CN105177170B - 五种食源性致病菌的检测试剂盒及检测方法 - Google Patents
五种食源性致病菌的检测试剂盒及检测方法 Download PDFInfo
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Abstract
本发明公开了五种食源性致病菌的检测试剂盒及检测方法,属于分子生物学和食品安全检测领域,该试剂盒主要包括以下组分:沙门氏菌的引物对;金黄色葡萄球菌的引物对;大肠杆菌O157:H7的引物对;副溶血弧菌的引物对;单核细胞增生李斯特菌的引物对;Taq DNA聚合酶;10×聚合酶链式反应缓冲液;脱氧核糖核苷三磷酸;MgCl2。检测时,制备待测样品聚合酶链式反应模板,加入到试剂盒中进行聚合酶链式反应扩增,再将扩增产物进行电泳与阳性对照即可。本发明的检测方法可同时检出食品中的5种致病菌,检测灵敏度高;检测程序简单、易推广。
Description
技术领域
本发明涉及分子生物学和食品安全检测领域,具体为五种食源性致病菌的检测试剂盒及检测方法。
背景技术
食品安全问题关系到国计民生,随着经济社会的不断发展,对食源性致病菌的检测提出越来越多的要求。沙门氏菌(Salmonella)、金黄色葡萄球菌(Staphylococcusaureus)、大肠杆菌O157:H7(Escherichia coli O157:H7)、单核细胞增生李斯特菌(Listeria monocytogenes)、副溶血弧菌(Vibrio parahaemolyticus)均为食源性致病菌。在一般生鲜食品如果蔬类、肉禽类、海产类及其加工产品中普遍存在,危及人类健康,甚至引发大规模的传染性疾病。传统检测致病菌采用细菌培养、分离、鉴定等一系列步骤,不仅操作时间长,而且检出敏感性差,不利于对食品质量把关。所以发明一种耗时短,易推广的检测方法对食源性致病进行检测、监控,对保障食品生产、加工及物流过程中的质量安全具有积极的作用。
发明内容
为解决上述问题,本发明提供一种可同时检测五种致病菌的检测试剂盒及检测方法,具有检测时间短、灵敏度高的特点。本发明通过以下技术方案实现:五种食源性致病菌的检测试剂盒,该试剂盒包括以下组分:
(1)沙门氏菌的引物对:上游引物5’-GTC CAG CTC TGT CGC CTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’,浓度为0.3-0.5μmol/L;
(2)金黄色葡萄球菌的引物对:上游引物5’-TCT GAT TCA ATT CGT GTT TAC TATGA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’,浓度为0.3-0.5μmol/L;
(3)大肠杆菌O157:H7的引物对:上游引物5’-TAT TTG ATG ATG ATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’,浓度为0.3-0.5μmol/L;
(4)副溶血弧菌的引物对:上游引物5’-ACT AAG TTG GTC GGT TCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’,浓度为0.3-0.5μmol/L;
(5)单核细胞增生李斯特菌的引物对:上游引物5’-GTT AAT GAA CCT ACA AGACCT TCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’,浓度为0.7-0.9μmol/L;
(6)Taq DNA聚合酶浓度为:0.5-1.5U/μL;
(7)10×聚合酶链式反应缓冲液:8-12vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.1-0.3mmol/L;
(9)MgCl2浓度为:1-3mmol/L。
优选的,该试剂盒包括以下组分:
(1)沙门氏菌的引物对:上游引物5’-GTC CAG CTC TGT CGC CTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’,浓度为0.4μmol/L;
(2)金黄色葡萄球菌的引物对:上游引物5’-TCT GAT TCA ATT CGT GTT TAC TATGA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’,浓度为0.4μmol/L;
(3)大肠杆菌O157:H7的引物对:上游引物5’-TAT TTG ATG ATG ATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’,浓度为0.4μmol/L;
(4)副溶血弧菌的引物对:上游引物5’-ACT AAG TTG GTC GGT TCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’,浓度为0.4μmol/L;
