摘要
猪链球菌(Streptococcus suis)是猪的重要致病菌,能够引发猪的脑膜炎、败血症、关节炎等多种疾病,给养猪业带来严重的经济损失。此外,该菌也能感染人类,导致疾病甚至死亡,是重要的人兽共患病原菌。因此,建立准确、快速、灵敏且简便的检测技术对于猪链球菌病的防控至关重要。目前,国内外已开发出多种猪链球菌检测技术,包括传统的微生物学、分子生物学、免疫学方法,以及基于纳米材料的免疫传感器、CRISPR-Cas12a系统、基质辅助激光解吸/电离飞行时间质谱等新型检测技术。本综述概述了上述技术的原理、应用及其优缺点,并探讨了猪链球菌检测技术未来的发展方向,旨在为猪链球菌病的防控提供重要参考。
猪链球菌是一种革兰阳性菌,可引起猪的脑膜炎、败血症、关节炎等疾病,是猪的重要致病菌,对养猪业造成严重的经济损失;同时,该菌也能感染人类,导致脑膜炎、败血症甚至死亡,是一种重要的人兽共患病原
目前,国内外已经开发了多种用于猪链球菌检测的技术,包括传统的微生物学、分子生物学和免疫学方法,以及新兴的基于纳米材料的免疫传感器和CRISPR-Cas12a系统等技术。本文综述了这些技术的原理、应用及其优劣势,并展望了猪链球菌检测技术的未来发展方向,旨在为猪链球菌病的防控提供参考。
1 传统微生物学检测技术
传统微生物学检测方法主要涉及对细菌的分离培养、生化鉴定和血清分型。将来自健康或发病动物的样品进行低温冷藏,并运送至实验室进行处理;样品经过匀浆后,将匀浆液加入到猪链球菌选择性液体培养基中,在37 ℃条件下培养;随后将培养物划线接种于加入5%脱纤维绵羊血的猪链球菌选择性固体培养基上,37 ℃培养后,挑取呈现草绿色α溶血环、针尖大小的菌落进行进一步的鉴
2 分子生物学检测技术
2.1 聚合酶链式反应(PCR)
聚合酶链式反应(PCR)技术具有特异性强、灵敏度高、重复性好、操作简便等优点,可在短时间内提供准确的检测结果。该技术在猪链球菌检测中得到了广泛应用。
2.1.1 普通PCR
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2.1.2 多重PCR (m-PCR)
多重PCR (multiplex PCR, m-PCR)技术是在常规PCR基础上发展起来的,能同时扩增多个DNA片段,快速确定多种病原体或不同血清型,显著提高了检测效率。Silva
相较于普通PCR法,多重PCR法检测范围更广,能显著提高检测效率,并降低检测成本,更适用于临床检测。然而,由于同一反应体系中引物的竞争作用,其灵敏度可能会有所降低。
2.1.3 荧光定量PCR检测技术
荧光定量PCR技术不仅扩增效率高,而且探针特异性强、灵敏度高,该技术已广泛应用于病原微生物的检
实时荧光定量PCR技术的发明实现了从定性到定量的飞跃,但该技术对仪器和检测人员的技术要求较高。
2.2 环介导等温扩增技术(LAMP)
环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)是一种由日本学者Notomi于2000年开发的核苷酸扩增技术,具有特异性强、灵敏度高、简单快速等特点,且不需要昂贵设
相较于PCR方法,LAMP技术显著提高了扩增效率,缩短了检测时间,且设备简单、检测成本低、人员技术要求低,但引物设计要求严格,且有可能产生非特异性扩
2.3 可视化重组酶聚合酶扩增-侧流层析技术(RPA-LFD)
重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA)是一种以DNA为模板的等温扩增技术(

