副溶血性弧菌微滴数字PCR定量方法的建立
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摘要
目的:采用微滴式数字PCR技术(Droplet digital PCR,ddPCR),建立副溶血性弧菌快速定量检测。方法:以副溶血性弧菌TLH基因为靶基因,验证确定ddPCR方法的特异性及定量检测的线性范围,以常检出副溶血性弧菌的海产品鳕鱼为样品进行阳性添加,确定方法的可行性。结果:本试验中采用ddPCR检测得到副溶血性弧菌特异性好,有效基因组DNA浓度范围为2~19440拷贝/20μL,分析得菌悬液浓度为50~4.86×105CFU/m L,与3M测试片所得菌悬液浓度无显著性差异(p> 0.05),与荧光PCR相比,可以进行更低浓度检测且能准确定量。结论:副溶血性弧菌ddPCR定量检测技术特异性良好、灵敏度高、结果准确,具有实际应用价值。
Objective: To establish a quantitative accuracy method to detect Vibrio parahemolyticus based on droplet digital PCR.Methods: With the specific sets of primers and probe synthesized to target genes including TLH genes,specificity and sensitivity were tested based on ddPCR and quantitative accuracy analysis was performed on the results.In order to verify the feasibility of the method,positive strain was added to seafood in which was often detected Vibrio parahaemolyticus.Conclusion: Vibrio parahaemolyticus performed good specificity in ddPCR detection in this experiment. The range of effective DNA concentration was 2~19440 copies/20μL.Bacterial suspension concentration was 50~4.86 × 105 CFU/m L,which had no significant difference with 3 M test( p > 0.05). Compared with real-time PCR,the concentration limit of detection was enhanced by two orders of magnitude.The results showed that quantitative detection of Vibrio parahaemolyticus by ddPCR technology had good specificity,high sensitivity and accuracy,which had practical application value.
Objective: To establish a quantitative accuracy method to detect Vibrio parahemolyticus based on droplet digital PCR.Methods: With the specific sets of primers and probe synthesized to target genes including TLH genes,specificity and sensitivity were tested based on ddPCR and quantitative accuracy analysis was performed on the results.In order to verify the feasibility of the method,positive strain was added to seafood in which was often detected Vibrio parahaemolyticus.Conclusion: Vibrio parahaemolyticus performed good specificity in ddPCR detection in this experiment. The range of effective DNA concentration was 2~19440 copies/20μL.Bacterial suspension concentration was 50~4.86 × 105 CFU/m L,which had no significant difference with 3 M test( p > 0.05). Compared with real-time PCR,the concentration limit of detection was enhanced by two orders of magnitude.The results showed that quantitative detection of Vibrio parahaemolyticus by ddPCR technology had good specificity,high sensitivity and accuracy,which had practical application value.
引文
[1]谢晓红,沈莉,王仁刚,等.荧光定量PCR技术快速检测食物中副溶血性弧菌[J].预防医学情报杂志,2013,29(2):171-173.
[2]张蕾,张海予,曾静,等.海产品中副溶血性弧菌免疫磁分离和环介导等温扩增快速检测方法的建立[J].中国食品卫生杂志,2013,25(5):401-405.
[3]陈刚,邵彪,许蓓蓓,等.副溶血性弧菌实时荧光定量PCR标准模板的构建和检测方法的建立[J].肉类研究,2012,26(12):8-11.
[4]郭钦,陈会,徐海堂,等.副溶血性弧菌快速检测技术研究进展[J].食品工业科技,2016,37(24):395-400.
[5]Mccarthy S A,Depaola A,Cook D W,et al. Evaluation of alkaline phosphatase-and digoxigenin-labelled probes for detection of the thermolabile hemolysin(tlh)gene of Vibrio parahaemolyticus[J]. Letters in Applied Microbiology,1999,28(1):66.
[6]秦国民,白雪松,毕可然,等.基于tlh基因病原副溶血弧菌LAMP检测方法的建立[J].黑龙江畜牧兽医(科技版),2014,56(3):39-42.
[7] Wong S K,Zhang X H,Nys W. Vibrio alginolyticus thermolabile hemolysin(TLH)induces apoptosis,membrane vesiculation and necrosis in sea bream erythrocytes[J].Aquaculture,2012,330-333(1):29-36.
[8]Taniguchi H,Hirano H,Kubomura S,et al.Comparison of thenucleotide sequences of the genes for thermostable direct hemolysin and thermolabile hemolysin genes from Vibrio parahaemolyticus[J].Microb Pathog,1986,1(5):425-432.
[9]GB 29921-2013,食品安全国家标准食品中致病菌限量[S].2013.
[10]相兴伟,郑斌,顾丽霞,等.双重LAMP技术快速检测水产品中副溶血性弧菌和霍乱弧菌的方法学研究[J].现代食品科技,2017,33(1):253-260.
[11]韩小龙,张海燕,曹明秀,等.基于环介导等温扩增法(LAMP)检测上海市售贝类产品中副溶血性弧菌的毒力菌株[J].现代食品科技,2015,31(9):278-283.
