LAMP技术检测肉及肉制品中沙门氏菌的研究
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摘要
食品安全突发事件频率高是近年来食品安全问题的一个显著特点,沙门氏菌病是公共卫生学上具有重要意义的人畜共患病之一,其病原沙门氏菌属肠道细菌科,包括那些引起食物中毒,导致胃肠炎、伤寒和副伤寒的细菌。人畜感染后可呈无症状带菌状态,也可表现为有临床症状的致死疾病,它可能加重病态或死亡率,或者降低动物的繁殖生产力。为确保食品安全与食品贸易,需要大力加强食品检验的力度,迫切需要建立快速、灵敏的沙门氏菌的检验方法。
环介导等温扩增(loop-mediated isothermal amplification,简称LAMP)是利用4条特殊设计的引物和具有链置换活性的DNA聚合酶,在恒温条件下特异、高效、快速地扩增DNA的新技术。该技术以其特异性强、灵敏度高、快速、准确和操作简便等优点在核酸的科学研究、疾病的诊断和转基因食品检测等领域得到了日益广泛的应用。
本研究针对已报道的沙门氏菌(Salmonella)保守的invA基因设计了2对特异性强的引物,对反应体系中的Mg~(2+)浓度、dNTP浓度、甜菜碱浓度、反应温度以及外引物与内引物浓度比进行优化,以确定适宜LAMP反应体系和LAMP扩增程序,其适宜反应体系为:2.5μL10×Bst buffer,4μLdNTPs混合物,FIP、BIP引物各4μL,F3、B3引物各0.5μL,适宜镁离子浓度为2.5mmol/L,1μLBst链置换聚合酶,4μL甜菜碱,模板DNA2μL,双蒸水2μL,总反应体系25μL。63℃保温1h,观察反应结果。
比较了LAMP检测肉及肉制品中沙门氏菌的7种模板制备方法,表明FTA滤膜法可高效从样品中提取沙门氏菌DNA,可有效的消除LAMP反应的抑制因子,灵敏度高、操作简便、耗时短,该方法明显优于其他方法。比较了LAMP方法与PCR方法检测人工污染肉制品的检出限,LAMP方法检测沙门氏菌的检出限为3cfu/μL,而PCR法的检出限为300 cfu/μL,LAMP法的检出限比PCR法的检出限低100倍,是高效的检测沙门氏菌的方法。
实际检测了48份样品,同时与PCR及国标检测沙门氏菌的方法做了比较,结果表明:国标法检出率为85.4%,检出时间为5d,PCR方法的检出率为89.5%,敏感性为100%,特异性为83.8%,符合率为95.8%,检出时间为6h,LAMP方法的检出率为91.6%,敏感性为100%,特异性为70.0%,符合率为93.8%,检出时间为1.5h。
本研究成功建立了LAMP检测沙门氏菌的新方法,该方法特异性好、敏感性高而且节省时间,为食品中致病菌的检测提供了技术平台,为检测机构推广此技术提供了技术支持。
Food safety emergencies frequently occurred in recent years.Salmonellosis is one of the most significant zoonosis,which is caused by salmonella belonging to intestinal bacteria branch,including bacterium leading to gastroenteritis,typhoid and paratyphoid. Human and animal infections can be a silent carrier state,or a lethal state manifesting clinical symptoms.It may increase the rate of mortality or death,or reduce the propagable productivity of animal.To ensure food safety and business,it is required to establish a rapid,sensitive salmonella testing method in food realm.
A novel nucleic acid amplification method,termed loop-mediated isothermal amplification(LAMP),which amplifies DNA with high specificity,efficiency, andrapidity under isothermal conditions,may be a valuable tool for the rapid detection ofinfectious diseases.This method employs a DNA polymerase that have activity ofstrand displacement DNA synthesis and a set of four specially designed primers thatrecognize a total of six distinct sequences on the target DNA.LAMP can amplify afew copies of DNA to 10~9 in less than an hour.The final products are stem-loop DNAwith several inverted repeats of the target and cauliflower-Iike structures with multipleloops.A positive reaction would be shown as a ladder-like pattern in a gelelectrophoresis analysis.Because of the advantage,the LAMP method will be widelyapplied to research of nucleic acid,clinical diagnosis of infectious diseases anddetection of genetically modif ed organisms etc.
