食源性致病菌高通量悬浮芯片检测技术研究
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摘要
食源性致病菌的污染不仅在世界范围内危害巨大，而且也严重影响我国进出口贸易，而致病菌的传统检测技术工作流程长、自动化水平低，难以适应快速检测的实际需求，其他常用的检测方法又不能够满足多元检测的要求。基于此，本研究旨在应用高通量悬浮芯片技术，以六种常见的食源性致病菌包括大肠杆菌O157:H7、志贺氏菌、沙门氏菌、副溶血性弧菌、金黄色葡萄球菌和单核增生李斯特菌为检测靶标物，建立一种新型快速、灵敏、高通量的检测方法，以弥补现有方法不足，实现对食品中致病菌的快速、准确和高效多元检测，进一步完善高通量悬浮芯片食品安全检测技术平台。
本研究以几种常见的食源性致病菌为靶标物，基于核酸特异识别和抗体特异识别的原理，实现了对多种致病菌靶标物的同时多元检测；通过对检测过程的优化和摸索实验条件，进一步提高了悬浮芯片检测的灵敏度、稳定性；通过与PCR检测方法和双抗夹心ELISA法的比对实验，确定了悬浮芯片法检测的准确性，与常规检测方法相比，悬浮芯片法检测简便、快捷，操作简单，结果重复性高，稳定可靠。
本研究的研究内容主要包括以下几个方面：
1、基于致病菌16S rDNA的悬浮芯片多重检测
以四种常见的食源性致病菌大肠杆菌、沙门氏菌、副溶血性弧菌和金黄色葡萄球菌为靶标物，以16SrRNA为靶基因，分别设计基于上述四种目标物的特异性引物及探针，将设计的探针与羧基化的聚苯乙烯微球偶联，特异性地捕获PCR扩增片段，从而达到检测目标菌的目的。
对悬浮芯片检测致病菌核酸的探针特异性及最佳杂交温度进行了优化，经探针特异性验证，所设计探针特异性较好，无明显交叉反应；对偶联探针的微球与目标物杂交温度条件进行了摸索，确定最佳杂交温度为42℃。同时还对单通道和多通道检测探针的灵敏度进行了验证，最终单通道检测探针灵敏度结果如下：大肠杆菌、沙门氏菌、副溶血性弧菌为5×10-6mmol/L，金黄色葡萄球菌为5×10-5mmol/L，多通道检测探针的灵敏度结果如下：大肠杆菌、沙门氏菌、副溶血性弧菌和金黄色葡萄球菌均为1.25×10-4mmol/L。优化了多重PCR检测的样品加入量，加入15μl多重扩增产物产生的荧光信号与单一PCR产物的荧光信号大致相当。将四种致病菌计数后分别添加到四种蔬菜中，利用悬浮芯片检测，结果表明大肠杆菌在油菜中的检测限和副溶血性弧菌在生菜中的检出限均为103CFU/ml，沙门氏菌在黄瓜中和金黄色葡萄球菌在辣椒中的检出限为100CFU/ml。
2、基于致病菌特有基因的悬浮芯片多重检测
针对大肠杆菌O157:H7、志贺氏菌、沙门氏菌、副溶血性弧菌、金黄色葡萄球菌和单核增生李斯特菌的各自特有基因，设计引物，在引物扩增区内设计能够区别其他菌株的特异性探针，通过探针特异性验证、探针灵敏度实验、方法特异性实验等证明该方法可以特异性的检测六种致病菌，与常规PCR方法比较，检测灵敏度明显高于常规方法，多通道检测探针灵敏度为1.6×10-6mmol/L,对单一PCR扩增产物的检测最低检出限为20-4×103CFU/ml，多重PCR扩增产物的检测最低检出限为1-10CFU/ml，不仅可以满足日常食品安全检测的要求，甚至可以满足临床样品的检测要求。
3、基于双抗夹心法的致病菌免疫悬浮芯片检测技术研究
分别制备了志贺氏菌、沙门氏菌和单核增生李斯特菌的多克隆抗体并生物素标记；建立了病原体蛋白悬浮芯片定量检测方法；优化了实验的条件；讨论了方法的特异性、灵敏度、检测范围、定量检测能力，并与临床常用的双抗夹心ELISA方法进行了系统地对比。
本研究通过建立高通量致病菌检测的悬浮芯片技术，通过对反应条件的摸索，最终实现了可用于食源性致病菌的高通量检测分析。检测效果灵敏、快速、高效、稳定，为食品中病原微生物快速检测提供了新方法，具有广阔的应用和发展前景。
Food contaminated with pathogens can lead to various diseases and is a serious threat tohuman health. Pathogens that cause food-borne diseases are the most important factors affectingfood safety. Recently, numerous food poisoning cases have been caused by bacteria.
