film, the detection strips are located on the side close to the gold standard pad, the quality control strips are located on the side close to the water-absorbing pad, and the water-absorbing filter paper pads and the water-absorbing pads are respectively arranged on the lateral layer The two ends of the matrix membrane are separated, the conjugate pad is arranged on the water-absorbing filter paper pad, and there is a certain interval between the gold standard pad and the water-absorbing pad.
在一些较为具体的实施方案中,在以包含核酸免疫层析试纸条的试剂盒进行大肠杆菌、金黄色葡萄球菌、停乳链球菌和无乳链球菌的检测时,将第一抗体(例如FITC抗体)与胶体金颗粒一起孵育,通过静电结合力使第一抗体包被在胶体金颗粒表面形成有色的交联物,并将交联物滴加在金标垫上烘干备用,将第二抗体以线条状固定于硝酸纤维素膜上形成检测带 (线)、将第三抗体以线条状固定于硝酸纤维素膜上形成质控带(线)。In some more specific embodiments, the primary antibody (eg FITC antibody) and colloidal gold particles were incubated together, and the first antibody was coated on the surface of colloidal gold particles to form colored cross-links by electrostatic binding force, and the cross-links were dropped on the gold pad to dry for use. The antibody was immobilized on the nitrocellulose membrane in the shape of a line to form a detection band (line), and the third antibody was immobilized in the shape of a line on the nitrocellulose membrane to form a quality control band (line).
优选的,所述吸水滤纸垫为玻纤RB 65,优选的,所述侧向层析基质膜为硝酸纤维素膜 Sartorius CN 140,优选的,所述吸水垫为CH 37,优选的,所述底板为SM 31-40。Preferably, the water-absorbing filter paper pad is glass fiber RB 65, preferably, the lateral chromatography matrix membrane is a nitrocellulose membrane Sartorius CN 140, preferably, the water-absorbing pad is CH 37, preferably, the The base plate is SM 31-40.
进一步的,本发明实施例还提供了用于核酸序列检测的展开液(0.4%NaCl),该展开液可以有效展开PCR扩增产物,使抗原抗体有效结合。Further, the embodiment of the present invention also provides a developing solution (0.4% NaCl) for nucleic acid sequence detection, and the developing solution can effectively develop PCR amplification products, so that the antigen and antibody can be effectively combined.
本发明实施例还提供了如所述的检测试剂或者所述试剂盒于制备检测致病菌的产品中的应用。The embodiments of the present invention also provide the application of the detection reagent or the kit in preparing a product for detecting pathogenic bacteria.
本发明实施例还提供了一种应用于致病菌检测方法的产品,所述产品包括权利要求5-7中任一项所述的试剂盒,并且,所述检测方法包括:The embodiment of the present invention also provides a product applied to a pathogenic bacteria detection method, the product includes the kit according to any one of claims 5-7, and the detection method includes:
利用所述四个引物对中的至少一个引物对,通过PCR扩增反应对从待测样品中提取到的核酸进行扩增,Using at least one primer pair among the four primer pairs, the nucleic acid extracted from the sample to be tested is amplified by PCR amplification reaction,
将扩增后的产物滴加到所述试剂盒的核酸免疫层析试纸上,并观察检测带和指控带上是否形成有色条带;若在检测带和质控带上均形成肉眼可见的有色条带,则判断为阳性;若检测带上未形成有色条带,且质控带上形成肉眼可见的有色条带,则判断为阴性;若质控带上未形成有色条带,则判断为无效。Add the amplified product dropwise to the nucleic acid immunochromatography test paper of the kit, and observe whether a colored band is formed on the detection band and the accusation band; if a visible colored band is formed on both the detection band and the quality control band If there is no colored band on the detection band, and there is a visible colored band on the quality control band, it is judged as negative; if there is no colored band on the quality control band, it is judged as invalid.
进一步的,所述致病菌包括大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌中的任意一种或两种以上。Further, the pathogenic bacteria include any one or two or more of Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae.
与现有技术相比,本发明的优点包括:Compared with the prior art, the advantages of the present invention include:
1)本发明通过结合PCR扩增技术和抗原抗体的特异性结合技术,提高了检测食源性致病菌的灵敏度,使用胶体金标记抗体技术及PCR产物放大双重策略实现最终信号的放大,从而实现对大肠杆菌的可视化检测;1) The present invention improves the sensitivity of detecting food-borne pathogens by combining PCR amplification technology and antigen-antibody specific binding technology, and uses colloidal gold-labeled antibody technology and PCR product amplification dual strategy to achieve final signal amplification, thereby Realize the visual detection of Escherichia coli;
2)本发明提供的方法具有快速、可视化、超灵敏等优点;对于提高食源性致病菌的检测灵敏度、降低成本和便捷性的实现具有重要意义,对于在实验环境较为不足的发明实施例进行普及或开展大肠杆菌的快速检测有巨大帮助;2) The method provided by the present invention has the advantages of rapidity, visualization, and ultra-sensitivity; it is of great significance for improving the detection sensitivity of food-borne pathogenic bacteria, reducing costs and realizing the convenience, and is relatively insufficient in the experimental environment. Popularization or rapid testing of E. coli would be of great help;
3)作为一个核酸扩增产物检测的通用技术平台,本发明提供的方法及其核酸免疫层析试纸条可广泛用于农牧业和食品工业,食源性致病菌临床检测,海关检验检验,环境监测,传染性疾病防控等领域。金黄色葡萄球菌、沙门氏菌、肠出血性大肠杆菌、单核细胞增生李斯特氏菌、志贺氏菌、副溶血性弧菌等常见食源性致病菌,这些都是本发明的核酸免疫层析试纸条的检测对象。3) As a general technology platform for nucleic acid amplification product detection, the method provided by the invention and its nucleic acid immunochromatography test strip can be widely used in agriculture, animal husbandry and food industry, clinical detection of food-borne pathogens, customs inspection Inspection, environmental monitoring, infectious disease prevention and control and other fields. Common food-borne pathogens such as Staphylococcus aureus, Salmonella, Enterohemorrhagic Escherichia coli, Listeria monocytogenes, Shigella, and Vibrio parahaemolyticus are the nucleic acid immune layers of the present invention Analysis of the detection object of the test strip.
