环介导等温扩增技术快速检测肉中沙门氏菌的研究
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摘要
沙门氏菌(Salmonella,SM)是一种普遍存在于自然界中的人兽共患病菌。它广泛分布于自然界中,是对人类和动物健康有极大危害的一类致病菌。沙门氏菌食物中毒是所有食物中毒中最常见的一种,对食品安全构成了严重威胁,因此加强食品中沙门氏菌的检查与控制,成为食品安全和公共卫生等方面亟待解决的重要问题。目前,检测食品中沙门氏菌仍采用传统的方法,其操作繁琐,检测时间长,通常需要4~7d,不能满足食品中沙门氏菌快速检测的需要。因此需要建立一种快速、方便的沙门氏菌的检验方法。
环介导等温扩增(loop-mediated isothermal amplification,简称LAMP) 2000年, Notomi等开发出一种新的核酸扩增技术。该技术以其灵敏度高、特异性强、快速、操作简便、检测成本低等优点在病毒、真菌、细菌以及转基因食品的检测中得到应用。因此,建立LAMP技术检测沙门氏菌对于控制沙门氏菌的食物中毒具有重要意义。
本研究针NCBI公布的沙门氏菌特异invA基因序(GenBank EU348367)中的保守序列,使用在线软件(https://primerexplorer.jp/lamp3.0.0/index.html)设计LAMP引物。对反应时间、反应温度、Bst酶的量、镁离子浓度、dNTP的浓度等扩增条件进行优化,建立了LAMP的反应体系。
建立的25μL的LAMP反应体系为:25μL反应体系为外引物(F3和B3)各0.2μM,内引物(FIP和BIP)各0.8μM,环引物(LF和LB)各0.4μM, 2.0 mmol/L Mg2+ ,1.6 mmol/L dNTP,2.5μLBst DNA Polymerase Buffe(r20 mM Tris-HCl (pH8.8,25°C), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% .Triton X-100), lμl (8U) BstDNA polymerase,1.6 M Betaine, 1μL模板DNA,灭菌双蒸水补足体系到25μl。扩增反应条件:62℃水浴30min,再在80℃水浴锅中10min使酶灭活。检测结果可以通过观察体系中否产生肉眼可见焦磷酸镁白色沉淀,或将5μL产物在1.5%琼脂糖凝胶上进行电泳,利用凝胶成像系统观察结果并成像,或者在扩增产物中加入SYBR GreenⅠ荧光染料后,观察反应体系管内颜色变化判断检测结果。
本研究对12株常见致病菌分别进行LAMP反应来验证引物的特异性,结果表明:沙门氏菌为阳性结果,其它菌株均为阴性结果。
本研究对LAMP法快速检测沙门氏菌进行了研究,确定了检测纯菌的灵敏度,人工污染检出限,特异性,并与普通PCR检测方法进行了对比。结果表明:LAMP检沙门氏菌纯菌的灵敏度为7.8 CFU/ml,人工污染沙门氏菌的肉中检出限为5.2×102 CFU/g。采用试剂盒提取DNA,从样品处理到报告结果,耗时2h。而对照,PCR检测沙门氏菌纯菌的灵敏度为7.8×102 CFU/ml,人工污染沙门氏菌的检出限为5.2×104 CFU/g。采用同样方法提取DNA,从样品处理到报告结果,耗时4 h。
本研究建立LAMP检测沙门氏菌的快速检测方法,该方法灵敏度高、耗时短、方法简便,便于在基层推广,为检测食源性致病菌构建了一个技术平台。
Salmonella was a widespread pathogenic bacteria that can infect animals and human beings, it is widely distributed in nature, and is a great health hazard to human and animals. Salmonella food poisoning was the most common of all kinds of food poisoning,therefore to strengthen the assessment and control of Salmonella in food, become a veterinary, food safety and public health aspects of important issues be solved. Traditional method for routine detection of Salmonella was complex and time-consuming. It takes from 4 to 7 days. The method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive.
Loop-mediated isothermal amplification(LAMP) 2000, Notomi developed a novle nucleic acid amplification technology. LAMP assay was a specific, rapid and sensitive method of detecting viruses, fungi, bacteria and Genetically modified food. Therefore, the establishment of LAMP technology for the detection of Salmonella was very important.