This study designed two pairs of gene-specific primers of conservative invA gene which have been reported by the salmonella,Mg~(2+)concentration,dNTP concentration, betaine concentration,the temperature of the reaction and the propotion of FIP/BIP than F3/B3 are optimized to determine the optimal LAMP.The reaction mixture consisted of 2.5μL of 10×Bst buffer,4μL of dNTPs(1 mM each in solution),each of FIP/BIP primer 4μL(10μM stock solution),each of F3/B3 primer 0.5μL(10μM stock solution),the concentration of Mgcl2 is 2.5mmol/L,1μL Bst DNA polymerase(SU/μL of stock solution),4μL of betaine and 2μL of double-distilled water.The reaction was run under 62℃for 1h,the LAMP products were examined by electrophoresis with 2%agarose gel.
The study compared the detection limit of the LAMP and PCR,the detection limit of the LAMP is 3cfu/μL,while 300 cfu/μL of PCR,so the detection limit of the LAMP is 100 times lower than PCR.The effect of seven methods of extracting DNA from Salmonella in meat were compared.An efficient extraction procedure was confirmed for extraction of Salmonella DNA from meat without enrichment.Salmonella DNA was directly extracted using FTA filter.The extracted DNA was suitable for LAMP detection. The LAMP method is high sensitivity and simple operation,Short time-consuming,this method is superior to other methods.
48 samples were analyzed and the detection rate using the GB method was 85.4%, detection time was 5d.The detection rate of PCR amplification was 89.5%,100%for sensitivity and specificity of 83.8%,the according rate was 95.8%,the detection time was 6h.The detection rate of LAMP amplification was 91.6%,100%for sensitivity and specificity of 70.0%,the according rate was 93.8%,the detection time was 1.5h.
This study establish the LAMP assay of detection of Salmonella successfully,which is high specifical and sensitive,also save time for the detection of pathogens in food.So it provides the technology platform and technical support for the general detection organizations to promote this technology.
环介导等温扩增(loop-mediated isothermal amplification,简称LAMP)是利用4条特殊设计的引物和具有链置换活性的DNA聚合酶,在恒温条件下特异、高效、快速地扩增DNA的新技术。该技术以其特异性强、灵敏度高、快速、准确和操作简便等优点在核酸的科学研究、疾病的诊断和转基因食品检测等领域得到了日益广泛的应用。
本研究针对已报道的沙门氏菌(Salmonella)保守的invA基因设计了2对特异性强的引物,对反应体系中的Mg~(2+)浓度、dNTP浓度、甜菜碱浓度、反应温度以及外引物与内引物浓度比进行优化,以确定适宜LAMP反应体系和LAMP扩增程序,其适宜反应体系为:2.5μL10×Bst buffer,4μLdNTPs混合物,FIP、BIP引物各4μL,F3、B3引物各0.5μL,适宜镁离子浓度为2.5mmol/L,1μLBst链置换聚合酶,4μL甜菜碱,模板DNA2μL,双蒸水2μL,总反应体系25μL。63℃保温1h,观察反应结果。
比较了LAMP检测肉及肉制品中沙门氏菌的7种模板制备方法,表明FTA滤膜法可高效从样品中提取沙门氏菌DNA,可有效的消除LAMP反应的抑制因子,灵敏度高、操作简便、耗时短,该方法明显优于其他方法。比较了LAMP方法与PCR方法检测人工污染肉制品的检出限,LAMP方法检测沙门氏菌的检出限为3cfu/μL,而PCR法的检出限为300 cfu/μL,LAMP法的检出限比PCR法的检出限低100倍,是高效的检测沙门氏菌的方法。
实际检测了48份样品,同时与PCR及国标检测沙门氏菌的方法做了比较,结果表明:国标法检出率为85.4%,检出时间为5d,PCR方法的检出率为89.5%,敏感性为100%,特异性为83.8%,符合率为95.8%,检出时间为6h,LAMP方法的检出率为91.6%,敏感性为100%,特异性为70.0%,符合率为93.8%,检出时间为1.5h。
本研究成功建立了LAMP检测沙门氏菌的新方法,该方法特异性好、敏感性高而且节省时间,为食品中致病菌的检测提供了技术平台,为检测机构推广此技术提供了技术支持。
Food safety emergencies frequently occurred in recent years.Salmonellosis is one of the most significant zoonosis,which is caused by salmonella belonging to intestinal bacteria branch,including bacterium leading to gastroenteritis,typhoid and paratyphoid. Human and animal infections can be a silent carrier state,or a lethal state manifesting clinical symptoms.It may increase the rate of mortality or death,or reduce the propagable productivity of animal.To ensure food safety and business,it is required to establish a rapid,sensitive salmonella testing method in food realm.