[12]叶盛,樊学军,裴晓方,等.Taq Man-实时荧光PCR快速检测副溶血性弧菌[J].现代预防医学,2008,35(22):4444-4445.
[13]胡兴娟,沈飚,余辉,等.多重荧光定量PCR法检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌[J].中国食品卫生杂志,2016,28(4):440-444.
[14]Hayden R T,Gu Z,Ingersoll J,et al.Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus[J]. Journal of Clinical Microbiology,2013,51(2):540.
[15]Kelley K,Cosman A,Belgrader P,et al. Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay[J]. Journal of Clinical Microbiology,2013,51(7):2033-2039.
[16]Zhao W,Li Y,Gao P,et al.Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.[J]. Journal of Industrial Microbiology&Biotechnology,2011,38(9):1279-1286.
[17]杨纯佳,张娟,周臣清,等.数字PCR在食源性致病微生物检测中的应用研究进展[J].食品安全质量检测学报,2016,7(12):4726-4730.
[18]MORISSET D,TEBIH D,MILAVEC M,et al.Quantitative analysis of food and feed samples with droplet digital PCR[J].Plos One,2013,8(5):e62583.
[19]董莲华,张玲,姜君,等.大肠杆菌O157:H7微滴数字PCR定量方法的建立[J].分析化学研究报告,2015,43(3):319-324.
[20]周巍,李月华,李勇,等.微滴式数字PCR技术定量检测发酵乳中金黄色葡萄球菌[J].食品科学,38(16):287-291.
[21]赵新,兰青阔,陈锐,等.应用微滴数字PCR技术快速检测食用菌中沙门氏菌[J].食品与生物技术学报,2017,36(3):315-321.
[22]禹思宇,唐连飞,陈雪琴,等.检测阴沟肠杆菌的数字PCR定量方法建立[J].畜牧与兽医,2016,48(3):1-4.
[23]王静,刘玉敏,李春喜,等.SD-PMA-dd PCR检测食品中单核细胞增生李斯特氏菌[J].微生物学通报,2016,43(10):2306-2313.
[24]Garrido-maestu A,Chapela M J,Peňaranda E,et al. Inhouse validation of novel multiplex real-time PCR gene combination for the simultaneous detection of the main human pathogenic vibrios(Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus)[J].Food Control,2014,37(1):371-379.
[25]赵丽青,方佩佩,唐静,等.数字PCR定量检测食品中单核细胞增生李斯特氏菌方法的研究[J].食品安全质量检测学报,2017,8(11):4133-4138.
[26]中华人民共和国国家质量监督检验检疫总局.SN/T 1870-2016,出口食品中食源性致病菌检测方法实时荧光PCR法[S].2016.
[27]中华人民共和国国家卫生和计划生育委员会.GB 4789.7-2013,食品安全国家标准食品微生物学检验副溶血性弧菌检验[S].2013.
[28]韩丽娟,遇婷,廖文,等.BAX实时荧光定量PCR法快速检测水产品中副溶血性弧菌[J].中国卫生检验杂志,2016,26(17):2492-2493.
[29]靳梦曈,孙文烁,李沁,等.基于反转录实时荧光定量PCR的3种标准曲线定量活副溶血性弧菌的差值分析[J].微生物学报,2014,54(4):383-390.
[30]王忠发,顾松叶,王虹玲,快速荧光定量PCR检测副溶血性弧菌及其毒力基因方法建立与应用效果评价的研究[J].中国卫生检验杂志,2009,19(8):1745-1748.
[31]王建昌,王金凤,李静,等.基于内参的副溶血性弧菌实时荧光定量PCR快速检测方法的建立[J].中国食品卫生杂志,2015,27(4):408-413.
[32]龚永平,郝中香,陈珍荣,等.微滴式数字PCR绝对定量应用研究进展[J].中国预防兽医学报,2017,39(8):686-690.
[33]Roberts C H,Last A,Molinagonzalez S,et al. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections.[J].Journal of Clinical Microbiology,2013,51(7):2195-2203.
[2]张蕾,张海予,曾静,等.海产品中副溶血性弧菌免疫磁分离和环介导等温扩增快速检测方法的建立[J].中国食品卫生杂志,2013,25(5):401-405.
[3]陈刚,邵彪,许蓓蓓,等.副溶血性弧菌实时荧光定量PCR标准模板的构建和检测方法的建立[J].肉类研究,2012,26(12):8-11.
[4]郭钦,陈会,徐海堂,等.副溶血性弧菌快速检测技术研究进展[J].食品工业科技,2016,37(24):395-400.
[5]Mccarthy S A,Depaola A,Cook D W,et al. Evaluation of alkaline phosphatase-and digoxigenin-labelled probes for detection of the thermolabile hemolysin(tlh)gene of Vibrio parahaemolyticus[J]. Letters in Applied Microbiology,1999,28(1):66.
[6]秦国民,白雪松,毕可然,等.基于tlh基因病原副溶血弧菌LAMP检测方法的建立[J].黑龙江畜牧兽医(科技版),2014,56(3):39-42.
