基于聚合酶螺旋反应技术快速检测铜绿假单胞菌
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摘要
【背景】铜绿假单胞菌是一种重要的水源和食源性致病菌,可引起急性肠道炎、脑膜炎、败血症和皮肤炎症等疾病。加强铜绿假单胞菌的快速检测,对保障食品安全具有重要的意义。【目的】建立聚合酶螺旋反应(Polymerasespiralreaction,PSR)方法快速检测铜绿假单胞菌。【方法】针对铜绿假单胞菌外毒素A调控基因——ETA基因(toxA)设计引物,通过引入加速引物、优化反应条件和筛选颜色指示剂,建立快速检测铜绿假单胞菌的PSR方法,并研究方法的特异性、敏感性和可靠性。【结果】建立的方法在等温65°C条件下,40 min内可完成PSR反应,且可通过钙黄绿素和羟基萘酚蓝直接判读结果。方法特异性强、灵敏度高,最低检出限分别为20 CFU/mL细菌和1.011 5 pg/μL基因组DNA。可视化PSR方法检测包装饮用水来源的分离菌株与传统生化方法检测结果一致。【结论】研究建立的可视化PSR方法为铜绿假单胞菌DNA快速检测提供了一种可行的有效手段。
[Background] Pseudomonas aeruginosa is an important water-and food-borne pathogenic bacterium causing acute enteritis, meningitis, sepsis and skin inflammation. It is important to detect rapidly P. aeruginosa for food safety. [Objective] A rapid and simple method for detection of P.aeruginosa using polymerase spiral reaction(PSR) technique was established. [Methods] The primers were designed according to the P. aeruginosa exotoxin A gene—ETA gene(toxA), the visual PSR method for rapid detection of P. aeruginosa comprised primer screening, accelerated primer introduction, reaction conditions optimization and color indicator screening. The specificity, sensitivity and reliability of the method were evaluated. [Results] The PSR assay to detect toxA of P. aeruginosa could fulfill within40 min at 65 °C, and visualize the results by Calcein and HNB. This method specifically detected P.aeruginosa without showing cross-reaction with closely related Pseudomonas species or other bacteria.The sensitivity of the method was high, with detection limit 20 CFU/mL bacteria and 1.011 5 pg/μL genomic DNA. The detection of isolated strain of packaged drinking water showed that the PSR method was consistent with the results of traditional biochemical methods for the P. aeruginosa detection.[Conclusion] The established visual PSR method provides a potential method to be used for rapid detection in the field.
[Background] Pseudomonas aeruginosa is an important water-and food-borne pathogenic bacterium causing acute enteritis, meningitis, sepsis and skin inflammation. It is important to detect rapidly P. aeruginosa for food safety. [Objective] A rapid and simple method for detection of P.aeruginosa using polymerase spiral reaction(PSR) technique was established. [Methods] The primers were designed according to the P. aeruginosa exotoxin A gene—ETA gene(toxA), the visual PSR method for rapid detection of P. aeruginosa comprised primer screening, accelerated primer introduction, reaction conditions optimization and color indicator screening. The specificity, sensitivity and reliability of the method were evaluated. [Results] The PSR assay to detect toxA of P. aeruginosa could fulfill within40 min at 65 °C, and visualize the results by Calcein and HNB. This method specifically detected P.aeruginosa without showing cross-reaction with closely related Pseudomonas species or other bacteria.The sensitivity of the method was high, with detection limit 20 CFU/mL bacteria and 1.011 5 pg/μL genomic DNA. The detection of isolated strain of packaged drinking water showed that the PSR method was consistent with the results of traditional biochemical methods for the P. aeruginosa detection.[Conclusion] The established visual PSR method provides a potential method to be used for rapid detection in the field.