Aside from the conventional techniques employed for detecting pathogens, newimmunologic methods and molecular biological methods have been described. Although theseapproaches have been successful for pathogen identification at the species and genotype levels,they are costly, time consuming, complicated, have poor sensitivity, or due to the amplificationprocess is not easy to control, likely to cause pollution false positive. Therefore, a rapid,accurate, sensitive multichannel method for detecting pathogens needs to be employed.Suspension array, an expeditious method for the simultaneous detection of several pathogens,was applied in the present study.
We reported the development of a protocol to simultaneously detection of the pathogenswas constructed using a suspension array. This protocol reached the requirements of clinicaldetection limit.
In first part, a new suspension array technology is proposed for the simultaneousquantitative determination of four pathogens in food. Four sets of primers and species-specificcapture probes were designed based on the16S rDNA gene sequences of Escherichia coli,Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella downloaded from GenBank.The specific nucleic acid probes were covalently bound to the surface of fluorescentmicrospheres for liquid suspension hybridization. The biotin-labeled PCR products obtainedfrom the samples were hybridized with their complementary nucleic acid probes, which weredetected after the addition of streptavidin phycoerythrin. No cross-reaction occurred among theproducts, and the sensitivity of the probe in the single-channel method was5×10-6mmol/L. Inthe multi-channel method, the sensitivities were as follows:5×10-4mmol/L for E. coli andSalmonella, and5×10-5mmol/L for S. aureus and V. parahaemolyticus. The multi-channelmethod achieved multidetection of several pathogens. For real sample detection, the methodsshowed a sensitivity of103CFU/g to100CFU/g of vegetables after enrichment for24h at37°C.This study demonstrates the utility of the suspension array specific for the16S rDNA gene fordetermining the presence of the four pathogens in food samples.
In second part, multiplex polymerase chain reaction (PCR) was coupled to a suspension array to construct an assay for simultaneous identification of six kinds of pathogens. Thesuspension array allows the simultaneous detection of different target sequences in a multiplexand high-throughput format. The assay uses a liquid suspension hybridization format withspecific oligonucleotide probes covalently bound to the surface of fluorescent color-codedmicrospheres. Biotinylated target amplicons, hybridized to their complementary probesequences, are quantified by adding the conjugate, streptavidin R-phycoerythrin. Six probesderived from the sequence analysis of a specific gene were developed and validated. There waslittle cross-reaction among the various probes. In the multi-channel method, the detectionsensitivity was1.6×10-6mmol/L. Results of nucleic acid detection assays show detectionsensitivity for20to4×103CFU/ml, which is one to five orders of magnitude higher than thoseof PCR-agarose method in different bacteria. Aside from single channel capability, the assayallows the simultaneous detection of target genes in a single reaction, with detection limits of1-10CFU/ml. The accuracy, speed, flexibility, and sensitivity of the proposed assay arebeneficial for the diagnosis of pathogenic diseases.
In third part, we prepared polyclonal antibodies of Shigella, Salmonella, and monocytesListeria, and antibodies labeled with biotin. Establish a quantitative detection of pathogenprotein suspension array; to optimize the experimental conditions; discussed the specificity,sensitivity, detection range, quantitative detection capabilities of the method and system tocompare with the commonly used in clinical double-antibody sandwich ELISA method. Thenewly developed suspension array appeared to be specific and sensitive, with the detectionsensitivities of about103CFU/ml,2.5×103CFU/ml,20ng/ml for Shigella, Salmonella, andmonocytes Listeria respectively, and in multiplex, the the detection sensitivities of about105CFU/ml,2.5×105CFU/ml,20ng/ml for Shigella, Salmonella, and monocytes Listeriarespectively, which were higher than those of corresponding conventional ELISA tests.