附图说明Description of drawings
图1a是本发明一典型实施案例中大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌检测试剂盒的检测原理示意图;1a is a schematic diagram of the detection principle of the detection kit for Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae in a typical implementation case of the present invention;
图1b是本发明典型实施案例中大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌检测试剂盒的判断结果示意图;Figure 1b is a schematic diagram of the judgment results of the detection kits for Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae in a typical implementation case of the present invention;
图2a是本发明实施例1中不同粒径的胶体金溶液表观图;Fig. 2a is the colloidal gold solution appearance figure of different particle diameters in the embodiment of the
图2b是本发明实施例1中不同粒径的胶体金溶液紫外分光光度计扫描图;Fig. 2b is the scanning diagram of ultraviolet spectrophotometer of colloidal gold solution of different particle sizes in the embodiment of the
图2c是本发明实施例1中不同粒径的胶体金溶液试纸条显色效果图;Fig. 2c is the color rendering effect diagram of the colloidal gold solution test strips of different particle sizes in the embodiment of the
图3a是本发明实施例2中大肠杆菌的特异性检测核酸电泳分析图;Fig. 3a is the specific detection nucleic acid electrophoresis analysis diagram of Escherichia coli in the
图3b是本发明实施例2中大肠杆菌的特异性检测核酸免疫层析试纸条分析图;Fig. 3b is the specific detection nucleic acid immunochromatographic test strip analysis diagram of Escherichia coli in the embodiment of the
图4a是本发明实施例3中大肠杆菌的灵敏度检测核酸电泳分析图;Fig. 4a is the nucleic acid electrophoresis analysis diagram of sensitivity detection of Escherichia coli in Example 3 of the present invention;
图4b是本发明实施例3中大肠杆菌的灵敏度检测核酸免疫层析试纸条分析图;Fig. 4b is the sensitivity detection nucleic acid immunochromatographic test strip analysis diagram of Escherichia coli in Example 3 of the present invention;
图5a是本发明实施例4中不同粒径的胶体金溶液表观图;Fig. 5a is the colloidal gold solution appearance figure of different particle diameters in the embodiment of the
图5b是本发明实施例4中不同粒径的胶体金溶液紫外分光光度计扫描图;Fig. 5b is the colloidal gold solution ultraviolet spectrophotometer scanning diagram of different particle diameters in the embodiment of the
图5c是本发明实施例4中不同粒径的胶体金溶液试纸条显色效果图;Fig. 5c is the color rendering effect diagram of the colloidal gold solution test strips of different particle diameters in the embodiment of the
图6a是本发明实施例5中金黄色葡萄球菌的特异性检测核酸电泳分析图;Fig. 6a is the specific detection nucleic acid electrophoresis analysis figure of Staphylococcus aureus in the embodiment of the
图6b是本发明实施例5中金黄色葡萄球菌的特异性检测核酸免疫层析试纸条分析图;Fig. 6b is the specific detection nucleic acid immunochromatographic test strip analysis diagram of Staphylococcus aureus in the embodiment of the
图7a是本发明实施例6中金黄色葡萄球菌的灵敏度检测核酸电泳分析图;Fig. 7a is the nucleic acid electrophoresis analysis diagram of sensitivity detection of Staphylococcus aureus in the embodiment of the
图7b是本发明实施例6中金黄色葡萄球菌的灵敏度检测核酸免疫层析试纸条分析图;Figure 7b is an analysis diagram of a nucleic acid immunochromatographic test strip for the sensitivity detection of Staphylococcus aureus in Example 6 of the present invention;
图8a是本发明实施例7中不同粒径的胶体金溶液表观图;Fig. 8a is the appearance diagram of the colloidal gold solution of different particle diameters in the embodiment of the
图8b是本发明实施例7中不同粒径的胶体金溶液紫外分光光度计扫描图;Fig. 8b is the ultraviolet spectrophotometer scanning diagram of the colloidal gold solution of different particle diameters in the embodiment of the
图8c是本发明实施例7中不同粒径的胶体金溶液试纸条显色效果图;Figure 8c is a color rendering effect diagram of the colloidal gold solution test strips of different particle sizes in the embodiment of the
图9a是本发明实施例8中停乳链球菌的特异性检测核酸电泳分析图;Fig. 9a is the specificity detection nucleic acid electrophoresis analysis figure of Streptococcus dysgalactiae in the embodiment of the
图9b是本发明实施例8中停乳链球菌的特异性检测核酸免疫层析试纸条分析图;Fig. 9b is the specific detection nucleic acid immunochromatographic test strip analysis diagram of Streptococcus dysgalactiae in the embodiment of the
图10a是本发明实施例9中停乳链球菌的灵敏度检测核酸电泳分析图;Fig. 10a is the nucleic acid electrophoresis analysis diagram of sensitivity detection of Streptococcus dysgalactiae in Example 9 of the present invention;
图10b是本发明实施例9中停乳链球菌的灵敏度检测核酸免疫层析试纸条分析图;Figure 10b is an analysis diagram of a nucleic acid immunochromatography test strip for the sensitivity detection of Streptococcus dysgalactiae in Example 9 of the present invention;
图11a是本发明实施例10中不同粒径的胶体金溶液表观图;Figure 11a is the appearance diagram of the colloidal gold solution of different particle sizes in Example 10 of the present invention;
图11b是本发明实施例10中不同粒径的胶体金溶液紫外分光光度计扫描图;Fig. 11b is the scanning diagram of ultraviolet spectrophotometer of colloidal gold solution with different particle sizes in Example 10 of the present invention;
图11c是本发明实施例10中不同粒径的胶体金溶液试纸条显色效果图;Figure 11c is a color rendering effect diagram of colloidal gold solution test strips with different particle sizes in Example 10 of the present invention;
图12a是本发明实施例11中无乳链球菌的特异性检测核酸电泳分析图;Figure 12a is a specific detection nucleic acid electrophoresis analysis diagram of Streptococcus agalactiae in Example 11 of the present invention;
图12b是本发明实施例11中无乳链球菌的特异性检测核酸免疫层析试纸条分析图;Figure 12b is an analysis diagram of a nucleic acid immunochromatographic test strip for the specific detection of Streptococcus agalactiae in Example 11 of the present invention;
图13a是本发明实施例12中无乳链球菌的灵敏度检测核酸电泳分析图;Fig. 13a is a nucleic acid electrophoresis analysis diagram of sensitivity detection of Streptococcus agalactiae in Example 12 of the present invention;
图13b是本发明实施例12中无乳链球菌的灵敏度检测核酸免疫层析试纸条分析图。Figure 13b is an analysis diagram of nucleic acid immunochromatographic test strips for the sensitivity detection of Streptococcus agalactiae in Example 12 of the present invention.
具体实施方式Detailed ways
鉴于现有技术中的不足,本案发明人经长期研究和大量实践,得以提出本发明的技术方案。如下将对该技术方案、其实施过程及原理等作进一步的解释说明。In view of the deficiencies in the prior art, the inventor of the present application was able to propose the technical solution of the present invention after long-term research and extensive practice. The technical solution, its implementation process and principle will be further explained as follows.
本发明说明书中所使用的技术术语解释:Explanation of technical terms used in the description of the present invention:
胶体金:金溶胶又称胶体金,是金盐被还原成金单质后形成的稳定、均匀、呈单一分散状态悬浮在液体中的金颗粒悬浮液。Colloidal gold: Gold sol, also known as colloidal gold, is a stable, uniform and monodispersed gold particle suspension in liquid formed after gold salt is reduced to gold.
核酸聚合酶:合成核酸长链的酶类;分为DNA聚合酶和RNA聚合酶两大类。Nucleic acid polymerases: Enzymes that synthesize long nucleic acid chains; are divided into two categories: DNA polymerases and RNA polymerases.
引物:DNA合成的起始物。一般为一对单链寡核苷酸,在与模板杂交后,DNA合成从其 3’末端开始。Primer: The starting material for DNA synthesis. Typically a pair of single-stranded oligonucleotides, after hybridization to a template, DNA synthesis begins at the 3' end.
标记:将可检测的信号分子(如半抗原,荧光,放射性等)与单链寡聚核苷酸相偶联的方法。Labeling: A method of conjugating a detectable signal molecule (eg, hapten, fluorescence, radioactivity, etc.) to a single-stranded oligonucleotide.
杂交:特指互补的DNA单链通过碱基配对形成双链结构。Hybridization: In particular, complementary DNA single strands form a double-stranded structure through base pairing.
展开:经扩增反应的PCR产物在展开缓冲液的层析作用下由试纸条吸水垫底端开始向检测线、质控线方向移动的过程。Development: The PCR product after the amplification reaction starts to move towards the detection line and the quality control line from the bottom end of the water-absorbing pad of the test strip under the chromatographic action of the development buffer.
核酸:脱氧核糖核酸(DNA)和核糖核酸(RNA)的通称。Nucleic acid: Generic term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
抗原和半抗原:具备免疫原性的物质。通常为大分子蛋白质或细胞组分。但有些小分子也具备免疫原性,被称为半抗原(Hapten)。半抗原常被用于标记探针。Antigens and Haptens: Substances that are immunogenic. Usually macromolecular proteins or cellular components. But some small molecules are also immunogenic, called hapten. Haptens are often used to label probes.
抗体:能与抗原或半抗原特异性结合的蛋白质分子。Antibody: A protein molecule that can specifically bind to an antigen or hapten.