(5)单核细胞增生李斯特菌的引物对:上游引物5’-GTT AAT GAA CCT ACA AGACCT TCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’,浓度为0.8μmol/L;
(6)Taq DNA聚合酶浓度为:1U/μL;
(7)10×聚合酶链式反应缓冲液:10vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.2mmol/L;
(9)MgCl2浓度为:2mmol/L。
本发明同时请求保护所述五种食源性致病菌的检测试剂盒的检测方法,该方法包括以下顺序的步骤:
(一)制备阳性对照
(1)将沙门氏菌、金黄色葡萄球菌、大肠杆菌O157:H7、副溶血弧菌和单核细胞增生李斯特菌进行活化培养,分别得到五种菌的培养液;
(2)分别取步骤(1)所得五种菌的培养液与Lysis Buffer细胞裂解液混合均匀后,置于80℃的水浴中,裂解30min,取出后离心4min,取上清液作为聚合酶链式反应模板;
(3)分别取步骤(2)所得聚合酶链式反应模板,添加到试剂盒中,在聚合酶链式反应扩增仪上孵育,其参数为95℃预变性3min;95℃变性30s,56℃退火30s,72℃延伸30s,32个循环;72℃终延伸10min,结束聚合酶链式反应扩增,分别得到五种菌的扩增产物;
(4)分别取步骤(3)得到五种菌的扩增产物与6×Loading buffer缓冲液混匀,在95V恒压下于3wt%的琼脂糖凝胶中,电泳50min;在GelRed染料稀释液里染色20min,所述的GelRed染料稀释液为:15微升GelRed和5mL 1M NaCl混匀后加到45ml蒸馏水中;可分别得到五种菌的条带,作为阳性对照;
(二)检测样品
(1)将待测样品放入装有蛋白胨水的无菌均质袋中,在均质器上拍打均匀,得到样液;
(2)取样液加入到Lysis Buffer细胞裂解液中,80℃水浴裂解20min后,离心10min,取上清液作为聚合酶链式反应模板;
(3)取步骤(2)所得聚合酶链式反应模板,添加到试剂盒中,在聚合酶链式反应扩增仪上孵育,其参数为95℃预变性3min;95℃变性30s,56℃退火30s,72℃延伸30s,32个循环;72℃终延伸10min,结束聚合酶链式反应扩增,得到样品扩增产物;
(4)取步骤(3)得到的样品扩增产物与6×Loading buffer缓冲液混匀,在95V恒压下于3wt%的琼脂糖凝胶中,电泳50min;在GelRed染料稀释液里染色20min,将所得条带分别与阳性对照。
优选的,所述的活化培养为:将沙门氏菌、金黄色葡萄球菌、大肠杆菌O157:H7、副溶血弧菌和单核细胞增生李斯特菌分别接种于营养肉汤培养基,胰酪胨大豆肉汤培养基,LB液体培养基,3wt%NaCl碱性蛋白胨水和胰酪胨大豆酵母浸膏肉汤培养基中,置于37℃,培养24h。
本发明的有益效果如下:
(1)本发明提供的试剂盒和检测方法可同时检出食品中的5种致病菌,检测灵敏度高;
(2)本发明提供的试剂盒和检测方法可在3-6小时内完成检测,耗时短,检测速度快;
(3)本发明提供的试剂盒和检测方法相比测序、杂交及芯片等传统检测技术,本发明方法的检测程序简单、易推广,可以推广至食品生产企业及相关检测机构使用。
附图说明
图1为本发明检测结果电泳图;
其中:1、1000bp DNA marker,2、待检测样品,3、大肠杆菌O157:H7,4、副溶血弧菌,5、金黄色葡萄球菌,6、单核细胞增生李斯特菌,7、沙门氏菌,8、阴性对照。
具体实施方式
以下通过实施例对本发明做进一步说明:
如无特殊说明,本发明所用原料均市售可得,作为优选,本实施例中所用试剂均购自宝生物工程(大连)有限公司;聚合酶链式反应扩增仪:Thermo Scientific Arktik系列多功能PCR仪购自美国赛默飞科技有限公司;金黄色葡萄球菌ATCC 6538、副溶血弧菌ATCC17802、沙门氏菌ATCC14028、单核细胞增生李斯特菌ATCC19115、大肠杆菌O157:H7CICC21530均购自中国工业微生物菌种保藏中心;本实施例试剂盒的反应体系为25μL;
实施例通过以下方法制备阳性对照:
(1)将沙门氏菌、金黄色葡萄球菌、大肠杆菌O157:H7、副溶血弧菌和单核细胞增生李斯特菌分别接种于营养肉汤培养基,胰酪胨大豆肉汤培养基,LB液体培养基,3wt%NaCl碱性蛋白胨水和胰酪胨大豆酵母浸膏肉汤培养基中,置于37℃,培养24h,分别得到五种菌的培养液;
(2)分别取步骤(1)所得五种菌的培养液培养液1000μL与80μL Lysis Buffer细胞裂解液混合均匀后,置于80℃的水浴中,裂解30min,取出后离心4min,取上清液作为聚合酶链式反应模板;
(3)分别取步骤(2)所得聚合酶链式反应模板1μL,添加到试剂盒中,在聚合酶链式反应扩增仪上孵育,其参数为95℃预变性3min;95℃变性30s,56℃退火30s,72℃延伸30s,32个循环;72℃终延伸10min,结束聚合酶链式反应扩增,分别得到五种菌的扩增产物;(4)分别取步骤(3)得到五种菌的扩增产物5μL与1μL 6×Loading buffer缓冲液混匀,在95V恒压下于3wt%的琼脂糖凝胶中,电泳50min;在GelRed染料稀释液里染色20min,可分别得到五种菌的条带,作为阳性对照:350bp的条带,为大肠杆菌O157:H7;440p的条带,为副溶血弧菌;547bp的条带,为金黄色葡萄球菌;708bp的条带,为单核细胞增生李斯特菌;932bp的条带,为沙门氏菌。