Hence, a high-throughput technology has been adopted to identify pathogens. Data fromthe current study proved to be specific, sensitive, and flexible. The proposed method allowed acomplete array of different target species to be identified in a multiplex format. The assay can beexecuted in short time. Thus, the proposed assay is beneficial for applications in clinical settingsand epidemiological studies where there is a demand for a high-throughput system that allowsthe creation of multiple-testing platforms for routine testing. Further studies involving clinicalcultures and clinical specimens are under way.
本研究以几种常见的食源性致病菌为靶标物，基于核酸特异识别和抗体特异识别的原理，实现了对多种致病菌靶标物的同时多元检测；通过对检测过程的优化和摸索实验条件，进一步提高了悬浮芯片检测的灵敏度、稳定性；通过与PCR检测方法和双抗夹心ELISA法的比对实验，确定了悬浮芯片法检测的准确性，与常规检测方法相比，悬浮芯片法检测简便、快捷，操作简单，结果重复性高，稳定可靠。
本研究的研究内容主要包括以下几个方面：
1、基于致病菌16S rDNA的悬浮芯片多重检测
以四种常见的食源性致病菌大肠杆菌、沙门氏菌、副溶血性弧菌和金黄色葡萄球菌为靶标物，以16SrRNA为靶基因，分别设计基于上述四种目标物的特异性引物及探针，将设计的探针与羧基化的聚苯乙烯微球偶联，特异性地捕获PCR扩增片段，从而达到检测目标菌的目的。
对悬浮芯片检测致病菌核酸的探针特异性及最佳杂交温度进行了优化，经探针特异性验证，所设计探针特异性较好，无明显交叉反应；对偶联探针的微球与目标物杂交温度条件进行了摸索，确定最佳杂交温度为42℃。同时还对单通道和多通道检测探针的灵敏度进行了验证，最终单通道检测探针灵敏度结果如下：大肠杆菌、沙门氏菌、副溶血性弧菌为5×10-6mmol/L，金黄色葡萄球菌为5×10-5mmol/L，多通道检测探针的灵敏度结果如下：大肠杆菌、沙门氏菌、副溶血性弧菌和金黄色葡萄球菌均为1.25×10-4mmol/L。优化了多重PCR检测的样品加入量，加入15μl多重扩增产物产生的荧光信号与单一PCR产物的荧光信号大致相当。将四种致病菌计数后分别添加到四种蔬菜中，利用悬浮芯片检测，结果表明大肠杆菌在油菜中的检测限和副溶血性弧菌在生菜中的检出限均为103CFU/ml，沙门氏菌在黄瓜中和金黄色葡萄球菌在辣椒中的检出限为100CFU/ml。
2、基于致病菌特有基因的悬浮芯片多重检测
针对大肠杆菌O157:H7、志贺氏菌、沙门氏菌、副溶血性弧菌、金黄色葡萄球菌和单核增生李斯特菌的各自特有基因，设计引物，在引物扩增区内设计能够区别其他菌株的特异性探针，通过探针特异性验证、探针灵敏度实验、方法特异性实验等证明该方法可以特异性的检测六种致病菌，与常规PCR方法比较，检测灵敏度明显高于常规方法，多通道检测探针灵敏度为1.6×10-6mmol/L,对单一PCR扩增产物的检测最低检出限为20-4×103CFU/ml，多重PCR扩增产物的检测最低检出限为1-10CFU/ml，不仅可以满足日常食品安全检测的要求，甚至可以满足临床样品的检测要求。
3、基于双抗夹心法的致病菌免疫悬浮芯片检测技术研究
分别制备了志贺氏菌、沙门氏菌和单核增生李斯特菌的多克隆抗体并生物素标记；建立了病原体蛋白悬浮芯片定量检测方法；优化了实验的条件；讨论了方法的特异性、灵敏度、检测范围、定量检测能力，并与临床常用的双抗夹心ELISA方法进行了系统地对比。
本研究通过建立高通量致病菌检测的悬浮芯片技术，通过对反应条件的摸索，最终实现了可用于食源性致病菌的高通量检测分析。检测效果灵敏、快速、高效、稳定，为食品中病原微生物快速检测提供了新方法，具有广阔的应用和发展前景。
Food contaminated with pathogens can lead to various diseases and is a serious threat tohuman health. Pathogens that cause food-borne diseases are the most important factors affectingfood safety. Recently, numerous food poisoning cases have been caused by bacteria.
