六种食源性致病微生物PCR检测及固相化试剂盒的研究
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摘要
食品安全是一个重大的世界性公共卫生问题,不仅影响到人们的健康,更是关系到国计民生的大事,越来越受到人们的重视,其中细菌性食物中毒是引起食源性疾病的主要因素。传统的细菌检测方法耗时长,操作繁琐,越来越不能满足实际检测的需要。
本研究通过设计食源性致病微生物的特异性引物、DNA提取方法的优化、以及退火温度和Mg2+浓度等PCR主要反应条件优化后,建立了沙门氏菌(Salmonella spp.)、单核细胞增生性李斯特菌(Listeria monocytogenes)、金黄色葡萄球菌(Staphylococcus aureus)、志贺氏菌(Shigella spp.)、大肠杆菌O157:H7(Escherichia coli O157:H7)和蜡样芽孢杆菌(Bacillus cereus)的单重及多重PCR检测体系。扩增结果表明这6株致病菌均有清晰、特异的目标条带,经测序验证其同源性高达99%,DNA灵敏度可达pg级。因此,本研究建立的PCR检测体系特异性强、灵敏度高。同时以国标法为对照,对人为接种致病菌的牛奶样品、生牛肉以及市场采集的200份样品进行了检测。结果表明,采用多重PCR方法简单快速且灵敏度高,整个检测时间在24 h以内,在肉品中的检测灵敏度可达1 CFU/g。与国标法相比,应用多重PCR在200份样品检测中无假阴性,假阳性低于2%。建立的多重PCR方法可作为现有国家标准方法的有效补充,从而提高现有检测方法的准确性和敏感性。此外,本研究在建立了多重PCR方法的基础上,通过固相化技术,构建了多重PCR检测试剂盒,并对其性能进行了分析。集成的固相化试剂盒稳定性和实用性较好,可常温保存3个月以上而不影响检测效果,具有较大的应用价值,可推广应用于食品卫生检测以及临床检验等领域。
The food security is a major public health problem attracting more and more attention in the world. It is associated closely with not only people’s health but also the national development. Bacterial food poisoning is the main factor which causes the food-borne diseases. The traditional detection of pathogenic bacterium was trivial and time-consuming, and can not satify with detection in practice.
The monoplex PCR and multiplex PCR detection of six food-borne pathogenic bacteria Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Shigella spp., Escherichia coli O157:H7 and Bacillus cereus, were developed on the basis of specific primers, optimization of DNA extracted methods and PCR reaction conditions. All these six pairs of primers were species-specific and did not interfere with each other. DNA sequencing showed that the sequences of PCR products had more than 99% identities with expected sequences. The sensitivity of PCR reaction reached a level of pg DNA. These results demonstrated the developed PCR assay had high specificity and sensitivity for detection of six food-borne pathogenic bacteria. Milk samples artificially inoculated with these six pathogenic bacteria, raw-beef and 200 samples collected from market were detected by multiplex PCR using traditional detection method as control. The result showed that the multiplex PCR method was simple and rapid, the whole detection time was less than 24 hours and the sensitivity was as low as 1 CFU/g. Compared to traditional method, the false negative rate of multiplex PCR was 0 and the false positive rate was below 2% in 200 samples. Therefore, the multiplex PCR method can be used as the supplement of traditional method to increase the accuracy and sensitivity of detection. In addition, the multiplex PCR detection kit with high stability and practicability was developed on the basis of the multiplex PCR method and solidified technology. It can be stored at room temperature for at least three months without affecting the detection results. Thus, it will be wildly applied in the fields of food sanitation detection, clinical inspection and so on.
本研究通过设计食源性致病微生物的特异性引物、DNA提取方法的优化、以及退火温度和Mg2+浓度等PCR主要反应条件优化后,建立了沙门氏菌(Salmonella spp.)、单核细胞增生性李斯特菌(Listeria monocytogenes)、金黄色葡萄球菌(Staphylococcus aureus)、志贺氏菌(Shigella spp.)、大肠杆菌O157:H7(Escherichia coli O157:H7)和蜡样芽孢杆菌(Bacillus cereus)的单重及多重PCR检测体系。扩增结果表明这6株致病菌均有清晰、特异的目标条带,经测序验证其同源性高达99%,DNA灵敏度可达pg级。因此,本研究建立的PCR检测体系特异性强、灵敏度高。同时以国标法为对照,对人为接种致病菌的牛奶样品、生牛肉以及市场采集的200份样品进行了检测。结果表明,采用多重PCR方法简单快速且灵敏度高,整个检测时间在24 h以内,在肉品中的检测灵敏度可达1 CFU/g。与国标法相比,应用多重PCR在200份样品检测中无假阴性,假阳性低于2%。建立的多重PCR方法可作为现有国家标准方法的有效补充,从而提高现有检测方法的准确性和敏感性。此外,本研究在建立了多重PCR方法的基础上,通过固相化技术,构建了多重PCR检测试剂盒,并对其性能进行了分析。集成的固相化试剂盒稳定性和实用性较好,可常温保存3个月以上而不影响检测效果,具有较大的应用价值,可推广应用于食品卫生检测以及临床检验等领域。
The food security is a major public health problem attracting more and more attention in the world. It is associated closely with not only people’s health but also the national development. Bacterial food poisoning is the main factor which causes the food-borne diseases. The traditional detection of pathogenic bacterium was trivial and time-consuming, and can not satify with detection in practice.
The monoplex PCR and multiplex PCR detection of six food-borne pathogenic bacteria Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Shigella spp., Escherichia coli O157:H7 and Bacillus cereus, were developed on the basis of specific primers, optimization of DNA extracted methods and PCR reaction conditions. All these six pairs of primers were species-specific and did not interfere with each other. DNA sequencing showed that the sequences of PCR products had more than 99% identities with expected sequences. The sensitivity of PCR reaction reached a level of pg DNA. These results demonstrated the developed PCR assay had high specificity and sensitivity for detection of six food-borne pathogenic bacteria. Milk samples artificially inoculated with these six pathogenic bacteria, raw-beef and 200 samples collected from market were detected by multiplex PCR using traditional detection method as control. The result showed that the multiplex PCR method was simple and rapid, the whole detection time was less than 24 hours and the sensitivity was as low as 1 CFU/g. Compared to traditional method, the false negative rate of multiplex PCR was 0 and the false positive rate was below 2% in 200 samples. Therefore, the multiplex PCR method can be used as the supplement of traditional method to increase the accuracy and sensitivity of detection. In addition, the multiplex PCR detection kit with high stability and practicability was developed on the basis of the multiplex PCR method and solidified technology. It can be stored at room temperature for at least three months without affecting the detection results. Thus, it will be wildly applied in the fields of food sanitation detection, clinical inspection and so on.
引文
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