复合体:由两种或两种以上分子特异性结成的结合物。Complex: A specific combination of two or more molecules.
引物二聚体:在聚合酶链式反应(PCR)过程中,因引物对间、或单条引物相互退火形成的二聚体分子,由两条不同引物构成的二聚体为异二聚体,因带有两种标记而可能引起与种抗体的结合而造成假阳性,由单一引物退火形成的二聚体为同二聚体,仅带有一种标记而不会引起假阳性,但过多的二聚体形成会降低扩增效率。Primer-dimer: In the polymerase chain reaction (PCR) process, a dimer molecule formed by the annealing of primer pairs or a single primer to each other, the dimer composed of two different primers is a heterodimer, It may cause false positives due to the binding of the antibody with two labels, and the dimer formed by the annealing of a single primer is a homodimer, and only one label will not cause false positives, but too many Dimer formation reduces amplification efficiency.
免疫层析试纸条:用于快速检测的医学工具,又称为呈色薄膜层析。Immunochromatographic test strips: Medical tools for rapid detection, also known as Chromogenic Film Chromatography.
本发明实施例提供的检测大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌的方法大大简化核酸扩增后的检测程序,节约检测成本;结果判读简单明了、直观(检测原理以及结果如附图1a、图ab所示);本发明实施例提供的检测大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌的方法作为一种新型的核酸扩增后检测技术,具有以下优点:The method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae provided in the embodiment of the present invention greatly simplifies the detection procedure after nucleic acid amplification and saves the detection cost; the interpretation of the results is simple, clear and intuitive (the detection principle and The results are shown in accompanying drawings 1a and ab); the method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae provided in the embodiment of the present invention is used as a novel detection technology after nucleic acid amplification, Has the following advantages:
应用广泛:作为一个核酸扩增产物检测的通用技术平台,本发明提供的方法及其试纸条可广泛用于农牧业和食品工业,食源性致病菌临床检测,海关检验检验,环境监测,传染性疾病防控等领域。金黄色葡萄球菌、沙门氏菌、肠出血性大肠杆菌、单核细胞增生李斯特氏菌、志贺氏菌、副溶血性弧菌等常见食源性致病菌,这些都是本发明的核酸扩增产物检测试纸条的对象。Wide application: as a general technology platform for nucleic acid amplification product detection, the method and test strip provided by the present invention can be widely used in agriculture, animal husbandry and food industry, clinical detection of food-borne pathogens, customs inspection and inspection, environmental surveillance, infectious disease prevention and control, etc. Common food-borne pathogens such as Staphylococcus aureus, Salmonella, Enterohemorrhagic Escherichia coli, Listeria monocytogenes, Shigella, and Vibrio parahaemolyticus are all nucleic acid amplifications of the present invention The object of the product detection test strip.
操作简单:只需要把核酸扩增后的样品直接滴到核酸试剂检测板上即可,不需要专业人员操作,不仅发明实施例可用,也可用于一线防控机构及养殖场;快速:检测5分钟后判读结果;便捷:无需电泳也无需凝胶成像系统,可通过肉眼直接观察;灵敏:与琼脂糖凝胶电泳相比,检测灵敏度提高近10-100倍;特异性高:检测过程中使用特异性标记引物,使结果更加准确;成本低:检测费用大大低于凝胶电泳和ELISA检测。Simple operation: just drop the amplified nucleic acid sample directly on the nucleic acid reagent detection plate, no professional operation is required, not only the invention embodiment can be used, but also can be used in front-line prevention and control institutions and farms; fast:
在本发明一典型实施案例中,一种采用核酸免疫层析试纸条法检测大肠杆菌或金黄色葡萄球菌或停乳链球菌或无乳链球菌的方法包括如下过程:In a typical implementation case of the present invention, a method for detecting Escherichia coli or Staphylococcus aureus or Streptococcus dysgalactiae or Streptococcus agalactiae using a nucleic acid immunochromatographic test strip method comprises the following steps:
1)提供两条独特的扩增引物:1) Provide two unique amplification primers:
其中,检测大肠杆菌时采用的上游引物Ecoil-FITC-F(即前述第一引物)的序列(SEQ ID NO:1)为5’-ATCAACCGAGATTCCCCCAGT-3’,5’端用异硫氰酸荧光素(FITC)等核酸标记物标记;下游引物Ecoil-DIG-R(即前述第二引物)的序列(SEQ ID NO:2)为5’-TCACTATCGGTCAGTCAGGAG-3’,5’端用诸如地高辛(DIG)等核酸标记物标记;在适宜的扩增条件下,可扩增一段长度为254bp的DNA片段;当无被检核酸模板存在时,无特异性核酸片段扩增产物;Wherein, the sequence (SEQ ID NO: 1) of the upstream primer Ecoil-FITC-F (that is, the aforementioned first primer) used in the detection of Escherichia coli is 5'-ATCAACCGAGATTCCCCCAGT-3', and the 5' end uses fluorescein isothiocyanate. (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 2) of the downstream primer Ecoil-DIG-R (that is, the aforementioned second primer) is 5'-TCACTATCGGTCAGTCAGGAG-3', and the 5' end is labeled with such as digoxigenin ( DIG) and other nucleic acid markers; under suitable amplification conditions, a DNA fragment with a length of 254 bp can be amplified; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;
检测金黄色葡萄球菌时采用的上游引物nuc-FITC-F(即前述第三引物)的序列(SEQ ID NO:3)为5’-CGATTGATGGTGATACGGTT-3’,5’端用异硫氰酸荧光素(FITC)等核酸标记物标记;下游引物nuc-DIG-R(即第四引物)的序列(SEQ ID NO:4)为5’-CTCTTTTTTCGCTTGTGCTT-3’,5’端用地高辛(DIG)等核酸标记物标记,在适宜的扩增条件下,可扩增一段长度为356bp的DNA片段;当无被检核酸模板存在时,无特异性核酸片段扩增产物;The sequence (SEQ ID NO: 3) of the upstream primer nuc-FITC-F (that is, the aforementioned third primer) used in the detection of Staphylococcus aureus is 5'-CGATTGATGGTGATACGGTT-3', and the 5' end uses fluorescein isothiocyanate. (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 4) of the downstream primer nuc-DIG-R (ie, the fourth primer) is 5'-CTCTTTTTTCGCTTGTGCTT-3', and the 5' end uses digoxigenin (DIG), etc. Nucleic acid marker labeling, under suitable amplification conditions, can amplify a DNA fragment with a length of 356bp; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;
检测停乳链球菌时采用的上游引物sdy-FITC-F(即第五引物)的序列(SEQ ID NO:5)为 5’-TAAAGGTGC AACTGCATCACTA-3’,5’端用异硫氰酸荧光素(FITC)等核酸标记物标记;下游引物sdy- DIG-R(即第六引物)的序列(SEQ ID NO:6)为5’-AGTCACATGGTGGATTTTCCA-3’,5’端用地高辛(DIG)等核酸标记物标记,在适宜的扩增条件下,可扩增一段长度为282bp的 DNA片段;当无被检核酸模板存在时,无特异性核酸片段扩增产物;The sequence (SEQ ID NO: 5) of the upstream primer sdy-FITC-F (that is, the fifth primer) used in the detection of Streptococcus dysgalactiae is 5'-TAAAGGTGC AACTGCATCACTA-3', and the 5' end uses fluorescein isothiocyanate. (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 6) of the downstream primer sdy-DIG-R (ie, the sixth primer) is 5'-AGTCACATGGTGGATTTTCCA-3', and the 5' end uses digoxigenin (DIG), etc. Nucleic acid marker labeling, under suitable amplification conditions, can amplify a DNA fragment with a length of 282bp; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;