A novel nucleic acid amplification method,termed loop-mediated isothermal amplification(LAMP),which amplifies DNA with high specificity,efficiency, andrapidity under isothermal conditions,may be a valuable tool for the rapid detection ofinfectious diseases.This method employs a DNA polymerase that have activity ofstrand displacement DNA synthesis and a set of four specially designed primers thatrecognize a total of six distinct sequences on the target DNA.LAMP can amplify afew copies of DNA to 10~9 in less than an hour.The final products are stem-loop DNAwith several inverted repeats of the target and cauliflower-Iike structures with multipleloops.A positive reaction would be shown as a ladder-like pattern in a gelelectrophoresis analysis.Because of the advantage,the LAMP method will be widelyapplied to research of nucleic acid,clinical diagnosis of infectious diseases anddetection of genetically modif ed organisms etc.
This study designed two pairs of gene-specific primers of conservative invA gene which have been reported by the salmonella,Mg~(2+)concentration,dNTP concentration, betaine concentration,the temperature of the reaction and the propotion of FIP/BIP than F3/B3 are optimized to determine the optimal LAMP.The reaction mixture consisted of 2.5μL of 10×Bst buffer,4μL of dNTPs(1 mM each in solution),each of FIP/BIP primer 4μL(10μM stock solution),each of F3/B3 primer 0.5μL(10μM stock solution),the concentration of Mgcl2 is 2.5mmol/L,1μL Bst DNA polymerase(SU/μL of stock solution),4μL of betaine and 2μL of double-distilled water.The reaction was run under 62℃for 1h,the LAMP products were examined by electrophoresis with 2%agarose gel.
The study compared the detection limit of the LAMP and PCR,the detection limit of the LAMP is 3cfu/μL,while 300 cfu/μL of PCR,so the detection limit of the LAMP is 100 times lower than PCR.The effect of seven methods of extracting DNA from Salmonella in meat were compared.An efficient extraction procedure was confirmed for extraction of Salmonella DNA from meat without enrichment.Salmonella DNA was directly extracted using FTA filter.The extracted DNA was suitable for LAMP detection. The LAMP method is high sensitivity and simple operation,Short time-consuming,this method is superior to other methods.
48 samples were analyzed and the detection rate using the GB method was 85.4%, detection time was 5d.The detection rate of PCR amplification was 89.5%,100%for sensitivity and specificity of 83.8%,the according rate was 95.8%,the detection time was 6h.The detection rate of LAMP amplification was 91.6%,100%for sensitivity and specificity of 70.0%,the according rate was 93.8%,the detection time was 1.5h.
This study establish the LAMP assay of detection of Salmonella successfully,which is high specifical and sensitive,also save time for the detection of pathogens in food.So it provides the technology platform and technical support for the general detection organizations to promote this technology.
引文
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