The study aimed the specific invA gene sequences of salmonella published in NCBI used Primer Explorer software,Version3, to design six LAMP primers. The reaction conditions were optimized including temperature, time and Mg2+ concentration, Bst concentration etc,established a set of LAMP reaction.
The established in 25μL of LAMP reaction system:0.8μM concentration of each inner primer(FIP and BIP), 0.2μM concentration of each outer primer(F3 and B3), 0.4μM concentration of the loop primer(LF and LB), 2.0 mmol/L Mg2+,1.6mmol/L dNTP,1.6 M betaine, , 2.5μL 10×Bst DNA polymerase reaction buffer (20 mM Tris-Hcl (pH8.8), 10 mM Kcl, 10 mM (NH4)2SO4, 2mM MgSO4, 0.1% Triton X-100), 1μL 8U of the Bst DNA polymerase large fragment, and 1μL isolated DNA templates distilled water was added to 25μL. Amplification reaction conditions: incubated at water bath 62℃, 30 minutes, then heated to 80℃for 10min to terminate the reaction. The detection results can be observed the system whether has visible white white precipitate of magnesium pyrophosphate, or 5μL products in 1.5% agarose gel electrophoresis, or to add product SYBR GreenⅠfluorescent dye, observed colour change reaction tube to judge the results.
There were 12 bacterial strains to be detected by LAMP in order to evaluate the specificity of primers. The result of salmonella was positive and the other strains were negative.
This study explored LAMP technology rapidly detected Salmonella. Determined the sensitivity, artificial contamination detection limit, specificity, and compared with the ordinary PCR detection method. The results showed: the sensitivity of LAMP detection of Salmonella stains was 7.8 CFU/ml, the detection limit of Salmonella in artificially contaminated samples were 5.6×102 CFU/g. Using the DNA extraction kit according to the manufacturer’s instructions, from samples processing to report results, time-consuming was 2 h.In the control, the results of the sensitivity of PCR detection of Salmonella stains was7.8 CFU/ml, the PCR detection limits of Salmonella in meat artificially contaminated samples were 5.6×104 CFU/g. Using the same method of extracting DNA, from samples processing to report results, time-consuming was 4 h.
This study established a rapid method of LAMP detection of Salmonella, the LAMP method is very rapid, sensitive and convenient, for the rapid detection of food-borne pathogens built a technology platform.
环介导等温扩增(loop-mediated isothermal amplification,简称LAMP) 2000年, Notomi等开发出一种新的核酸扩增技术。该技术以其灵敏度高、特异性强、快速、操作简便、检测成本低等优点在病毒、真菌、细菌以及转基因食品的检测中得到应用。因此,建立LAMP技术检测沙门氏菌对于控制沙门氏菌的食物中毒具有重要意义。
本研究针NCBI公布的沙门氏菌特异invA基因序(GenBank EU348367)中的保守序列,使用在线软件(https://primerexplorer.jp/lamp3.0.0/index.html)设计LAMP引物。对反应时间、反应温度、Bst酶的量、镁离子浓度、dNTP的浓度等扩增条件进行优化,建立了LAMP的反应体系。
建立的25μL的LAMP反应体系为:25μL反应体系为外引物(F3和B3)各0.2μM,内引物(FIP和BIP)各0.8μM,环引物(LF和LB)各0.4μM, 2.0 mmol/L Mg2+ ,1.6 mmol/L dNTP,2.5μLBst DNA Polymerase Buffe(r20 mM Tris-HCl (pH8.8,25°C), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% .Triton X-100), lμl (8U) BstDNA polymerase,1.6 M Betaine, 1μL模板DNA,灭菌双蒸水补足体系到25μl。扩增反应条件:62℃水浴30min,再在80℃水浴锅中10min使酶灭活。检测结果可以通过观察体系中否产生肉眼可见焦磷酸镁白色沉淀,或将5μL产物在1.5%琼脂糖凝胶上进行电泳,利用凝胶成像系统观察结果并成像,或者在扩增产物中加入SYBR GreenⅠ荧光染料后,观察反应体系管内颜色变化判断检测结果。
本研究对12株常见致病菌分别进行LAMP反应来验证引物的特异性,结果表明:沙门氏菌为阳性结果,其它菌株均为阴性结果。
本研究对LAMP法快速检测沙门氏菌进行了研究,确定了检测纯菌的灵敏度,人工污染检出限,特异性,并与普通PCR检测方法进行了对比。结果表明:LAMP检沙门氏菌纯菌的灵敏度为7.8 CFU/ml,人工污染沙门氏菌的肉中检出限为5.2×102 CFU/g。采用试剂盒提取DNA,从样品处理到报告结果,耗时2h。而对照,PCR检测沙门氏菌纯菌的灵敏度为7.8×102 CFU/ml,人工污染沙门氏菌的检出限为5.2×104 CFU/g。采用同样方法提取DNA,从样品处理到报告结果,耗时4 h。
