LAMP和PCR检测单核细胞增生性李斯特氏菌的研究
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摘要
单核细胞增生性李斯特氏菌(Listeria monocytogenes)简称单增李斯特,它是一种能够引起人畜共患的食源性致病菌,属李斯特菌属。它在自然界中分布广泛,如土壤、腐败的蔬菜及多种食品中。食用了被单增李斯特污染的食物后可以引起“李氏杆菌病”,使人和动物患脑膜炎、败血症、流产等,死亡率可达20%～30%。该菌主要侵袭孕妇、新生儿、老年人和免疫力低的人群。近年来由该菌引起的食物中毒事件越来越多,引起了人们的广泛关注。传统的检测单增李斯特的方法操作繁琐、检测时间较长,通常需要5～7d才能完成且灵敏度较低。不能满足食品中致病菌快速、灵敏的检测需要。因此需要建立快速、灵敏的单增李斯特的检验方法。
目前,快速检测单增李斯特的方法报道最多的是应用PCR方法。环介导等温扩增技术(Loop-mediated isothermal amplification,简称LAMP)是2000年由日本的荣研株式会独自开发出来的一种新的核酸扩增方法。它是针对靶基因的6个区域设计4种特异的引物,利用一种链置换DNA聚合酶(Bst DNApolymerase)在恒温条件(65℃左右)下进行扩增。可以在1h之内,将靶DNA片段扩增10~9～10~(10)倍。
本研究以单增李斯特的李氏溶血素基因(hly)为靶基因,选择特异性引物进行PCR扩增,同时成功设计了LAMP引物进行LAMP反应,并与PCR进行比较。
对PCR反应体系中镁离子浓度、退火温度和循环数等PCR影响要素进行优化,以确定适宜PCR反应体系和PCR扩增程序。PCR反应的总反应体系为50μL,其中含有5μL 10×PCR buffer,4μL dNTPs混合物,2μL 10μmol/L上游引物,2μL 10μmol/L下游引物,4μL氯化镁,0.5μL(5U/μL)Taq DNA聚合酶,32.5μL ddH_2O;PCR扩增程序为:PCR反应采用冷启动,94℃预变性4min,再按94℃变性45s～60℃退火30s～72℃延伸45s进行35个循环,最后72℃再延伸5min。PCR反应产物在2%的琼脂糖凝胶中进行电泳,凝胶成像系统观察结果,产生预期大小的片段(702bp)。
基于单增李斯特的hly基因利用官方设计软件设计出LAMP反应的外引物F3、B3和内引物FIP、BIP。并对反应时间、反应温度、镁离子浓度、内外引物浓度比等扩增条件进行优化。LAMP反应的总反应体系为25μL,其中含有0.8μmol/L的FIP和BIP,0.2μmol/L的F3和B3,400μmol/L的每种dNTP,1mol/L的甜菜碱,20mM Tris-HCl(pH8.8),10mM KCI,10mM(NH_4)_2SO_4,4mM MgSO_4,0.1%Triton-100和一定量的模版DNA。将反应混合物在95℃加热5min后放置冰上冷却,然后加入8U的Bst DNA聚合酶大片段,在63℃孵育60min,然后在80℃加热10min终止反应。LAMP反应产物在2%的琼脂糖凝胶中进行电泳,凝胶成像系统观察结果,产生预期的梯形条带。对12株菌分别进行了PCR反应和LAMP反应来验证引物的特异性,结果表明:单增李斯特均为阳性结果,其它菌株均为阴性结果。采用FTA滤膜制备模板进行PCR和LAMP反应,其方法的灵敏度分别为1.4×10~2CFU/mL和7.3×10CFU/mL。利用PCR技术和LAMP技术直接检测人工污染的鸡肉中的单增李斯特,检出限分别为9.6×10~2CFU/g和8.9×10CFU/g。
通过两种方法的比较,可知LAMP比PCR更快速、灵敏。有着更为广泛的发展前景。通过本课题的研究,为单增李斯特氏菌的快速检验构建了一个新的技术平台。
Listeria monocytogenes,belonging to Listeria genus,is a kind of food-borne pathogenic bacteria which can cause human and animal diseases.It is widely distributed in the environment,where its primary habitat may be soil,decaying vegetation and foods. Ingestion of foods contaminated with L.monocytogenes can result in listeriosis,a severe infectious disease characterized by meningitis,septicemia and abortion etc.The fatality rate is high(30%～40%).Listeriosis predominantly affects certain risk groups,including pregnant women,newborns,elderly people,and immunocompromised patients.The increasing incidences of L.monocytogenes in food-borne outbreaks draw people's attention. Traditional method for routine detection of L.monocytogenes is complex and time-consuming.It takes from 5 to 7 days with low sensitivity.This method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive.