引文
[1]Micek ST,Lloyd AE,Ritchie DJ,et al.Pseudomonas aeruginosa bloodstream infection:importance of appropriate initial antimicrobial treatment[J].Antimicrobial Agents and Chemotherapy,2005,49(4):1306-1311
[2]Niu ST.Microbiological quality of bottled water on the market and related regulations[J].Foreign Medical Sciences(Section of Hygiene),1994,21(1):31-34(in Chinese)牛胜田.市售瓶装水的微生物学质量及有关条例[J].国外医学卫生学分册,1994,21(1):31-34
[3]Zhang SH,Wu QP,Xu XK,et al.Comparison of detection of Pseudomonas aeruginosa in bottled water[J].Modern Food Science and Technology,2011,27(11):1403-1405,1335(in Chinese)张淑红,吴清平,徐晓可,等.桶装水中铜绿假单胞菌检测方法的比较[J].现代食品科技,2011,27(11):1403-1405,1335
[4]Zhang XD.Interpretation of National Food Safety Standard Packaged drinking water(GB 19298-2014)[J].The Beverage Industry,2016,19(2):7-9(in Chinese)张旭东.《食品安全国家标准包装饮用水》(GB 19298-2014)标准解读[J].饮料工业,2016,19(2):7-9
[5]Guo LP,Ren YH,Niu SW.Pollution investigation of Pseudomonas aeruginosa in bottled drinking water of Luohe City between 2013 and 2014[J].Chinese Journal of Public Health Engineering,2017,16(1):33-35(in Chinese)郭辽朴,仁蕴慧,牛世文.2013-2014年漯河市桶装饮用水中铜绿假单胞菌污染状况调查[J].中国卫生工程学,2017,16(1):33-35
[6]Wang WJ,Yan Y,Luo YB,et al.Analysis of the contamination status of Pseudomonas aeruginosa in mineral water and packed drinking water in Jiangxi province[J].Chinese Journal of Health Laboratory Technology,2017,27(3):419-421(in Chinese)王文娟,颜瑛,罗玉彬,等.江西省矿泉水和包装饮用水中铜绿假单胞菌污染情况分析[J].中国卫生检验杂志,2017,27(3):419-421
[7]Chen S,Li H,Li DH,et al.Analysis of the contamination of barreled drinking water with P.aeruginosa[J].Modern Preventive Medicine,2015,42(6):1129-1130,1135(in Chinese)陈松,李红,李德华,等.桶装饮用水中铜绿假单胞菌污染情况分析[J].现代预防医学,2015,42(6):1129-1130,1135
[8]Wang YM,Tang Z,Qiao X,et al.Survey of Pseudomonas aeruginosa contamination in barreled water in Jiangsu province[J].Chinese Journal of Health Laboratory Technology,2015,25(12):2019-2020(in Chinese)王燕梅,唐震,乔昕,等.江苏省桶装饮用水中铜绿假单胞菌污染情况调查[J].中国卫生检验杂志,2015,25(12):2019-2020
[9]Luo YQ.Analysis of the microbial contaminations of packaged drinking purified water[J].Journal of Food Safety and Quality,2017,8(1):360-363(in Chinese)骆业巧.包装饮用纯净水微生物污染情况分析[J].食品安全质量检测学报,2017,8(1):360-363
[10]Xiao XL,Zhang JW,Gong J,et al.Rapid detection of Pseudomonas aernginosa by the fluorescence quantitative TaqMan PCR assay targetting ETA gene[J].Chinese Journal of Biotechnology,2008,24(4):581-585(in Chinese)肖性龙,张经纬,龚俊,等.ETA基因作为荧光定量PCR靶基因设计TaqMan探针快速检测铜绿假单胞菌的研究[J].生物工程学报,2008,24(4):581-585
[11]Tomita N,Mori Y,Kanda H,et al.Loop-mediated isothermal amplification(LAMP)of gene sequences and simple visual detection of products[J].Nature Protocols,2008,3(5):877-882
[12]Wang H,Huang Q,Huang JF,et al.Rapid detections of OprD2gene in Pseudomoas aeruginosa using loop-mediated isothermal amplification[J].Chinese Journal of Laboratory Medicine,2013,36(6):543-547(in Chinese)王欢,黄庆,黄君富,等.铜绿假单胞菌OprD2基因环介导等温扩增快速检测技术的研究[J].中华检验医学杂志,2013,36(6):543-547
[13]Xu YM,Zou DY,Tang F,et al.Design and analysis of LAMPprimers used in differentially detecting Yersinia enterocolitica and Brucella spp.[J].Chinese Journal of Laboratory Diagnosis,2013,17(1):19-22(in Chinese)徐云明,邹德颖,唐峰,等.区别检测小肠结肠炎耶尔森氏菌与布鲁氏菌LAMP引物的设计与分析[J].中国实验诊断学,2013,17(1):19-22