检测无乳链球菌时采用的上游引物cfb-F(即第七引物)的序列(SEQ ID NO:7)为5’- TAATAATCAAGCCCAGC-3’,5’端用异硫氰酸荧光素(FITC)等核酸标记物标记;下游引物cfb-R(即第八引物)的序列(SEQ ID NO:8)为5’-CCTTTTGTTCTAATGCC-3’, 5’端用地高辛(DIG)等核酸标记物标记,在适宜的扩增条件下,可扩增一段长度为388bp 的DNA片段;当无被检核酸模板存在时,无特异性核酸片段扩增产物;The sequence (SEQ ID NO: 7) of the upstream primer cfb-F (that is, the seventh primer) used in the detection of Streptococcus agalactiae is 5'-TAATAATCAAGCCCAGC-3', and the 5' end uses fluorescein isothiocyanate (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 8) of the downstream primer cfb-R (ie, the eighth primer) is 5'-CCTTTTGTTCTAATGCC-3', and the 5' end is marked with nucleic acid markers such as digoxigenin (DIG), Under suitable amplification conditions, a DNA fragment with a length of 388bp can be amplified; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;
2).将一种标志物(如前述的核酸标记物,命名为标记物1)的通用抗体(即前述第一抗体如抗地高辛抗体)与胶体金颗粒交联,在胶体颗粒表面形成包被的抗体;2). Cross-linking a universal antibody of a marker (such as the aforementioned nucleic acid marker, named as marker 1) (ie, the aforementioned first antibody such as anti-digoxigenin antibody) and colloidal gold particles, forming on the surface of the colloidal particles. coated antibody;
3)将另一种标志物(如前述的核酸标记物,命名为标记物2)的抗体或配体(即第二抗体,例如FITC抗体)以线条状固定于试纸条上的膜(例如硝酸纤维膜)上形成检测带(线);3) An antibody or ligand (ie, a second antibody, such as FITC antibody) of another marker (such as the aforementioned nucleic acid marker, named as marker 2) is immobilized in a line shape on the membrane on the test strip (such as A detection band (line) is formed on the nitrocellulose membrane);
4)当存在待检的特异性扩增产物时,因上、下游引物的作用,扩增产物中同时带有上述三种标记物质的两种,形成标志物1-扩增产物-标志物2的复合物,其中的标志物1即所述的第一核酸标记物,标志物2即所述的第二核酸标记物;4) When there is a specific amplification product to be detected, due to the action of the upstream and downstream primers, the amplification product simultaneously carries two of the above three labeling substances, forming Marker 1-Amplification Product-
5)将步骤4)形成的标志物1-扩增产物-标志物2与胶体金颗粒表面包被的标志物1的抗体结合,形成抗标志物1抗体-标志物1-扩增产物-标志物2有色颗粒复合物;5) Combine the marker 1-amplification product-
6)将步骤5)得到的有色颗粒复合物在溶液中通过毛细现象沿纤维向上流动至涂布有标志物 2的抗体或配体的线条上,复合物因与标志物2的抗体(配体)结合而沉积,滞留在检测线上,形成肉眼可见的有色线条,判断为阳性;6) The colored particle complex obtained in step 5) flows upward along the fiber through the capillary phenomenon in the solution to the line coated with the antibody or ligand of Marker 2. ) combined and deposited, stayed on the detection line, formed a colored line visible to the naked eye, and judged as positive;
7)当特异性扩增产物不存在时,不发生上述步骤4)-步骤6),不能形成标志物1-扩增产物- 标志物2复合物,也就不能形成沉积于检测线上标志物2的抗体(配体)之上,不形成可见的条带,判为阴性。7) When the specific amplification product does not exist, the above step 4)-step 6) does not occur, the marker 1-amplification product-
本发明提供的检测大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌的方法由筛选的特异性引物保证扩增产物的特异性,同时引物两端的修饰物与试纸条上的抗原抗体特异性结合,双重保证了检测结果的准确性;并且本发明的检测方法避免了核酸扩增后的凝胶电泳复杂操作环节,融入试纸条检测技术使检测方法更加的安全、简单、快速、便捷、廉价。In the method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus agalactiae provided by the present invention, the specificity of the amplified product is ensured by the specific primers screened, and the modifiers at both ends of the primers are compatible with those on the test strip. The antigen-antibody specific binding doubles to ensure the accuracy of the detection results; and the detection method of the present invention avoids the complex operation link of gel electrophoresis after nucleic acid amplification, and integrates the test strip detection technology to make the detection method more safe, simple, and convenient. Fast, easy and cheap.
本发明提供的检测大肠杆菌、金黄色葡萄球菌、停乳链球菌、无乳链球菌的方法是以标记的特异性引物扩增适当长度的基因片段,能够保证检测的高度特异性,提高了检测结果的准确性;保持了免疫胶体金(试纸条)的直观特性,具有快速,方便,成熟,廉价等优点。The method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus agalactiae provided by the present invention is to amplify gene fragments of appropriate length with labeled specific primers, which can ensure high specificity of detection and improve detection The accuracy of the results; the intuitive characteristics of the immunocolloidal gold (test strip) are maintained, and it has the advantages of rapidity, convenience, maturity, and cheapness.
如下将结合附图以及具体实施例对该技术方案、其实施过程及原理等作进一步的解释说明。The technical solution, its implementation process and principle will be further explained below with reference to the accompanying drawings and specific embodiments.
实施例1Example 1
测试不同胶体金粒径大小对试纸条的显色效果的影响。The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
由10nm到50nm不同粒径大小的胶体金溶液由本发明实施例制备。Colloidal gold solutions with different particle sizes from 10 nm to 50 nm are prepared by the examples of the present invention.
1.2不同柠檬酸加入量引起不同粒径的金纳米粒子的外观变化情况如下表所示:1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
1)取不同粒径的胶体金溶液各1ml(如附图2a所示);1) Get 1 ml of colloidal gold solutions of different particle sizes (as shown in accompanying drawing 2a);
2)在1ml胶体金溶液中加入1M碳酸钾溶液10ul调节溶液pH,混匀;加入4ul FITC抗体在四度混匀孵育1h;再加入封闭液室温孵育30min;于7000r/min的条件下离心处理3min去除未结合蛋白,取上清液再次离心,于9400r/min条件下离心20min,取上清液;加入80ul复溶液,于4℃条件下老化2h;取出后各取6ul滴在金标垫上,于25℃条件下烘干;将烘干的金标垫组装到试纸条上;2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min 3min to remove unbound protein, take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, take the supernatant; add 80ul of reconstituted solution, age at 4°C for 2h; after taking out, take 6ul and drop them on the gold pad , dried at 25°C; assemble the dried gold label pads onto the test strips;
3)将保存的大肠杆菌,培养至对数期,取菌液进行核酸扩增,用上述(1.3)PCR反应体系进行PCR扩增;将PCR扩增产物进行不同梯度的稀释;分别滴加到不同粒径的胶体金溶液制备的试纸条上,观察结果。3) Culture the preserved Escherichia coli to the logarithmic phase, take the bacterial solution for nucleic acid amplification, and carry out PCR amplification with the above-mentioned (1.3) PCR reaction system; carry out different gradient dilutions of the PCR amplification products; Observe the results on test strips prepared from colloidal gold solutions of different particle sizes.
