单核细胞增生李斯特菌hlyA基因实时荧光PCR的建立与研究
|
摘要
目的建立单核细胞增生李斯特菌hlyA基因TaqMan探针实时荧光PCR,用于食品卫生检验的快速筛查。方法根据单核细胞增生李斯特菌hlyA基因设计特异的引物与TaqMan探针,建立和优化实时荧光PCR反应体系,评估其特异性、灵敏度、稳定性,并在食品卫生检验中与细菌分离方法同步应用比较。结果建立了单核细胞增生李斯特菌TaqMan探针实时荧光PCR反应体系,经优化其最佳退火/延伸的温度为58°C,25μL反应体系中上、下游引物和探针最佳终浓度配比分别为0.7、0.7、0.4μmol/L;该实时荧光PCR反应体系可在35 min内完成检测,与沙门菌、绵羊李斯特菌、英诺克李斯特菌等16种非目标菌无交叉反应,对阳性克隆质粒的检测下限可达100拷贝/μL的稀释梯度,对同一核酸浓度样本20次检测的Ct值变异系数为0.46%;在对120份食品安全风险监测标本的快速检测应用中,6份标本单核细胞增生李斯特菌实时荧光PCR扩增阳性,从相应样本中分离出6株单核细胞增生李斯特菌,实时荧光PCR与分离培养方法检测结果相一致。结论针对单核细胞增生李斯特菌hlyA基因建立的TaqMan探针实时荧光PCR具有快速、特异、灵敏、稳定等优势,可用于食品卫生检验中单核细胞增生李斯特菌的快速、高效筛查。
Objective To establish a TaqMan probe-based real-time PCR for hlyA gene of Listeria monocytogenes andapply it in food hygiene inspection. Methods Based on the conservative sequence of hemolysin gene A of L. monocytogenes,the specific primers and TaqMan probe-based probe were designed to establish a real-time PCR assay. The optimization andassessment for the specificity, sensitivity, and stability of the real-time PCR assay were conducted and applied in food hygieneinspection by contrast with the bacteria isolation method. Results The TaqMan probe-based real-time PCR assay for L.monocytogenes was established. After optimization tests, the optimum annealing/extension temperature for the real-time PCRwas 58 °C, and the optimum concentration ratio for the upstream and downstream primers and the probe in 25 μl reactionvolume were 0.7 μmol/L, 0.7 μmol/L and 0.4 μmol/L, respectively. The real-time PCR assay could finish a test in 35 minutes,and there was no crossing reaction with the other 16 strains of non-targeted bacteria such as Salmonella, L. iuanuii, L. innocua,et al. The lowest detection limit for the assay could reach the dilution of 100 copies of recombinant plasmids per microlitre. Thecoefficient of variation for Ct value tested in twenty times for the same concentration of nucleic acid was 0.46%. Six out of 120 food samples were tested positive for L. monocytogenes by the rapid detection of real-time PCR, and 6 strains of L.monocytogenes were isolated from the corresponding samples. Conclusion The TaqMan probe-based real-time PCR assayfor hlyA gene of L. monocytogenes is rapid, specific, sensitive and stable, and it could have a good use in the rapid and efficientdetection of food hygiene inspection.
Objective To establish a TaqMan probe-based real-time PCR for hlyA gene of Listeria monocytogenes andapply it in food hygiene inspection. Methods Based on the conservative sequence of hemolysin gene A of L. monocytogenes,the specific primers and TaqMan probe-based probe were designed to establish a real-time PCR assay. The optimization andassessment for the specificity, sensitivity, and stability of the real-time PCR assay were conducted and applied in food hygieneinspection by contrast with the bacteria isolation method. Results The TaqMan probe-based real-time PCR assay for L.monocytogenes was established. After optimization tests, the optimum annealing/extension temperature for the real-time PCRwas 58 °C, and the optimum concentration ratio for the upstream and downstream primers and the probe in 25 μl reactionvolume were 0.7 μmol/L, 0.7 μmol/L and 0.4 μmol/L, respectively. The real-time PCR assay could finish a test in 35 minutes,and there was no crossing reaction with the other 16 strains of non-targeted bacteria such as Salmonella, L. iuanuii, L. innocua,et al. The lowest detection limit for the assay could reach the dilution of 100 copies of recombinant plasmids per microlitre. Thecoefficient of variation for Ct value tested in twenty times for the same concentration of nucleic acid was 0.46%. Six out of 120 food samples were tested positive for L. monocytogenes by the rapid detection of real-time PCR, and 6 strains of L.monocytogenes were isolated from the corresponding samples. Conclusion The TaqMan probe-based real-time PCR assayfor hlyA gene of L. monocytogenes is rapid, specific, sensitive and stable, and it could have a good use in the rapid and efficientdetection of food hygiene inspection.