本研究建立LAMP检测沙门氏菌的快速检测方法,该方法灵敏度高、耗时短、方法简便,便于在基层推广,为检测食源性致病菌构建了一个技术平台。
Salmonella was a widespread pathogenic bacteria that can infect animals and human beings, it is widely distributed in nature, and is a great health hazard to human and animals. Salmonella food poisoning was the most common of all kinds of food poisoning,therefore to strengthen the assessment and control of Salmonella in food, become a veterinary, food safety and public health aspects of important issues be solved. Traditional method for routine detection of Salmonella was complex and time-consuming. It takes from 4 to 7 days. The method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive.
Loop-mediated isothermal amplification(LAMP) 2000, Notomi developed a novle nucleic acid amplification technology. LAMP assay was a specific, rapid and sensitive method of detecting viruses, fungi, bacteria and Genetically modified food. Therefore, the establishment of LAMP technology for the detection of Salmonella was very important.
The study aimed the specific invA gene sequences of salmonella published in NCBI used Primer Explorer software,Version3, to design six LAMP primers. The reaction conditions were optimized including temperature, time and Mg2+ concentration, Bst concentration etc,established a set of LAMP reaction.
The established in 25μL of LAMP reaction system:0.8μM concentration of each inner primer(FIP and BIP), 0.2μM concentration of each outer primer(F3 and B3), 0.4μM concentration of the loop primer(LF and LB), 2.0 mmol/L Mg2+,1.6mmol/L dNTP,1.6 M betaine, , 2.5μL 10×Bst DNA polymerase reaction buffer (20 mM Tris-Hcl (pH8.8), 10 mM Kcl, 10 mM (NH4)2SO4, 2mM MgSO4, 0.1% Triton X-100), 1μL 8U of the Bst DNA polymerase large fragment, and 1μL isolated DNA templates distilled water was added to 25μL. Amplification reaction conditions: incubated at water bath 62℃, 30 minutes, then heated to 80℃for 10min to terminate the reaction. The detection results can be observed the system whether has visible white white precipitate of magnesium pyrophosphate, or 5μL products in 1.5% agarose gel electrophoresis, or to add product SYBR GreenⅠfluorescent dye, observed colour change reaction tube to judge the results.
There were 12 bacterial strains to be detected by LAMP in order to evaluate the specificity of primers. The result of salmonella was positive and the other strains were negative.
This study explored LAMP technology rapidly detected Salmonella. Determined the sensitivity, artificial contamination detection limit, specificity, and compared with the ordinary PCR detection method. The results showed: the sensitivity of LAMP detection of Salmonella stains was 7.8 CFU/ml, the detection limit of Salmonella in artificially contaminated samples were 5.6×102 CFU/g. Using the DNA extraction kit according to the manufacturer’s instructions, from samples processing to report results, time-consuming was 2 h.In the control, the results of the sensitivity of PCR detection of Salmonella stains was7.8 CFU/ml, the PCR detection limits of Salmonella in meat artificially contaminated samples were 5.6×104 CFU/g. Using the same method of extracting DNA, from samples processing to report results, time-consuming was 4 h.
This study established a rapid method of LAMP detection of Salmonella, the LAMP method is very rapid, sensitive and convenient, for the rapid detection of food-borne pathogens built a technology platform.
引文
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