2结果2 results
不同粒径的胶体金溶液的外观结果和紫外分光光度计扫描结果如图2a、2b所示,本发明实施例所制备的胶体金溶液性状稳定、粒径分布均匀满足实验所需;不同粒径及梯度稀释核酸免疫层析试纸条结果如图2c所示,金颗粒粒径越大越容易发生聚集沉淀,因此在处理过程中大于 35nm的胶体金溶液容易聚集无法获得稳定分散的溶剂,不适于制备试纸条使用,在图2c所示的显色结果中10nm对应的试纸条从金标垫释放程度、背景清晰度、显色强度、显色均匀性等判断显色效果最好。The appearance results and UV spectrophotometer scanning results of colloidal gold solutions with different particle sizes are shown in Figures 2a and 2b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; different particle sizes And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 2c. The larger the particle size of gold particles, the easier aggregation and precipitation will occur. Therefore, the colloidal gold solution larger than 35 nm is easy to aggregate during the treatment process and cannot obtain a stable and dispersed solvent, which is not suitable for The test strip was prepared for use. In the color development results shown in Figure 2c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.
实施例2Example 2
测试大肠杆菌在不同食源性致病菌污染的特异性鉴定。Test for the specific identification of Escherichia coli in contamination with different foodborne pathogens.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
本实施例中涉及的各种人畜共患病菌株见表1。The various zoonotic strains involved in this example are shown in Table 1.
表1 实验菌株Table 1 Experimental strains
Table 1 Bacterial strains for detectionTable 1 Bacterial strains for detection
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将不同菌种复苏培养,使用平板计数菌落数,提取细菌DNA,用上述(1.3)PCR反应体系对大肠杆菌、金黄色葡萄球菌、无乳链球菌、停乳链球菌、粪肠球菌、表皮葡萄球菌、酵母菌、沙门氏菌、蜡样芽孢杆菌、单核李斯特菌和纯水进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其不同菌种间的特异性。The different strains were recovered and cultured, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Vitis epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果;1) Take 4 μL of PCR product, mix it with 196ul developing solution, add 60ul dropwise to the test strip, and use a smartphone or camera to photograph the color development result of the test strip after five minutes of color development;
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为: 1×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.
2结果2 results
大肠杆菌不同菌种间特异性鉴定电泳结果如图3a所示,大肠杆菌不同菌种间特异性鉴定核酸免疫层析试纸条鉴定结果如图3b所示,结果显示:本发明建立的PCR扩增后检测方法对大肠杆菌具有较好的特异性,在不同菌种间也未发生明显的试纸条阳性反应,具有很好的辨识度,在未借助其他实验仪器的情况下肉眼便可观察,可以方便基层工作者们的使用,更有助于检出大肠杆菌。The electrophoresis results of specific identification between different strains of E. coli are shown in Figure 3a, and the identification results of nucleic acid immunochromatography test strips for the specific identification of different strains of E. coli are shown in Figure 3b. The results show that: the PCR amplification method established in the present invention The post-increase detection method has good specificity for Escherichia coli, and there is no obvious positive reaction of the test strip between different strains. , which can facilitate the use of grassroots workers, and is more conducive to the detection of Escherichia coli.
实施例3Example 3
测试核酸免疫层析试纸条检测大肠杆菌的灵敏度。Test the sensitivity of nucleic acid immunochromatographic strips to detect Escherichia coli.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
大肠杆菌由南京农业大学动物医学院发明实施例分离鉴定保存。Escherichia coli was isolated, identified and preserved by the Invention Example of the School of Veterinary Medicine of Nanjing Agricultural University.
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将大肠杆菌复苏培养,取菌液梯度稀释后采用平板计数,图4a、4b中1-9分别对应的菌落数为2×108cfu/ml、2×107cfu/ml、2×106cfu/ml、2×105cfu/ml、2×104cfu/ml、2×103cfu/ml、2×102 cfu/ml、2×101cfu/ml、2cfu/ml;同时提取细菌DNA作为PCR模板,用上述(1.3)PCR反应体系对大肠杆菌进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其灵敏度。The Escherichia coli were recovered and cultured, and the bacterial solution was diluted by gradient and counted on a plate. The number of colonies corresponding to 1-9 in Figures 4a and 4b were 2 × 10 8 cfu/ml, 2 × 10 7 cfu/ml, and 2 × 10 6 respectively. cfu/ml, 2×10 5 cfu/ml, 2×10 4 cfu/ml, 2×10 3 cfu/ml, 2×10 2 cfu/ml, 2×10 1 cfu/ml, 2cfu/ml; simultaneous extraction Bacterial DNA was used as a PCR template, and E. coli was amplified by PCR using the PCR reaction system (1.3) above, and its sensitivity was verified by gel electrophoresis and nucleic acid immunochromatography test strips, respectively.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果。1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为: 1×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.
2结果2 results
大肠杆菌检测灵敏度鉴定电泳结果如图4a所示,大肠杆菌检测灵敏度鉴定核酸免疫层析试纸条鉴定结果如图4b所示,结果显示:可见本发明建立的检测大肠杆菌的核酸免疫层析试纸条检测方法检测灵敏度高,可达到最低检测限为2×102cfu/mL,比核酸凝胶电泳的检测灵敏度高 10倍。The electrophoresis results of Escherichia coli detection sensitivity identification are shown in Figure 4a, and the identification results of Escherichia coli detection sensitivity identification nucleic acid immunochromatography test strips are shown in Figure 4b. The paper strip detection method has high detection sensitivity, and the lowest detection limit is 2×10 2 cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.
实施例1-实施例3中的Ecoil-FITC-F序列(即第一引物序列)ATCAACCGAGATTCCCCCAGT;Ecoil-DIG-R序列(即第二引物序列)为:TCACTATCGGTCAGTCAGGAG。The Ecoil-FITC-F sequence (ie the first primer sequence) in Example 1-Example 3 is ATCAACCGAGATTCCCCCAGT; the Ecoil-DIG-R sequence (ie the second primer sequence) is: TCACTATCGGTCAGTCAGGAG.
实施例4Example 4
测试不同胶体金粒径大小对试纸条的显色效果的影响。The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
由10nm到50nm不同粒径大小到的胶体金溶液由本发明实施例制备。Colloidal gold solutions with different particle sizes ranging from 10 nm to 50 nm are prepared by the examples of the present invention.
1.2不同柠檬酸加入量引起不同粒径的金纳米粒子的外观变化情况如下表所示:1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
1)取不同粒径的胶体金溶液各1ml(如附图5a所示);1) get 1ml each of colloidal gold solutions of different particle sizes (as shown in accompanying drawing 5a);
2)在1ml胶体金溶液中加入1M碳酸钾溶液10ul调节溶液pH,混匀;加入4ul FITC抗体在四度混匀孵育1h;再加入封闭液室温孵育30min;7000r/min离心3min去除未结合蛋白,取上清再次离心,9400r/min离心20min,去上清;加入80ul复溶液,放置4度老化2h;取出后各取6ul滴在金标垫上,于25度烘干;将烘干的金标垫组装到试纸条上。2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min for 3 min to remove unbound proteins , take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, remove the supernatant; add 80ul of reconstituted solution, place it at 4 degrees for aging for 2h; after taking out, take 6ul of each drop on the gold pad, and dry at 25 degrees; The pads are assembled to the test strips.
3)将保存的金黄色葡萄球菌复苏,培养至对数期,取菌液进行核酸扩增,用上述(1.3) PCR反应体系进行PCR扩增;将PCR扩增产物进行不同梯度的稀释;分别滴加到不同粒径胶体金溶液制备的试纸条上,观察结果。3) Resuscitate the preserved Staphylococcus aureus, cultivate it to logarithmic phase, take the bacterial solution to carry out nucleic acid amplification, and carry out PCR amplification with the above-mentioned (1.3) PCR reaction system; carry out different gradient dilutions of the PCR amplification products; respectively; Add dropwise to test strips prepared from colloidal gold solutions with different particle sizes and observe the results.