图1 RPA-LFD技术原理图。A:RPA原理图;B:LFD原理图。
Figure 1 RPA-LFD schematic diagram. A: RPA schematic diagram; B: LFD schematic diagram.
RPA-LFD作为一种快速、灵敏的恒温核酸扩增技术,无需特殊仪器,对检测人员技术要求低,相较于PCR技术,更适用于临床快速检
3 免疫学检测技术
免疫学检测技术是基于抗原抗体特异性结合的原理,通过猪链球菌表面特异性抗原刺激机体产生特异性抗体,从而达到检测和诊断的目的。各种检测技术的特异性、敏感性、优缺点各不相同,因此根据技术特点及检测对象选择合适的免疫学检测技术,更适合临床快速检测(
免疫学检测技术 Immunological detection techniques | 特异性 Specificity | 灵敏度 Sensitivity | 优点 Advantages | 缺点 Disadvantages |
---|---|---|---|---|
ELISA |
强 High |
高 High |
操作简单;稳定性较好 Easy to operate; good stability |
样品制备难度大;耗时长 Difficult sample preparation; time-consuming |
GICA |
强 High |
较低 Low |
操作简单;适合临床现场观察结果 Easy to operate; suitable for on-site clinical observation of results |
稳定性较差;易受环境中离子浓度和pH值的影响 Poor stability; easily affected by ion concentration and pH in the environment |
IMS |
强 High |
高 High |
操作简便;从复杂样品中富集目标检测对象;稳定性好 Easy to operate; enrichment of target detection objects from complex samples; Good stability |
磁珠存在非特异性吸附;磁珠回收率低 Non-specific adsorption of magnetic beads; low recovery rate of magnetic beads |
3.1 酶联免疫吸附试验(ELISA)
酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)广泛用于多种病原微生物的检测,它根据抗原-抗体相互作用的原理,利用酶和比色检测来量化目标分子,主要用于检测和量化生物样品中特定的抗体或抗原。
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ELISA技术操作简单,其敏感性、特异性、稳定性较好,适用于临床大规模流行病学调查。然而,该技术也存在样品制备难度大和耗时较长等缺点。
3.2 胶体金免疫层析法(GICA)
胶体金免疫层析法(gold immunochromatographic assay, GICA)是以胶体金作为示踪标志物的一种免疫标记技术,具有操作方便、可视化、不需要复杂的专用设备等优点。Yang
尽管GICA具有诸多优点,但其稳定性易受环境中离子浓度和pH值的影响,且灵敏度不高,难以实现定量测定。
3.3 免疫磁珠技术(IMS)
免疫磁珠技术(immune microbead, IMS)是一种通过抗原抗体特异性反应及磁珠的磁性反应进行分离和富集的技术,由于免疫磁珠粒径小、比表面积大,因此可捕获较多的待测物,并直接在其表面进行酶显色、荧光或同位素显示,该技术具有快速、灵敏度高、重复性好等优点。此外,IMS还可以消除基质干扰并从复杂样品中富集目标检测对
免疫磁珠技术具有操作简便、分离效率高、稳定性良好等优点。然而,磁珠存在非特异性吸附、回收率较低等问题,需进一步优化IMS的应用效率及可靠性。
4 新型检测技术
4.1 基于纳米材料的免疫传感器
免疫传感器是基于抗原与抗体相互识别原理设计的生物传感器,由受体、转换器及放大器三部分构成;它借助抗体或抗原的高度特异性识别功能,将生物分子信息转化为电化学信号,实现对目标分子的快速、准确检测;与传统的培养方法相比,免疫传感器基于抗原-抗体特异性结合,具有更高的灵敏
一些纳米材料具有与生物酶相似的特性。例如,Pt-Pd纳米材料(hollow Pt-Pd bimetal alloy nanoparticles, HPtPd)具有大的比表面积、良好的生物相容性和高的催化能力,还能催化H2O2分解生成O2,作为辣根过氧化物酶(horseradish peroxidase, HRP)的模拟酶。因此,Wang

图2 ECL免疫传感器的制备及反应机理示意图
Figure 2 Preparation and reaction mechanism diagram of ECL immune sensor.
4.2 基于CRISPR-Cas平台的检测技术
微生物适应性免疫系统(CRISPR-Cas)是原核生物的一种天然免疫系统,存在6种CRISPR-Cas类型和至少29种亚

图3 Cards-SSJ/K方法原理图
Figure 3 Schematic diagram of the Cards-SSJ/K method.
4.3 融合机器学习的基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)
基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)是近年来发展起来的一种微生物诊断技术,其基本构造包括进样系统、离子源、飞行时间分析器和离子检测器。其工作原理是:离子源通过激光轰击待测样品,形成带电荷的生物分子从而发生电离,电离的生物分子在电场的作用下加速通过飞行管道,根据到达检测器的时间及离子的数量得到质荷比值(m/z)及信号值,进而形成相应的峰图(

图4 MALDI-TOF MS方法原理图
Figure 4 Schematic diagram of MALDI-TOF MS.
MALDI-TOF MS具有灵敏度高、分辨率高、图谱简明、速度快等优点,而且其制样简便,可实现微量化、大规模、并行化和高度自动化处理待检生物样品。然而,该技术的设备复杂,对实验室条件要求高,因此较难推广于基层兽医的使用中。此外,由于该技术依赖于数据库,对于缺乏质谱数据库的细菌,该方法无法进行鉴定。此外,不良的样品或基质极易造成污染,对检测结果产生严重影响。
5 总结与展望
当前,PCR技术依然是检测猪链球菌病原的主流手段。然而,该技术对专业人员、设备的依赖以及易污染等缺陷限制了其在基层的广泛应用。胶体金免疫层析法等免疫学检测技术虽然适用于临床快速诊断,但其灵敏度和准确性仍有待提升。免疫传感器结合纳米材料展现出灵敏度高、体积小、操作便捷、自动化等优势;CRISPR-Cas系统则以高灵敏度、强特异性及快速检测为特点;MALDI-TOF MS技术同样具备高灵敏度、高分辨率、图谱简明、检测速度快等优点,展现出良好的应用前景。尽管如此,这些新型检测技术目前价格较为昂贵,临床应用推广尚面临一定难度。在临床上,猪链球菌常与其他病原如胸膜肺炎放线杆菌、副猪格拉菌等混合感染,且不同血清型猪链球菌的混合感染也颇为常见(如血清型2、4、5、7、8、9型等)。因此,未来临床诊断的发展方向应根据猪链球菌混合感染的情况,建立准确、简便、快速、灵敏、特异且易于推广的检测方法。在实验室检测中,应考虑应用猪链球菌抗体或核酸类标准物质,以提高检测方法的准确性和可靠性。此外,微流控检测技术作为近年来发展起来的新型检测技术,具有样本需求量少、高通量、灵敏度高、易于集成和便携等优点,今后可考虑将该技术应用于猪链球菌检测。
作者贡献声明
李繁:论文设计;彭泽仁:论文撰写;刘荣启:论文设计;孙洁:论文撰写;吴宗福:论文审阅与修改。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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