实施例1
检测试剂盒包括以下组分:
(1)沙门氏菌的引物对浓度为0.3μmol/L:上游引物5’-GTC CAG CTC TGT CGCCTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’;
(2)金黄色葡萄球菌的引物对浓度为0.3μmol/L:上游引物5’-TCT GAT TCA ATTCGT GTT TAC TAT GA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’;
(3)大肠杆菌O157:H7的引物对浓度为0.5μmol/L:上游引物5’-TAT TTG ATG ATGATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’;
(4)副溶血弧菌的引物对浓度为0.5μmol/L:上游引物5’-ACT AAG TTG GTC GGTTCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’;
(5)单核细胞增生李斯特菌的引物对浓度为0.9μmol/L:上游引物5’-GTT AATGAA CCT ACA AGA CCT TCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’;
(6)Taq DNA聚合酶浓度为:0.5U/μL;
(7)10×聚合酶链式反应缓冲液:8vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.1mmol/L;
(9)MgCl2浓度为:3mmol/L;
样品检测:
(1)无菌操作取哈密瓜样品10g,分别按照109cfu/mL人工污染五种致病菌,在无菌操作台中吹干1h后,放入含有90mL 0.1wt%蛋白胨水的无菌均质袋中,在均质器上拍打混匀,取1.0mL样液分别加50μL Lysis Buffer细胞裂解液,80℃水浴裂解20min,1000r/min离心10min,取上清液作为聚合酶链式反应模板;
(2)将步骤(1)所得聚合酶链式反应模板添加到检测试剂盒中,在聚合酶链式反应扩增仪上孵育,其参数为95℃预变性3min;95℃变性30s,56℃退火30s,72℃延伸30s,32个循环;72℃终延伸10min,结束聚合酶链式反应扩增,得到样品扩增产物;
(3)将步骤(2)所得样品扩增产物5μL与1μL 6×Loading buffer缓冲液混匀,在95V恒压下于3wt%的琼脂糖凝胶中,电泳50min。在GelRed染料稀释液里染色20min,在凝胶成像系统下观察,与阳性对照对比。
实施例2
检测试剂盒包括以下组分:
(1)沙门氏菌的引物对浓度为0.5μmol/L:上游引物5’-GTC CAG CTC TGT CGCCTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’;
(2)金黄色葡萄球菌的引物对浓度为0.5μmol/L:上游引物5’-TCT GAT TCA ATTCGT GTT TAC TAT GA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’;
(3)大肠杆菌O157:H7的引物对浓度为0.3μmol/L:上游引物5’-TAT TTG ATG ATGATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’;
(4)副溶血弧菌的引物对浓度为0.3μmol/L:上游引物5’-ACT AAG TTG GTC GGTTCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’;
(5)单核细胞增生李斯特菌的引物对浓度为0.7μmol/L:上游引物5’-GTT AATGAA CCT ACA AGA CCT TCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’;
(6)Taq DNA聚合酶浓度为:1.5U/μL;
(7)10×聚合酶链式反应缓冲液:8vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.3mmol/L;
(9)MgCl2浓度为:1mmol/L;
样品检测:
(1)无菌操作取生菜样品10g,分别按照109cfu/mL人工污染五种致病菌,在无菌操作台中吹干1h后,放入含有90mL 0.1wt%蛋白胨水的无菌均质袋中,在均质器上拍打混匀,取1.0mL样液分别加50μL Lysis Buffer细胞裂解液,80℃水浴裂解20min,1000r/min离心10min,取上清液作为聚合酶链式反应模板;
