猪肉中沙门氏菌的分离鉴定及PCR快速检测
摘要
沙门氏菌是重要的病原菌,国内外食源性食品安全事故中,有很大一部分是由沙门氏菌引起的,所以对沙门氏菌的检测具有重要意义。本研究以沙门氏菌为出发菌株,验证肠杆菌科噬菌体的特异性及其在选择性增菌过程中消除非沙门氏菌的作用,分离、鉴定沙门氏菌,分析猪肉中肠杆菌科各属的分布;此外,根据沙门氏菌属特异基因FimY设计引物,检验该引物的特异性和在沙门氏菌属内的广谱性,优化普通PCR和Real-time PCR反应条件,建立快速、简便的沙门氏菌检测方法。通过研究得出以下结论:
(1)肠杆菌科噬菌体对其宿主具有很强的专嗜性,选择性增菌过程中加入噬菌体能够有效地消除肠杆菌科中非沙门氏菌的影响;同时,肠杆菌科噬菌体也可用于检测沙门氏菌及其他肠杆菌科细菌;
(2)肠杆菌科各属在猪肉中均有分布,来自市场的猪肉样品肠杆菌检出率低于来自小型屠宰场的猪肉样品;
(3)根据沙门氏菌属特异基因FimY设计的引物在沙门氏菌属内具有广谱性,能够检测出不同血清型的沙门氏菌;同时该引物也具有很高的特异性,大肠杆菌等非沙门氏菌不能检出。
(4)iQ5 PCR仪扩增鼠伤寒沙门氏菌DNA的最佳退火温度为57℃,引物最佳浓度约为0.4-1.0nmol/mL。在以上参数条件下可以得到良好的PCR产物溶解曲线,梯度稀释的DNA扩增曲线的CT值与初始DNA浓度相关性很高;
本研究所用的肠杆菌科噬菌体有很强的特异性,可以用于选择性增菌,消除特定的非目标宿主菌,也可用于食品生产加工过程,消除和控制特定的细菌,保障食品质量。
本研究建立的沙门氏菌PCR检测方法快速、准确,具有很高的特异性,可用于肉类及其制品沙门氏菌的快速检测和质量控制,也可用于食品安全事故中快速溯源。所获得的Real-time PCR的较优参数,可以作为其他微生物Real-time PCR检测时的方法学参考。
Salmonella is an important pathogen, which is a leading course of a great part of food safety accidents happened worldwide. So it is of great significance to detect salmonella. In this research, specificity of Enterobacteriaceae bacteriophages is detected to estimate the effect of bacteriophages to eliminate some non-salmonella species in the enrichment step is tested, and distribution of Enterobacteriaceae within pork is analyzed. At the same time, Primers are devised according to salmonella genus-specific gene FimY to detect salmonella, specificity and wide-adaptability of the primers are confirmed, and parameters for general PCR and Real-time PCR are optimized to develop a quick, handy detection method for salmonella. Conclusions from this research are as follows:
(1) Enterobacteriaceae bacteriophages are of high specificity to their host bacteria, and they can eliminate certain non-salmonella if applied in the enrichment step. Enterobacteriaceae bacteriophages can be used to detect salmonella and other Enterobacteriaceae.
(2)Each Genera of Enterobacteriaceae can be detected in pork samples; detection rate of Enterobacteriaceae within pork samples from markets is higher than that from tiny slaughters.
(3) A pair of primers devised according to salmonella FimY gene can be used to detect different salmonella serovars, which indicates that the primers are wide-adaptable within salmonella genus. This pair of primers is also of high specificity; non-salmonella bacteria, E.coli et al are not detectable when using these primers.
(4) Optimal annealing temperature is 57℃when using iQ5 PCR instrument to amplify S.typhimurium DNA, with optimal primer concentration at 0.4-1.0nmol/mL. With the parameters above, melting curve of PCR product if of good quality, and amplification curves of fold-dilution S .typhimurium DNA are highly correlated.
Enterobacteriaceae bacteriophages used in this study have high specificity, and can be used in selective enrichment to eliminate non-target host bacteria. These bacteriophages can also be used in food processing to control bacteria contamination, ensuring food quality.