引文
[1]王伟,闫韶飞,白莉,等.北京市售即食熟肉制品中单核细胞增生李斯特菌定量污染水平研究[J].卫生研究,2015,44(6):918-921.
[2]陈学兵.单核细胞增生李斯特菌血流感染并发脑膜炎脑梗死1例[J].中国感染与化疗杂志,2015,15(2):178-179.
[3]中华人民共和国卫生部.食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌检验:GB4789.30-2010[S].北京:中国标准出版社,2010.
[4]周莉,朱海华,关炳峰,等.免疫磁珠检测食品中单增李斯特菌的研究[J].中国食品添加剂,2016,(2):132-136.
[5]崔小辉,贾艳艳,王伟杰,等.针对单增李斯特菌iap基因PCR检测方法的建立及其应用[J].黑龙江畜牧兽医,2015,(23):160-162,172.
[6]李波,赵文胜,王国柱,等.单增李斯特菌的快速检测方法研究进展[J].现代农业科技,2016,(24):265-267,269.
[7]宫照龙,祝仁发,叶长芸,等.118株单核细胞增生李斯特菌的毒力基因检测[J].疾病监测,2007,22(5):299-301.
[8]姚琳,江艳华,李风铃,等.鲜活贝类中单核细胞增生李斯特菌的分离鉴定及毒力基因与耐药性分析[J].中国食品卫生杂志,2017,29(1):5-8.
[9]谭翰清,谭海芳,蔡建生,等.克罗诺杆菌TaqMan-MGB探针实时荧光PCR在食品安全风险监测中的应用[J].现代预防医学,2016,43(6):1005-1007.
[10]李丹丹,徐义刚,李梦圆,等.单核细胞增生李斯特氏菌实时荧光定量PCR快速检测方法的建立[J].中国畜牧兽医,2016,43(6):1453-1457.
[11]闫琳,王晓英,郭云昌,等.应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌[J].卫生研究,2014,43(2):177-183.
[12]许华青.模拟粪便标本中单增李斯特菌和伊氏李斯特菌TaqMan双重Real-time PCR检测方法及初步应用性评价[D].贵阳:贵阳医学院,2014.
[13]刘二龙,袁慕云,吕英姿,等.单增李斯特菌三重实时荧光PCR检测的建立及其毒力基因在分离菌株中分布[J].中国人兽共患病学报,2016,32(5):451-456.
[2]陈学兵.单核细胞增生李斯特菌血流感染并发脑膜炎脑梗死1例[J].中国感染与化疗杂志,2015,15(2):178-179.
[3]中华人民共和国卫生部.食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌检验:GB4789.30-2010[S].北京:中国标准出版社,2010.
[4]周莉,朱海华,关炳峰,等.免疫磁珠检测食品中单增李斯特菌的研究[J].中国食品添加剂,2016,(2):132-136.
[5]崔小辉,贾艳艳,王伟杰,等.针对单增李斯特菌iap基因PCR检测方法的建立及其应用[J].黑龙江畜牧兽医,2015,(23):160-162,172.
[6]李波,赵文胜,王国柱,等.单增李斯特菌的快速检测方法研究进展[J].现代农业科技,2016,(24):265-267,269.
[7]宫照龙,祝仁发,叶长芸,等.118株单核细胞增生李斯特菌的毒力基因检测[J].疾病监测,2007,22(5):299-301.
[8]姚琳,江艳华,李风铃,等.鲜活贝类中单核细胞增生李斯特菌的分离鉴定及毒力基因与耐药性分析[J].中国食品卫生杂志,2017,29(1):5-8.
[9]谭翰清,谭海芳,蔡建生,等.克罗诺杆菌TaqMan-MGB探针实时荧光PCR在食品安全风险监测中的应用[J].现代预防医学,2016,43(6):1005-1007.
[10]李丹丹,徐义刚,李梦圆,等.单核细胞增生李斯特氏菌实时荧光定量PCR快速检测方法的建立[J].中国畜牧兽医,2016,43(6):1453-1457.
[11]闫琳,王晓英,郭云昌,等.应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌[J].卫生研究,2014,43(2):177-183.
[12]许华青.模拟粪便标本中单增李斯特菌和伊氏李斯特菌TaqMan双重Real-time PCR检测方法及初步应用性评价[D].贵阳:贵阳医学院,2014.
[13]刘二龙,袁慕云,吕英姿,等.单增李斯特菌三重实时荧光PCR检测的建立及其毒力基因在分离菌株中分布[J].中国人兽共患病学报,2016,32(5):451-456.