(2)将步骤(1)所得聚合酶链式反应模板添加到检测试剂盒中,在聚合酶链式反应扩增仪上孵育,其参数为95℃预变性3min;95℃变性30s,56℃退火30s,72℃延伸30s,32个循环;72℃终延伸10min,结束聚合酶链式反应扩增,得到样品扩增产物;
(3)将步骤(2)所得样品扩增产物5μL与1μL 6×Loading buffer缓冲液混匀,在95V恒压下于3wt%的琼脂糖凝胶中,电泳50min。在GelRed染料稀释液里染色20min,在凝胶成像系统下观察,与阳性对照对比。
实施例3
检测试剂盒包括以下组分:
(1)沙门氏菌的引物对浓度为0.4μmol/L:上游引物5’-GTC CAG CTC TGT CGCCTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’;
(2)金黄色葡萄球菌的引物对浓度为0.4μmol/L:上游引物5’-TCT GAT TCA ATTCGT GTT TAC TAT GA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’;
(3)大肠杆菌O157:H7的引物对浓度为0.4μmol/L:上游引物5’-TAT TTG ATG ATGATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’;
(4)副溶血弧菌的引物对浓度为0.4μmol/L:上游引物5’-ACT AAG TTG GTC GGTTCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’;
(5)单核细胞增生李斯特菌的引物对浓度为0.8μmol/L:上游引物5’-GTT AATGAA CCT ACA AGA CCT TCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’;
(6)Taq DNA聚合酶浓度为:1.0U/μL;
(7)10×聚合酶链式反应缓冲液:12vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.2mmol/L;
(9)MgCl2浓度为:2mmol/L;
样品检测:
(1)无菌操作取牛肉样品10g,分别按照109cfu/mL人工污染五种致病菌,在无菌操作台中吹干1h后,放入含有90mL 0.1wt%蛋白胨水的无菌均质袋中,在均质器上拍打混匀,取1.0mL样液分别加50μL Lysis Buffer细胞裂解液,80℃水浴裂解20min,1000r/min离心10min,取上清液作为聚合酶链式反应模板;
(2)将步骤(1)所得聚合酶链式反应模板添加到检测试剂盒中,在聚合酶链式反应扩增仪上孵育,其参数为95℃预变性3min;95℃变性30s,56℃退火30s,72℃延伸30s,32个循环;72℃终延伸10min,结束聚合酶链式反应扩增,得到样品扩增产物;
(3)将步骤(2)所得样品扩增产物5μL与1μL 6×Loading buffer缓冲液混匀,在95V恒压下于3wt%的琼脂糖凝胶中,电泳50min。在GelRed染料稀释液里染色20min,在凝胶成像系统下观察,与阳性对照对比。
实施例4
检测试剂盒包括以下组分:
(1)沙门氏菌的引物对浓度为0.3μmol/L:上游引物5’-GTC CAG CTC TGT CGCCTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’;
(2)金黄色葡萄球菌的引物对浓度为0.5μmol/L:上游引物5’-TCT GAT TCA ATTCGT GTT TAC TAT GA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’;
(3)大肠杆菌O157:H7的引物对浓度为0.4μmol/L:上游引物5’-TAT TTG ATG ATGATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’;
(4)副溶血弧菌的引物对浓度为0.4μmol/L:上游引物5’-ACT AAG TTG GTC GGTTCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’;
(5)单核细胞增生李斯特菌的引物对浓度为0.7μmol/L:上游引物5’-GTT AATGAA CCT ACA AGA CCT TCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’;
(6)Taq DNA聚合酶浓度为:1.5U/μL;
(7)10×聚合酶链式反应缓冲液:10vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.1mmol/L;
(9)MgCl2浓度为:3mmol/L;
样品检测:
(1)无菌操作取三文鱼样品10g,分别按照109cfu/mL人工污染五种致病菌,在无菌操作台中吹干1h后,放入含有90mL 0.1wt%蛋白胨水的无菌均质袋中,在均质器上拍打混匀,取1.0mL样液分别加50μL Lysis Buffer细胞裂解液,80℃水浴裂解20min,1000r/min离心10min,取上清液作为聚合酶链式反应模板;