[14]Xiao LP.Application and study of LAMP technique in the detection of Salmonella in infectious diarrhea[J].Chinese Journal of Health Laboratory Technology,2015,25(24):4240-4242(in Chinese)肖丽萍.环介导技术在感染性腹泻沙门菌检测中的应用与研究[J].中国卫生检验杂志,2015,25(24):4240-4242
[15]Zhou Y,Wan Q,Cai ZH,et al.Loop-mediated isothermal amplification-based rapid detection for Shigella dysenteriae in food samples[J].Microbiology China,2017,44(9):2247-2254(in Chinese)周杨,万强,蔡芷荷,等.环介导等温扩增技术在食品中痢疾志贺氏菌快速检测[J].微生物学通报,2017,44(9):2247-2254
[16]Ding WC,Hu JR,Shi YH,et al.Establishment of loop-mediated isothermal amplification for detection of Vibrio alginolyticus[J].Journal of Molecular Cell Biology,2009,42(1):70-76(in Chinese)丁文超,胡健饶,史雨红,等.环介导恒温扩增技术快速检测溶藻弧菌[J].分子细胞生物学报,2009,42(1):70-76
[17]Dong DR,Zou DY,Liu H,et al.Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing,China[J].Frontiers in Microbiology,2015,6:1100
[18]Dong DR.The study of a new nucleic acid amplification technology termed Polymerase Spiral Reaction(PSR)and application in POCT detection[D].Beijing:Doctoral Dissertation of Academy of Military Medical Sciences,2016(in Chinese)董德荣.一种新型核酸恒温扩增方法的研究及其在现场检测中的应用[D].北京:中国人民解放军军事医学科学院博士学位论文,2016
[19]Liu W,Dong DR,Yang Z,et al.Polymerase Spiral Reaction(PSR):a novel isothermal nucleic acid amplification method[J].Scientific Reports,2015,5:12723
[20]Dong DR,Liu W,Li H,et al.Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing,China[J].Frontiers in Microbiology,2015,6:519
[21]Liu W,Xu YH,Dong DR,et al.Survey and rapid detection of Bordetella pertussis in clinical samples targeting the BP485 in China[J].Frontiers in Public Health,2015,3:39
[22]Kidd TJ,Grimwood K,Ramsay KA,et al.Comparison of three molecular techniques for typing Pseudomonas aeruginosa isolates in sputum samples from patients with cystic fibrosis[J].Journal of Clinical Microbiology,2011,49(1):263-268.
[23]Wang P,Zhang ZL,Qiao YS.Differentiation and identification of Pseudomonas aeruginosa and its interference strains by Fourier transform-infrared spectroscopy[J].Food Science and Technology,2017,42(4):302-305(in Chinese)王萍,张占林,乔勇升.傅立叶变换红外光谱技术对铜绿假单胞菌及其干扰菌的分类鉴定[J].食品科技,2017,42(4):302-305
[24]Goto M,Honda E,Ogura A,et al.Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue[J].Biotechniques,2009,46(3):167-172
[25]Iglewski BH,Kabat D.NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin[J].Proceedings of the National Academy of Sciences of the United States of America,1975,72(6):2284-2288
[26]Gray GL,Smith DH,Baldridge JS,et al.Cloning,nucleotide sequence,and expression in Escherichia coli of the exotoxin Astructural gene of Pseudomonas aeruginosa[J].Proceedings of the National Academy of Sciences of the United States of America,1984,81(9):2645-2649
[27]Zhang W,Li W,Zhang WW,et al.The methodological study on rapid identification of Pseudomonas aeruginosa based on PCRtechnique[J].Chinese Journal of Health Laboratory Technology,2005,15(9):1065-1067(in Chinese)张伟,李闻,张伟尉,等.基于PCR技术的绿脓杆菌快速检测方法研究[J].中国卫生检验杂志,2005,15(9):1065-1067
[28]Khan AA,Cerniglia CE.Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR[J].Applied and Environmental Microbiology,1994,60(10):3739-3745.