"LAMP" which stands for Loop-mediated Isothermal Amplification is a new nucleic acid amplification method solely developed by Eiken Chemical Co.,Ltd in 2000.It is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions on the target gene and the reaction process proceeds at a constant temperature (about 65℃)using a DNA polymerase with strand displacement activity.It provides high amplification efficiency,with DNA being amplified 10~9-10~10 times in less than 60 min.
In this study,the hly gene of L.monocytogenes was chose as target gene.The specific primers were chose for PCR,meanwhile,two sets of LAMP primers were designed for LAMP Compare the two methods.
The reaction conditions were optimized including Mg~(2+)concentration,annealing temperature,circulating parameter etc.PCR was carried out in a total 50μL reaction mixture containing 5μL10×PCR buffer,4μL mixture of dNTPs,2μL of each primer (10μmol/L),4μL MgCl_2(25mmol/L),0.5μL(5U/uL)Taq DNA polymerase and 32.5μL double-distilled water.Amplification conditions for PCR assay were:cool start,4 min at 94℃,35 cycles of 45s at 94℃,30s at 60℃,45s at 72℃and a final extension of five min at 72℃.PCR products were electrophoresed in a 2%agarose gel,The size of the fragment of the PCR product is in good agreement with the predicted size(702bp).
The outer primers(F3/B3)and the inner primers(FIP/BIP)for LAMP were designed to target the Hly gene from L.monocytogenes by using official primer explorer software. The reaction conditions were optimized including temperature、time、Mg~(2+)concentration and the ratio of the concentration of the outer primers and the inner primers etc.LAMP was carried out in a total 25μL reaction mixture containing 0.8μmol/L each FIP and BIP, 0.2μmol/L F3 and B3,400μmol/L each dNTP,1mol/L betaine,20mM Tris-HCl(pH8.8), 10 mM KCl,10 mM(NH_4)_2SO_4,4 mM MgSO_4,0.1%Triton-100 and amount of DNA. The mixture was heated at 95℃for 5min,then chilled on ice,8 U Bst DNA polymerase large fragment were added,followed by incubation at 63℃for 1 h and heating at 80℃for 10min to termination the reaction.There were 12 bacterial strains to be detected by PCR and LAMP respectively in order to evaluate the specificity of primers.The result of L.monocytogenes was positive and those of other strains were negative.With pure culture, The sensitivities of PCR and LAMP assays using the FTA filters as templates were 1.4×10~2CFU/mL and 7.3×10CFU/mL,respectively.The detection limits of PCR and LAMP assays obtained from artificially inoculated chicken samples were 9.6×10~2CFU/g and 8.9×10CFU/g,respectively.
Compare PCR and LAMP,LAMP is more sensitive and time-saving.
目前,快速检测单增李斯特的方法报道最多的是应用PCR方法。环介导等温扩增技术(Loop-mediated isothermal amplification,简称LAMP)是2000年由日本的荣研株式会独自开发出来的一种新的核酸扩增方法。它是针对靶基因的6个区域设计4种特异的引物,利用一种链置换DNA聚合酶(Bst DNApolymerase)在恒温条件(65℃左右)下进行扩增。可以在1h之内,将靶DNA片段扩增10~9～10~(10)倍。
本研究以单增李斯特的李氏溶血素基因(hly)为靶基因,选择特异性引物进行PCR扩增,同时成功设计了LAMP引物进行LAMP反应,并与PCR进行比较。
对PCR反应体系中镁离子浓度、退火温度和循环数等PCR影响要素进行优化,以确定适宜PCR反应体系和PCR扩增程序。PCR反应的总反应体系为50μL,其中含有5μL 10×PCR buffer,4μL dNTPs混合物,2μL 10μmol/L上游引物,2μL 10μmol/L下游引物,4μL氯化镁,0.5μL(5U/μL)Taq DNA聚合酶,32.5μL ddH_2O;PCR扩增程序为:PCR反应采用冷启动,94℃预变性4min,再按94℃变性45s～60℃退火30s～72℃延伸45s进行35个循环,最后72℃再延伸5min。PCR反应产物在2%的琼脂糖凝胶中进行电泳,凝胶成像系统观察结果,产生预期大小的片段(702bp)。
基于单增李斯特的hly基因利用官方设计软件设计出LAMP反应的外引物F3、B3和内引物FIP、BIP。并对反应时间、反应温度、镁离子浓度、内外引物浓度比等扩增条件进行优化。LAMP反应的总反应体系为25μL,其中含有0.8μmol/L的FIP和BIP,0.2μmol/L的F3和B3,400μmol/L的每种dNTP,1mol/L的甜菜碱,20mM Tris-HCl(pH8.8),10mM KCI,10mM(NH_4)_2SO_4,4mM MgSO_4,0.1%Triton-100和一定量的模版DNA。将反应混合物在95℃加热5min后放置冰上冷却,然后加入8U的Bst DNA聚合酶大片段,在63℃孵育60min,然后在80℃加热10min终止反应。LAMP反应产物在2%的琼脂糖凝胶中进行电泳,凝胶成像系统观察结果,产生预期的梯形条带。对12株菌分别进行了PCR反应和LAMP反应来验证引物的特异性,结果表明:单增李斯特均为阳性结果,其它菌株均为阴性结果。采用FTA滤膜制备模板进行PCR和LAMP反应,其方法的灵敏度分别为1.4×10~2CFU/mL和7.3×10CFU/mL。利用PCR技术和LAMP技术直接检测人工污染的鸡肉中的单增李斯特,检出限分别为9.6×10~2CFU/g和8.9×10CFU/g。