(2)将步骤(1)所得聚合酶链式反应模板添加到检测试剂盒中,在聚合酶链式反应扩增仪上孵育,其参数为95℃预变性3min;95℃变性30s,56℃退火30s,72℃延伸30s,32个循环;72℃终延伸10min,结束聚合酶链式反应扩增,得到样品扩增产物;
(3)将步骤(2)所得样品扩增产物5μL与1μL 6×Loading buffer缓冲液混匀,在95V恒压下于3wt%的琼脂糖凝胶中,电泳50min。在GelRed染料稀释液里染色20min,在凝胶成像系统下观察,与阳性对照对比。
表1本发明试剂盒和检测方法的检测结果
污染菌 | 实施例1 | 实施例2 | 实施例3 | 实施例4 |
金黄色葡萄球菌 | + | + | + | + |
沙门氏菌 | + | + | + | + |
大肠杆菌O157:H7 | + | + | + | + |
单核细胞增生李斯特菌 | + | + | + | + |
副溶血弧菌 | + | + | + | + |
**“+”表示检出;“-”表示未检出。
由上表可知,从哈密瓜样品、生菜样品、牛肉样品、三文鱼样品中均同时检测出金黄色葡萄球菌、沙门氏菌、大肠杆菌O157:H7、单核细胞增生李斯特菌和副溶血弧菌。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
<110> 大连民族大学
<120> 五种食源性致病菌的检测试剂盒及检测方法
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<212> DNA
<213> 人工序列
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<223> 人工序列描述:沙门氏菌上游引物的序列
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GTC CAG CTC TGT CGC CTT AAT C
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<212> DNA
<213> 人工序列
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<223> 人工序列描述:沙门氏菌下游引物的序列
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ACG GCT CCC TGC TAC GCT
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<212> DNA
<213> 人工序列
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<223> 人工序列描述:金黄色葡萄球菌上游引物的序列
<400> 3
TCT GAT TCA ATT CGT GTT TAC TAT GA
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<213> 人工序列
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<223> 人工序列描述:金黄色葡萄球菌下游引物的序列
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CGC TAC CTT TAG GCA CTG ACA
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<212> DNA
<213> 人工序列
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<223> 人工序列描述:大肠杆菌O157:H7上游引物的序列
<400> 5
TAT TTG ATG ATG ATC CAG GGG
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<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列描述:大肠杆菌O157:H7下游引物的序列
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ATT TTG TTC TCC GTC TTG TCC TA
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<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列描述:副溶血弧菌上游引物的序列
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ACT AAG TTG GTC GGT TCG ATA TG