2结果2 results
不同粒径的胶体金溶液表观结果和紫外分光光度计扫描结果如图5a、5b所示,本发明实施例所制备的胶体金溶液性状稳定、粒径分布均匀满足实验所需;不同粒径及梯度稀释核酸免疫层析试纸条结果如图5c所示,金颗粒粒径越大越容易发生聚集沉淀,因此在处理过程中大于 35nm的胶体金溶液容易聚集无法获得稳定分散的溶剂,不适于制备试纸条使用,在图5c所示的显色结果中10nm对应的试纸条从金标垫释放程度、背景清晰度、显色强度、显色均匀性等判断显色效果最好。The apparent results of colloidal gold solutions with different particle sizes and the scanning results of UV spectrophotometers are shown in Figures 5a and 5b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 5c. The larger the particle size of gold particles, the more likely to cause aggregation and precipitation. Therefore, the colloidal gold solution larger than 35nm is easy to aggregate during the treatment process, and a stable dispersion solvent cannot be obtained, which is not suitable for Test strips were prepared for use. In the color development results shown in Figure 5c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.
实施例5Example 5
测试金黄色葡萄球菌在不同食源性致病菌污染的特异性鉴定。Test for specific identification of Staphylococcus aureus in different foodborne pathogenic contamination.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
本发明实施例涉及的各种人畜共患病菌株见表2。The various zoonotic strains involved in the embodiments of the present invention are shown in Table 2.
表2 实验菌株Table 2 Experimental strains
Table 2 Bacterial strains for detectionTable 2 Bacterial strains for detection
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将不同菌种复苏培养,使用平板计数菌落数,提取细菌DNA,用上述(1.3)PCR反应体系对金黄色葡萄球菌、大肠杆菌、无乳链球菌、停乳链球菌、粪肠球菌、表皮葡萄球菌、酵母菌、沙门氏菌、蜡样芽孢杆菌、单核李斯特菌和纯水进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其不同菌种间的特异性。The different strains were recovered and cultured, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Staphylococcus epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果。1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为: 1×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.
2结果2 results
金黄色葡萄球菌不同菌种间特异性鉴定电泳结果如图6a所示,金黄色葡萄球菌不同菌种间特异性鉴定核酸免疫层析试纸条鉴定结果如图6b所示,结果显示:本发明建立的PCR扩增后检测方法对金黄色葡萄球菌具有较好的特异性,在不同菌种间也未发生明显的试纸条阳性反应,具有很好的辨识度,在未借助其他实验仪器的情况下肉眼便可观察,可以方便基层工作者们的使用,更有助于检出金黄色葡萄球菌。The electrophoresis results for the specific identification of different strains of Staphylococcus aureus are shown in Figure 6a, and the identification results of nucleic acid immunochromatography test strips for the specific identification of different strains of Staphylococcus aureus are shown in Figure 6b. The results show that: the present invention The established PCR-amplified detection method has good specificity for Staphylococcus aureus, and there is no obvious positive test strip reaction between different strains, and it has a good degree of identification. It can be observed with the naked eye under the circumstances, which is convenient for grass-roots workers to use, and is more helpful for the detection of Staphylococcus aureus.
实施例6Example 6
测试核酸免疫层析试纸条检测金黄色葡萄球菌的灵敏度。Test the sensitivity of nucleic acid immunochromatographic strips to detect Staphylococcus aureus.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
本发明实施例涉及的各种人畜共患病菌株见表3。Various zoonotic strains involved in the embodiments of the present invention are shown in Table 3.
表3 实验菌株Table 3 Experimental strains
Table 3 Bacterial strains for detectionTable 3 Bacterial strains for detection
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将金黄色葡萄球菌复苏培养,取菌液梯度稀释后采用平板计数,图6a、6b中1-9分别对应的菌落数为4×108cfu/ml、4×107cfu/ml、4×106cfu/ml、4×105cfu/ml、4×104cfu/ml、4×103cfu/ml 、4×102cfu/ml、4×101cfu/ml、4cfu/ml;同时提取细菌DNA作为PCR模板,用上述(1.3) PCR反应体系对金黄色葡萄球菌进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其灵敏度。Staphylococcus aureus was revived and cultured, and the bacterial solution was serially diluted and counted on a plate. The number of colonies corresponding to 1-9 in Figures 6a and 6b were 4×10 8 cfu/ml, 4×10 7 cfu/ml, 4× 10 6 cfu/ml, 4 x 10 5 cfu/ml, 4 x 10 4 cfu/ml, 4 x 10 3 cfu/ml, 4 x 10 2 cfu/ml, 4 x 10 1 cfu/ml, 4 cfu/ml; At the same time, bacterial DNA was extracted as a PCR template, and the above-mentioned (1.3) PCR reaction system was used to carry out PCR amplification of Staphylococcus aureus, and then use gel electrophoresis and nucleic acid immunochromatography test strips to verify its sensitivity.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果。1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为: 1×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.
2结果2 results
金黄色葡萄球菌检测灵敏度鉴定电泳结果如图7a所示,金黄色葡萄球菌检测灵敏度鉴定核酸免疫层析试纸条鉴定结果如图7b所示,结果显示:可见本发明建立的检测金黄色葡萄球菌的核酸免疫层析试纸条检测方法检测灵敏度高,可达到最低检测限为4×102cfu/mL,比核酸凝胶电泳的检测灵敏度高10倍。The electrophoresis results of the detection sensitivity identification of Staphylococcus aureus are shown in Figure 7a, and the identification results of the nucleic acid immunochromatographic test strips for the detection sensitivity identification of Staphylococcus aureus are shown in Figure 7b. The results show: it can be seen that the detection sensitivity of Staphylococcus aureus established by the present invention The nucleic acid immunochromatography test strip detection method has high detection sensitivity and can reach the lowest detection limit of 4×10 2 cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.
实施例4-实施例6中的nuc-FITC-F序列(即第三引物序列)为:CGATTGATGGTGATACGGTT;nuc-DIG-R序列(即第四引物序列)为: CTCTTTTTTCGCTTGTGCTT。The nuc-FITC-F sequence (ie, the third primer sequence) in Example 4-Example 6 is: CGATTGATGGTGATACGGTT; the nuc-DIG-R sequence (ie, the fourth primer sequence) is: CTCTTTTTTCGCTTGTGCTT.
实施例7Example 7
测试不同胶体金粒径大小对试纸条的显色效果的影响。The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
由10nm到50nm不同粒径大小到的胶体金溶液由本发明实施例制备。Colloidal gold solutions with different particle sizes ranging from 10 nm to 50 nm are prepared by the examples of the present invention.
1.2不同柠檬酸加入量引起不同粒径的金纳米粒子的外观变化情况如下表所示:1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
1)取不同粒径的胶体金溶液各1ml(如附图8a所示);1) Take 1 ml of colloidal gold solutions of different particle sizes (as shown in accompanying drawing 8a);
2)在1ml胶体金溶液中加入1M碳酸钾溶液10ul调节溶液pH,混匀;加入4ul FITC抗体在四度混匀孵育1h;再加入封闭液室温孵育30min;7000r/min离心3min去除未结合蛋白,取上清再次离心,9400r/min离心20min,去上清;加入80ul复溶液,放置4度老化2h;取出后各取6ul滴在金标垫上,于25度烘干;将烘干的金标垫组装到试纸条上。2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min for 3 min to remove unbound proteins , take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, remove the supernatant; add 80ul of reconstituted solution, place it at 4 degrees for aging for 2h; after taking out, take 6ul of each drop on the gold pad, and dry at 25 degrees; The pads are assembled to the test strips.
3)将保存的停乳链球菌复苏,培养至对数期,取菌液进行核酸扩增,用上述(1.3)PCR 反应体系进行PCR扩增;将PCR扩增产物进行不同梯度的稀释;分别滴加到不同粒径胶体金溶液制备的试纸条上,观察结果。3) Resuscitate the preserved Streptococcus dysgalactiae, cultivate it to logarithmic phase, take the bacterial solution to carry out nucleic acid amplification, and use the above (1.3) PCR reaction system to carry out PCR amplification; carry out different gradient dilutions of the PCR amplification products; respectively; Add dropwise to test strips prepared from colloidal gold solutions with different particle sizes and observe the results.