通过两种方法的比较,可知LAMP比PCR更快速、灵敏。有着更为广泛的发展前景。通过本课题的研究,为单增李斯特氏菌的快速检验构建了一个新的技术平台。
Listeria monocytogenes,belonging to Listeria genus,is a kind of food-borne pathogenic bacteria which can cause human and animal diseases.It is widely distributed in the environment,where its primary habitat may be soil,decaying vegetation and foods. Ingestion of foods contaminated with L.monocytogenes can result in listeriosis,a severe infectious disease characterized by meningitis,septicemia and abortion etc.The fatality rate is high(30%～40%).Listeriosis predominantly affects certain risk groups,including pregnant women,newborns,elderly people,and immunocompromised patients.The increasing incidences of L.monocytogenes in food-borne outbreaks draw people's attention. Traditional method for routine detection of L.monocytogenes is complex and time-consuming.It takes from 5 to 7 days with low sensitivity.This method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive.
"LAMP" which stands for Loop-mediated Isothermal Amplification is a new nucleic acid amplification method solely developed by Eiken Chemical Co.,Ltd in 2000.It is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions on the target gene and the reaction process proceeds at a constant temperature (about 65℃)using a DNA polymerase with strand displacement activity.It provides high amplification efficiency,with DNA being amplified 10~9-10~10 times in less than 60 min.
In this study,the hly gene of L.monocytogenes was chose as target gene.The specific primers were chose for PCR,meanwhile,two sets of LAMP primers were designed for LAMP Compare the two methods.
The reaction conditions were optimized including Mg~(2+)concentration,annealing temperature,circulating parameter etc.PCR was carried out in a total 50μL reaction mixture containing 5μL10×PCR buffer,4μL mixture of dNTPs,2μL of each primer (10μmol/L),4μL MgCl_2(25mmol/L),0.5μL(5U/uL)Taq DNA polymerase and 32.5μL double-distilled water.Amplification conditions for PCR assay were:cool start,4 min at 94℃,35 cycles of 45s at 94℃,30s at 60℃,45s at 72℃and a final extension of five min at 72℃.PCR products were electrophoresed in a 2%agarose gel,The size of the fragment of the PCR product is in good agreement with the predicted size(702bp).
The outer primers(F3/B3)and the inner primers(FIP/BIP)for LAMP were designed to target the Hly gene from L.monocytogenes by using official primer explorer software. The reaction conditions were optimized including temperature、time、Mg~(2+)concentration and the ratio of the concentration of the outer primers and the inner primers etc.LAMP was carried out in a total 25μL reaction mixture containing 0.8μmol/L each FIP and BIP, 0.2μmol/L F3 and B3,400μmol/L each dNTP,1mol/L betaine,20mM Tris-HCl(pH8.8), 10 mM KCl,10 mM(NH_4)_2SO_4,4 mM MgSO_4,0.1%Triton-100 and amount of DNA. The mixture was heated at 95℃for 5min,then chilled on ice,8 U Bst DNA polymerase large fragment were added,followed by incubation at 63℃for 1 h and heating at 80℃for 10min to termination the reaction.There were 12 bacterial strains to be detected by PCR and LAMP respectively in order to evaluate the specificity of primers.The result of L.monocytogenes was positive and those of other strains were negative.With pure culture, The sensitivities of PCR and LAMP assays using the FTA filters as templates were 1.4×10~2CFU/mL and 7.3×10CFU/mL,respectively.The detection limits of PCR and LAMP assays obtained from artificially inoculated chicken samples were 9.6×10~2CFU/g and 8.9×10CFU/g,respectively.
Compare PCR and LAMP,LAMP is more sensitive and time-saving.
引文
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