Aside from the conventional techniques employed for detecting pathogens, newimmunologic methods and molecular biological methods have been described. Although theseapproaches have been successful for pathogen identification at the species and genotype levels,they are costly, time consuming, complicated, have poor sensitivity, or due to the amplificationprocess is not easy to control, likely to cause pollution false positive. Therefore, a rapid,accurate, sensitive multichannel method for detecting pathogens needs to be employed.Suspension array, an expeditious method for the simultaneous detection of several pathogens,was applied in the present study.
We reported the development of a protocol to simultaneously detection of the pathogenswas constructed using a suspension array. This protocol reached the requirements of clinicaldetection limit.
In first part, a new suspension array technology is proposed for the simultaneousquantitative determination of four pathogens in food. Four sets of primers and species-specificcapture probes were designed based on the16S rDNA gene sequences of Escherichia coli,Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella downloaded from GenBank.The specific nucleic acid probes were covalently bound to the surface of fluorescentmicrospheres for liquid suspension hybridization. The biotin-labeled PCR products obtainedfrom the samples were hybridized with their complementary nucleic acid probes, which weredetected after the addition of streptavidin phycoerythrin. No cross-reaction occurred among theproducts, and the sensitivity of the probe in the single-channel method was5×10-6mmol/L. Inthe multi-channel method, the sensitivities were as follows:5×10-4mmol/L for E. coli andSalmonella, and5×10-5mmol/L for S. aureus and V. parahaemolyticus. The multi-channelmethod achieved multidetection of several pathogens. For real sample detection, the methodsshowed a sensitivity of103CFU/g to100CFU/g of vegetables after enrichment for24h at37°C.This study demonstrates the utility of the suspension array specific for the16S rDNA gene fordetermining the presence of the four pathogens in food samples.
In second part, multiplex polymerase chain reaction (PCR) was coupled to a suspension array to construct an assay for simultaneous identification of six kinds of pathogens. Thesuspension array allows the simultaneous detection of different target sequences in a multiplexand high-throughput format. The assay uses a liquid suspension hybridization format withspecific oligonucleotide probes covalently bound to the surface of fluorescent color-codedmicrospheres. Biotinylated target amplicons, hybridized to their complementary probesequences, are quantified by adding the conjugate, streptavidin R-phycoerythrin. Six probesderived from the sequence analysis of a specific gene were developed and validated. There waslittle cross-reaction among the various probes. In the multi-channel method, the detectionsensitivity was1.6×10-6mmol/L. Results of nucleic acid detection assays show detectionsensitivity for20to4×103CFU/ml, which is one to five orders of magnitude higher than thoseof PCR-agarose method in different bacteria. Aside from single channel capability, the assayallows the simultaneous detection of target genes in a single reaction, with detection limits of1-10CFU/ml. The accuracy, speed, flexibility, and sensitivity of the proposed assay arebeneficial for the diagnosis of pathogenic diseases.
In third part, we prepared polyclonal antibodies of Shigella, Salmonella, and monocytesListeria, and antibodies labeled with biotin. Establish a quantitative detection of pathogenprotein suspension array; to optimize the experimental conditions; discussed the specificity,sensitivity, detection range, quantitative detection capabilities of the method and system tocompare with the commonly used in clinical double-antibody sandwich ELISA method. Thenewly developed suspension array appeared to be specific and sensitive, with the detectionsensitivities of about103CFU/ml,2.5×103CFU/ml,20ng/ml for Shigella, Salmonella, andmonocytes Listeria respectively, and in multiplex, the the detection sensitivities of about105CFU/ml,2.5×105CFU/ml,20ng/ml for Shigella, Salmonella, and monocytes Listeriarespectively, which were higher than those of corresponding conventional ELISA tests.
Hence, a high-throughput technology has been adopted to identify pathogens. Data fromthe current study proved to be specific, sensitive, and flexible. The proposed method allowed acomplete array of different target species to be identified in a multiplex format. The assay can beexecuted in short time. Thus, the proposed assay is beneficial for applications in clinical settingsand epidemiological studies where there is a demand for a high-throughput system that allowsthe creation of multiple-testing platforms for routine testing. Further studies involving clinicalcultures and clinical specimens are under way.
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