2结果2 results
不同粒径的胶体金溶液表观结果和紫外分光光度计扫描结果如图8a、8b所示,本发明实施例所制备的胶体金溶液性状稳定、粒径分布均匀满足实验所需;不同粒径及梯度稀释核酸免疫层析试纸条结果如图8c所示,金颗粒粒径越大越容易发生聚集沉淀,因此在处理过程中大于 35nm的胶体金溶液容易聚集无法获得稳定分散的溶剂,不适于制备试纸条使用,在图8c所示的显色结果中10nm对应的试纸条从金标垫释放程度、背景清晰度、显色强度、显色均匀性等判断显色效果最好。The apparent results of colloidal gold solutions with different particle sizes and the scanning results of UV spectrophotometers are shown in Figures 8a and 8b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 8c. The larger the particle size of gold particles, the more likely to cause aggregation and precipitation. Therefore, colloidal gold solutions larger than 35 nm are easily aggregated during the treatment process and cannot obtain stable dispersion. The solvent is not suitable for The test strip was prepared for use. In the color development results shown in Figure 8c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.
实施例8Example 8
测试停乳链球菌在不同食源性致病菌污染的特异性鉴定。Testing the specific identification of Streptococcus dysgalactiae in different foodborne pathogenic contamination.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
本发明实施例涉及的各种人畜共患病菌株见表4。Various zoonotic strains involved in the embodiments of the present invention are shown in Table 4.
表4 实验菌株Table 4 Experimental strains
Table 4 Bacterial strains for detectionTable 4 Bacterial strains for detection
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将不同菌种复苏培养,使用平板计数菌落数,提取细菌DNA,用上述(1.3)PCR反应体系对停乳链球菌、金黄色葡萄球菌、大肠杆菌、停乳链球菌、粪肠球菌、表皮葡萄球菌、酵母菌、沙门氏菌、蜡样芽孢杆菌、单核李斯特菌和纯水进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其不同菌种间的特异性。The different strains were recovered and cultivated, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Streptococcus dysgalactiae, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Enterococcus faecalis, and Staphylococcus epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果。1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为: 1×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.
2结果2 results
停乳链球菌不同菌种间特异性鉴定电泳结果如图9a所示,停乳链球菌不同菌种间特异性鉴定核酸免疫层析试纸条鉴定结果如图9b所示,结果显示:本发明建立的PCR扩增后检测方法对停乳链球菌具有较好的特异性,在不同菌种间也未发生明显的试纸条阳性反应,具有很好的辨识度,在未借助其他实验仪器的情况下肉眼便可观察,可以方便基层工作者们的使用,更有助于检出停乳链球菌。The electrophoresis results of specific identification between different strains of Streptococcus dysgalactiae are shown in Figure 9a, and the identification results of nucleic acid immunochromatographic test strips for the specific identification of different strains of Streptococcus dysgalactiae are shown in Figure 9b. The results show that: the present invention The established PCR amplification detection method has good specificity for Streptococcus dysgalactiae, and there is no obvious positive test strip reaction between different strains, and it has a good degree of identification. It can be observed with the naked eye under circumstances, which is convenient for grass-roots workers to use, and is more helpful for the detection of Streptococcus dysgalactiae.
实施例9Example 9
测试核酸免疫层析试纸条检测停乳链球菌的灵敏度。To test the sensitivity of nucleic acid immunochromatographic strips to detect Streptococcus dysgalactiae.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
停乳链球菌由南京农业大学动物医学院发明实施例分离鉴定保存。Streptococcus dysgalactiae was isolated, identified and preserved by the Invention Example of the School of Veterinary Medicine of Nanjing Agricultural University.
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将停乳链球菌复苏培养,取菌液梯度稀释后采用平板计数,图9a、9b中1-9分别对应的菌落数为1.6×108cfu/ml、1.6×107cfu/ml、1.6×106cfu/ml、1.6×105cfu/ml、1.6×104cfu/ml、1.6× 103cfu/ml、1.6×102cfu/ml、1.6×101cfu/ml、2cfu/ml;同时提取细菌DNA作为PCR模板,用上述(1.3)PCR反应体系对停乳链球菌进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其灵敏度。Streptococcus dysgalactiae was revived and cultured, and the bacterial solution was serially diluted and counted on a plate. The number of colonies corresponding to 1-9 in Figures 9a and 9b were 1.6×10 8 cfu/ml, 1.6×10 7 cfu/ml, 1.6× 10 6 cfu/ml, 1.6×10 5 cfu/ml, 1.6×10 4 cfu/ml, 1.6×10 3 cfu/ml, 1.6×10 2 cfu/ml, 1.6×10 1 cfu/ml, 2cfu/ml; At the same time, bacterial DNA was extracted as a PCR template, and the above-mentioned (1.3) PCR reaction system was used for PCR amplification of Streptococcus dysgalactiae, and then gel electrophoresis and nucleic acid immunochromatography test strips were used to verify its sensitivity.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果。1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为: 1×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis are photographed and saved.
2结果2 results
停乳链球菌检测灵敏度鉴定电泳结果如图10a所示,停乳链球菌检测灵敏度鉴定核酸免疫层析试纸条鉴定结果如图10b所示,结果显示:可见本发明建立的检测停乳链球菌的核酸免疫层析试纸条检测方法检测灵敏度高,可达到最低检测限为1.6×103cfu/mL,比核酸凝胶电泳的检测灵敏度高10倍。The electrophoresis results of the detection sensitivity identification of Streptococcus dysgalactiae are shown in Figure 10a, and the identification results of the nucleic acid immunochromatography test strips of the detection sensitivity of Streptococcus dysgalactiae are shown in Figure 10b. The nucleic acid immunochromatography test strip detection method has high detection sensitivity and can reach the lowest detection limit of 1.6×10 3 cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.
实施例7-实施例9中sdy-FITC-F序列(即第五引物序列)为:TAAAGGTGCAACTGCATACTA;sdy-DIG-R序列(即第六引物序列)为: AGTCACATGGTGGATTTTCCA。In Example 7-Example 9, the sdy-FITC-F sequence (ie, the fifth primer sequence) is: TAAAGGTGCAACTGCATACTA; the sdy-DIG-R sequence (ie, the sixth primer sequence) is: AGTCACATGGTGGATTTTCCA.
实施例10Example 10
测试不同胶体金粒径大小对试纸条的显色效果的影响。The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
由10nm到50nm不同粒径大小到的胶体金溶液由本发明实施例制备。Colloidal gold solutions with different particle sizes ranging from 10 nm to 50 nm are prepared by the examples of the present invention.
1.2不同柠檬酸加入量引起不同粒径的金纳米粒子的外观变化情况如下表所示:1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
1)取不同粒径的胶体金溶液各1ml(如附图11a所示);1) Take 1 ml of colloidal gold solutions of different particle sizes (as shown in Figure 11a);
2)在1ml胶体金溶液中加入1M碳酸钾溶液10ul调节溶液pH,混匀;加入4ul FITC抗体在四度混匀孵育1h;再加入封闭液室温孵育30min;7000r/min离心3min去除未结合蛋白,取上清再次离心,9400r/min离心20min,去上清;加入80ul复溶液,放置4度老化2h;取出后各取6ul滴在金标垫上,于25度烘干;将烘干的金标垫组装到试纸条上。2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min for 3 min to remove unbound proteins , take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, remove the supernatant; add 80ul of reconstituted solution, place it at 4 degrees for aging for 2h; after taking out, take 6ul of each drop on the gold pad, and dry at 25 degrees; The pads are assembled to the test strips.