[7] Wong S K,Zhang X H,Nys W. Vibrio alginolyticus thermolabile hemolysin(TLH)induces apoptosis,membrane vesiculation and necrosis in sea bream erythrocytes[J].Aquaculture,2012,330-333(1):29-36.
[8]Taniguchi H,Hirano H,Kubomura S,et al.Comparison of thenucleotide sequences of the genes for thermostable direct hemolysin and thermolabile hemolysin genes from Vibrio parahaemolyticus[J].Microb Pathog,1986,1(5):425-432.
[9]GB 29921-2013,食品安全国家标准食品中致病菌限量[S].2013.
[10]相兴伟,郑斌,顾丽霞,等.双重LAMP技术快速检测水产品中副溶血性弧菌和霍乱弧菌的方法学研究[J].现代食品科技,2017,33(1):253-260.
[11]韩小龙,张海燕,曹明秀,等.基于环介导等温扩增法(LAMP)检测上海市售贝类产品中副溶血性弧菌的毒力菌株[J].现代食品科技,2015,31(9):278-283.
[12]叶盛,樊学军,裴晓方,等.Taq Man-实时荧光PCR快速检测副溶血性弧菌[J].现代预防医学,2008,35(22):4444-4445.
[13]胡兴娟,沈飚,余辉,等.多重荧光定量PCR法检测海产品中副溶血性弧菌、沙门菌和单增李斯特菌[J].中国食品卫生杂志,2016,28(4):440-444.
[14]Hayden R T,Gu Z,Ingersoll J,et al.Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus[J]. Journal of Clinical Microbiology,2013,51(2):540.
[15]Kelley K,Cosman A,Belgrader P,et al. Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay[J]. Journal of Clinical Microbiology,2013,51(7):2033-2039.
[16]Zhao W,Li Y,Gao P,et al.Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.[J]. Journal of Industrial Microbiology&Biotechnology,2011,38(9):1279-1286.
[17]杨纯佳,张娟,周臣清,等.数字PCR在食源性致病微生物检测中的应用研究进展[J].食品安全质量检测学报,2016,7(12):4726-4730.
[18]MORISSET D,TEBIH D,MILAVEC M,et al.Quantitative analysis of food and feed samples with droplet digital PCR[J].Plos One,2013,8(5):e62583.
[19]董莲华,张玲,姜君,等.大肠杆菌O157:H7微滴数字PCR定量方法的建立[J].分析化学研究报告,2015,43(3):319-324.
[20]周巍,李月华,李勇,等.微滴式数字PCR技术定量检测发酵乳中金黄色葡萄球菌[J].食品科学,38(16):287-291.
[21]赵新,兰青阔,陈锐,等.应用微滴数字PCR技术快速检测食用菌中沙门氏菌[J].食品与生物技术学报,2017,36(3):315-321.
[22]禹思宇,唐连飞,陈雪琴,等.检测阴沟肠杆菌的数字PCR定量方法建立[J].畜牧与兽医,2016,48(3):1-4.
[23]王静,刘玉敏,李春喜,等.SD-PMA-dd PCR检测食品中单核细胞增生李斯特氏菌[J].微生物学通报,2016,43(10):2306-2313.
[24]Garrido-maestu A,Chapela M J,Peňaranda E,et al. Inhouse validation of novel multiplex real-time PCR gene combination for the simultaneous detection of the main human pathogenic vibrios(Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus)[J].Food Control,2014,37(1):371-379.
[25]赵丽青,方佩佩,唐静,等.数字PCR定量检测食品中单核细胞增生李斯特氏菌方法的研究[J].食品安全质量检测学报,2017,8(11):4133-4138.
[26]中华人民共和国国家质量监督检验检疫总局.SN/T 1870-2016,出口食品中食源性致病菌检测方法实时荧光PCR法[S].2016.
[27]中华人民共和国国家卫生和计划生育委员会.GB 4789.7-2013,食品安全国家标准食品微生物学检验副溶血性弧菌检验[S].2013.
[28]韩丽娟,遇婷,廖文,等.BAX实时荧光定量PCR法快速检测水产品中副溶血性弧菌[J].中国卫生检验杂志,2016,26(17):2492-2493.
[29]靳梦曈,孙文烁,李沁,等.基于反转录实时荧光定量PCR的3种标准曲线定量活副溶血性弧菌的差值分析[J].微生物学报,2014,54(4):383-390.
[30]王忠发,顾松叶,王虹玲,快速荧光定量PCR检测副溶血性弧菌及其毒力基因方法建立与应用效果评价的研究[J].中国卫生检验杂志,2009,19(8):1745-1748.
[31]王建昌,王金凤,李静,等.基于内参的副溶血性弧菌实时荧光定量PCR快速检测方法的建立[J].中国食品卫生杂志,2015,27(4):408-413.
[32]龚永平,郝中香,陈珍荣,等.微滴式数字PCR绝对定量应用研究进展[J].中国预防兽医学报,2017,39(8):686-690.
[33]Roberts C H,Last A,Molinagonzalez S,et al. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections.[J].Journal of Clinical Microbiology,2013,51(7):2195-2203.
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