The PCR method established in this research is fast, accurate and specific to detect salmonella. This method can be used in detection of salmonella and quality control in meat or meat products, and it can also be applied for tracing salmonella contamination in food safety accidents. The Real-time PCR parameters to detect salmonella obtained in this study can be a reference for other microorganisms.
(1)肠杆菌科噬菌体对其宿主具有很强的专嗜性,选择性增菌过程中加入噬菌体能够有效地消除肠杆菌科中非沙门氏菌的影响;同时,肠杆菌科噬菌体也可用于检测沙门氏菌及其他肠杆菌科细菌;
(2)肠杆菌科各属在猪肉中均有分布,来自市场的猪肉样品肠杆菌检出率低于来自小型屠宰场的猪肉样品;
(3)根据沙门氏菌属特异基因FimY设计的引物在沙门氏菌属内具有广谱性,能够检测出不同血清型的沙门氏菌;同时该引物也具有很高的特异性,大肠杆菌等非沙门氏菌不能检出。
(4)iQ5 PCR仪扩增鼠伤寒沙门氏菌DNA的最佳退火温度为57℃,引物最佳浓度约为0.4-1.0nmol/mL。在以上参数条件下可以得到良好的PCR产物溶解曲线,梯度稀释的DNA扩增曲线的CT值与初始DNA浓度相关性很高;
本研究所用的肠杆菌科噬菌体有很强的特异性,可以用于选择性增菌,消除特定的非目标宿主菌,也可用于食品生产加工过程,消除和控制特定的细菌,保障食品质量。
本研究建立的沙门氏菌PCR检测方法快速、准确,具有很高的特异性,可用于肉类及其制品沙门氏菌的快速检测和质量控制,也可用于食品安全事故中快速溯源。所获得的Real-time PCR的较优参数,可以作为其他微生物Real-time PCR检测时的方法学参考。
Salmonella is an important pathogen, which is a leading course of a great part of food safety accidents happened worldwide. So it is of great significance to detect salmonella. In this research, specificity of Enterobacteriaceae bacteriophages is detected to estimate the effect of bacteriophages to eliminate some non-salmonella species in the enrichment step is tested, and distribution of Enterobacteriaceae within pork is analyzed. At the same time, Primers are devised according to salmonella genus-specific gene FimY to detect salmonella, specificity and wide-adaptability of the primers are confirmed, and parameters for general PCR and Real-time PCR are optimized to develop a quick, handy detection method for salmonella. Conclusions from this research are as follows:
(1) Enterobacteriaceae bacteriophages are of high specificity to their host bacteria, and they can eliminate certain non-salmonella if applied in the enrichment step. Enterobacteriaceae bacteriophages can be used to detect salmonella and other Enterobacteriaceae.
(2)Each Genera of Enterobacteriaceae can be detected in pork samples; detection rate of Enterobacteriaceae within pork samples from markets is higher than that from tiny slaughters.
(3) A pair of primers devised according to salmonella FimY gene can be used to detect different salmonella serovars, which indicates that the primers are wide-adaptable within salmonella genus. This pair of primers is also of high specificity; non-salmonella bacteria, E.coli et al are not detectable when using these primers.
(4) Optimal annealing temperature is 57℃when using iQ5 PCR instrument to amplify S.typhimurium DNA, with optimal primer concentration at 0.4-1.0nmol/mL. With the parameters above, melting curve of PCR product if of good quality, and amplification curves of fold-dilution S .typhimurium DNA are highly correlated.
Enterobacteriaceae bacteriophages used in this study have high specificity, and can be used in selective enrichment to eliminate non-target host bacteria. These bacteriophages can also be used in food processing to control bacteria contamination, ensuring food quality.
The PCR method established in this research is fast, accurate and specific to detect salmonella. This method can be used in detection of salmonella and quality control in meat or meat products, and it can also be applied for tracing salmonella contamination in food safety accidents. The Real-time PCR parameters to detect salmonella obtained in this study can be a reference for other microorganisms.
引文
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