3)将保存的无乳链球菌复苏,培养至对数期,取菌液进行核酸扩增,用上述(1.3)PCR 反应体系进行PCR扩增;将PCR扩增产物进行不同梯度的稀释;分别滴加到不同粒径胶体金溶液制备的试纸条上,观察结果。3) Resuscitate the preserved Streptococcus agalactiae, cultivate it to logarithmic phase, take the bacterial solution to carry out nucleic acid amplification, and carry out PCR amplification with the above-mentioned (1.3) PCR reaction system; carry out different gradient dilutions of the PCR amplification products; Add dropwise to test strips prepared from colloidal gold solutions of different particle sizes, and observe the results.
2结果2 results
不同粒径的胶体金溶液表观结果和紫外分光光度计扫描结果如图11a、11b所示,本发明实施例所制备的胶体金溶液性状稳定、粒径分布均匀满足实验所需;不同粒径及梯度稀释核酸免疫层析试纸条结果如图11c所示,金颗粒粒径越大越容易发生聚集沉淀,因此在处理过程中大于35nm的胶体金溶液容易聚集无法获得稳定分散的溶剂,不适于制备试纸条使用,在图11c 所示的显色结果中10nm对应的试纸条从金标垫释放程度、背景清晰度、显色强度、显色均匀性等判断显色效果最好。The apparent results of colloidal gold solutions with different particle sizes and the scanning results of UV spectrophotometers are shown in Figures 11a and 11b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 11c. The larger the particle size of gold particles, the more likely to cause aggregation and precipitation. Therefore, colloidal gold solutions larger than 35 nm tend to aggregate during the treatment process and cannot obtain a stable dispersion solvent, which is not suitable for Test strips were prepared for use. In the color development results shown in Figure 11c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.
实施例11Example 11
测试无乳链球菌在不同食源性致病菌污染的特异性鉴定。Test for the specific identification of Streptococcus agalactiae in different foodborne pathogenic contamination.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
本发明实施例涉及的各种人畜共患病菌株见表5。The various zoonotic strains involved in the embodiments of the present invention are shown in Table 5.
表5 实验菌株Table 5 Experimental strains
Table 5 Bacterial strains for detectionTable 5 Bacterial strains for detection
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将不同菌种复苏培养,使用平板计数菌落数,提取细菌DNA,用上述(1.3)PCR反应体系对无乳链球菌、金黄色葡萄球菌、大肠杆菌、停乳链球菌、粪肠球菌、表皮葡萄球菌、酵母菌、沙门氏菌、蜡样芽孢杆菌、单核李斯特菌和纯水进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其不同菌种间的特异性。The different strains were recovered and cultured, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Enterococcus faecalis, Vitis epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果。1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为:1 ×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1 × TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.
2结果2 results
无乳链球菌不同菌种间特异性鉴定电泳结果如图12a所示,无乳链球菌不同菌种间特异性鉴定核酸免疫层析试纸条鉴定结果如图12b所示,结果显示:本发明建立的PCR扩增后检测方法对无乳链球菌具有较好的特异性,在不同菌种间也未发生明显的试纸条阳性反应,具有很好的辨识度,在未借助其他实验仪器的情况下肉眼便可观察,可以方便基层工作者们的使用,更有助于检出无乳链球菌。The electrophoresis results for the specific identification of different strains of Streptococcus agalactiae are shown in Figure 12a, and the identification results of nucleic acid immunochromatography test strips for the specific identification of different strains of Streptococcus agalactiae are shown in Figure 12b. The results show that: the present invention The established PCR amplification detection method has good specificity for Streptococcus agalactiae, and there is no obvious positive reaction of the test strip between different strains, and it has a good degree of identification. It can be observed with the naked eye under the circumstances, which is convenient for grass-roots workers to use, and is more helpful for the detection of Streptococcus agalactiae.
实施例12Example 12
测试核酸免疫层析试纸条检测无乳链球菌的灵敏度。To test the sensitivity of nucleic acid immunochromatographic strips for the detection of Streptococcus agalactiae.
1材料与方法1 Materials and methods
1.1材料1.1 Materials
无乳链球菌由南京农业大学动物医学院发明实施例分离鉴定保存。Streptococcus agalactiae was isolated, identified and preserved by the Invention Example of the School of Veterinary Medicine of Nanjing Agricultural University.
1.2引物设计1.2 Primer Design
1.3 PCR扩增体系:1.3 PCR amplification system:
反应条件:Reaction conditions:
1.4操作方法1.4 How to operate
将无乳链球菌复苏培养,取菌液梯度稀释后采用平板计数,同时提取细菌DNA作为PCR 模板,用上述(1.3)PCR反应体系对无乳链球菌进行PCR扩增后分别用凝胶电泳和核酸免疫层析试纸条验证其灵敏度。Resuscitate and cultivate Streptococcus agalactiae, take the bacterial solution for gradient dilution and count it on a plate, and extract bacterial DNA as a PCR template. The above (1.3) PCR reaction system is used to carry out PCR amplification of Streptococcus agalactiae, and then use gel electrophoresis and gel electrophoresis respectively. Nucleic acid immunochromatography test strips verify its sensitivity.
1)取4μL PCR产物,与196ul展开液混匀后,取60ul滴加到试纸条上,显色五分钟后使用智能手机或照相机拍摄试纸条显色结果。1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.
2)另取5μL PCR产物在含溴化乙锭的1.5%琼脂糖凝胶电泳中进行电泳,电泳条件为:1 ×TAE缓冲液,电压125V,电泳时间25min,并使用核酸凝胶成像仪观察跑完电泳后的凝胶结果,拍摄并保存。2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1 × TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.
2结果2 results
无乳链球菌检测灵敏度鉴定电泳结果如图13a所示,无乳链球菌检测灵敏度鉴定核酸免疫层析试纸条鉴定结果如图13b所示,结果显示:可见本发明建立的检测无乳链球菌的核酸免疫层析试纸条检测方法检测灵敏度高,可达到最低检测限为2×102cfu/mL,比核酸凝胶电泳的检测灵敏度高10倍。The electrophoresis results of the detection sensitivity identification of Streptococcus agalactiae are shown in Figure 13a, and the identification results of the nucleic acid immunochromatographic test strips of the detection sensitivity of Streptococcus agalactiae are shown in Figure 13b. The nucleic acid immunochromatography test strip detection method has high detection sensitivity, and the lowest detection limit is 2 × 102cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.
实施例10-12中的cfb-FITC-F序列(即第七引物序列)为:TAATAATCAAGCCCAGC;cfb-DIG-R序列(即第八引物序列)为:CCTTTTGTTCTAATGCC。The cfb-FITC-F sequence (ie, the seventh primer sequence) in Examples 10-12 is: TAATAATCAAGCCCAGC; the cfb-DIG-R sequence (ie, the eighth primer sequence) is: CCTTTTGTTCTAATGCC.
应当理解,上述实施例仅为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。It should be understood that the above-mentioned embodiments are only intended to illustrate the technical concept and characteristics of the present invention, and the purpose thereof is to enable those who are familiar with the art to understand the content of the present invention and implement it accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included within the protection scope of the present invention.
序列表sequence listing
<110> 南京农业大学<110> Nanjing Agricultural University
<120> 检测试剂、试剂盒及其应用<120> Detection reagents, kits and their applications
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<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列(人工序列)<213> Artificial Sequence (Artificial Sequence)
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<212> DNA<212> DNA
<213> 人工序列(人工序列)<213> Artificial Sequence (Artificial Sequence)
<400> 3<400> 3
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<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(人工序列)<213> Artificial Sequence (Artificial Sequence)
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<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列(人工序列)<213> Artificial Sequence (Artificial Sequence)
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<213> 人工序列(人工序列)<213> Artificial Sequence (Artificial Sequence)
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<213> 人工序列(人工序列)<213> Artificial Sequence (Artificial Sequence)
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