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<213> 人工序列
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<223> 人工序列描述:副溶血弧菌下游引物的序列
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TCT GGC TGC TTA GTT TGT GTT TAG
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<211> 24
<212> DNA
<213> 人工序列
<220>
<223>人工序列描述:单核细胞增生李斯特菌上游引物的序列
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GTT AAT GAA CCT ACA AGA CCT TCC
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<211> 21
<212> DNA
<213> 人工序列
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<223> 人工序列描述:单核细胞增生李斯特菌下游引物的序列
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AAC CGT TCT CCA CCA TTC CCA
21
Claims (2)
1.五种食源性致病菌的检测试剂盒,其特征在于,该试剂盒包括以下组分:
(1)沙门氏菌的引物对:上游引物5’-GTC CAG CTC TGT CGC CTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’,浓度为0.3-0.5μmol/L;
(2)金黄色葡萄球菌的引物对:上游引物5’-TCT GAT TCA ATT CGT GTT TAC TATGA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’,浓度为0.3-0.5μmol/L;
(3)大肠杆菌O157:H7的引物对:上游引物5’-TAT TTG ATG ATG ATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’,浓度为0.3-0.5μmol/L;
(4)副溶血弧菌的引物对:上游引物5’-ACT AAG TTG GTC GGT TCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’,浓度为0.3-0.5μmol/L;
(5)单核细胞增生李斯特菌的引物对:上游引物5’-GTT AAT GAA CCT ACA AGA CCTTCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’,浓度为0.7-0.9μmol/L;
(6)Taq DNA聚合酶浓度为:0.5-1.5 U/μL;
(7)10×聚合酶链式反应缓冲液:8-12vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.1-0.3 mmol/L;
(9)MgCl2浓度为:1-3 mmol/L。
2.如权利要求1所述的五种食源性致病菌的检测试剂盒,其特征在于,该试剂盒包括以下组分:
(1)沙门氏菌的引物对:上游引物5’-GTC CAG CTC TGT CGC CTT AAT C-3’,下游引物5’-ACG GCT CCC TGC TAC GCT-3’,浓度为0.4μmol/L;
(2)金黄色葡萄球菌的引物对:上游引物5’-TCT GAT TCA ATT CGT GTT TAC TATGA-3’,下游引物5’-CGC TAC CTT TAG GCA CTG ACA-3’,浓度为0.4μmol/L;
(3)大肠杆菌O157:H7的引物对:上游引物5’-TAT TTG ATG ATG ATC CAG GGG-3’,下游引物5’-ATT TTG TTC TCC GTC TTG TCC TA-3’,浓度为0.4μmol/L;
(4)副溶血弧菌的引物对:上游引物5’-ACT AAG TTG GTC GGT TCG ATA TG-3’,下游引物5’-TCT GGC TGC TTA GTT TGT GTT TAG-3’,浓度为0.4μmol/L;
(5)单核细胞增生李斯特菌的引物对:上游引物5’-GTT AAT GAA CCT ACA AGA CCTTCC-3’,下游引物5’-AAC CGT TCT CCA CCA TTC CCA-3’,浓度为0.8μmol/L;
(6)Taq DNA聚合酶浓度为:1 U/μL;
(7)10×聚合酶链式反应缓冲液:10vt%;
(8)脱氧核糖核苷三磷酸浓度为:0.2 mmol/L;
(9)MgCl2浓度为:2mmol/L。
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