[29]Qin X,Emerson J,Stapp J,et al.Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis[J].Journal of Clinical Microbiology,2003,41(9):4312-4317
[30]Xu JR,Moore JE,Murphy PG,et al.Early detection of Pseudomonas aeruginosa-comparison of conventional versus molecular(PCR)detection directly from adult patients with cystic fibrosis(CF)[J].Annals of Clinical Microbiology and Antimicrobials,2004,3:21
[2]Niu ST.Microbiological quality of bottled water on the market and related regulations[J].Foreign Medical Sciences(Section of Hygiene),1994,21(1):31-34(in Chinese)牛胜田.市售瓶装水的微生物学质量及有关条例[J].国外医学卫生学分册,1994,21(1):31-34
[3]Zhang SH,Wu QP,Xu XK,et al.Comparison of detection of Pseudomonas aeruginosa in bottled water[J].Modern Food Science and Technology,2011,27(11):1403-1405,1335(in Chinese)张淑红,吴清平,徐晓可,等.桶装水中铜绿假单胞菌检测方法的比较[J].现代食品科技,2011,27(11):1403-1405,1335
[4]Zhang XD.Interpretation of National Food Safety Standard Packaged drinking water(GB 19298-2014)[J].The Beverage Industry,2016,19(2):7-9(in Chinese)张旭东.《食品安全国家标准包装饮用水》(GB 19298-2014)标准解读[J].饮料工业,2016,19(2):7-9
[5]Guo LP,Ren YH,Niu SW.Pollution investigation of Pseudomonas aeruginosa in bottled drinking water of Luohe City between 2013 and 2014[J].Chinese Journal of Public Health Engineering,2017,16(1):33-35(in Chinese)郭辽朴,仁蕴慧,牛世文.2013-2014年漯河市桶装饮用水中铜绿假单胞菌污染状况调查[J].中国卫生工程学,2017,16(1):33-35
[6]Wang WJ,Yan Y,Luo YB,et al.Analysis of the contamination status of Pseudomonas aeruginosa in mineral water and packed drinking water in Jiangxi province[J].Chinese Journal of Health Laboratory Technology,2017,27(3):419-421(in Chinese)王文娟,颜瑛,罗玉彬,等.江西省矿泉水和包装饮用水中铜绿假单胞菌污染情况分析[J].中国卫生检验杂志,2017,27(3):419-421
[7]Chen S,Li H,Li DH,et al.Analysis of the contamination of barreled drinking water with P.aeruginosa[J].Modern Preventive Medicine,2015,42(6):1129-1130,1135(in Chinese)陈松,李红,李德华,等.桶装饮用水中铜绿假单胞菌污染情况分析[J].现代预防医学,2015,42(6):1129-1130,1135
[8]Wang YM,Tang Z,Qiao X,et al.Survey of Pseudomonas aeruginosa contamination in barreled water in Jiangsu province[J].Chinese Journal of Health Laboratory Technology,2015,25(12):2019-2020(in Chinese)王燕梅,唐震,乔昕,等.江苏省桶装饮用水中铜绿假单胞菌污染情况调查[J].中国卫生检验杂志,2015,25(12):2019-2020
[9]Luo YQ.Analysis of the microbial contaminations of packaged drinking purified water[J].Journal of Food Safety and Quality,2017,8(1):360-363(in Chinese)骆业巧.包装饮用纯净水微生物污染情况分析[J].食品安全质量检测学报,2017,8(1):360-363
[10]Xiao XL,Zhang JW,Gong J,et al.Rapid detection of Pseudomonas aernginosa by the fluorescence quantitative TaqMan PCR assay targetting ETA gene[J].Chinese Journal of Biotechnology,2008,24(4):581-585(in Chinese)肖性龙,张经纬,龚俊,等.ETA基因作为荧光定量PCR靶基因设计TaqMan探针快速检测铜绿假单胞菌的研究[J].生物工程学报,2008,24(4):581-585
[11]Tomita N,Mori Y,Kanda H,et al.Loop-mediated isothermal amplification(LAMP)of gene sequences and simple visual detection of products[J].Nature Protocols,2008,3(5):877-882
[12]Wang H,Huang Q,Huang JF,et al.Rapid detections of OprD2gene in Pseudomoas aeruginosa using loop-mediated isothermal amplification[J].Chinese Journal of Laboratory Medicine,2013,36(6):543-547(in Chinese)王欢,黄庆,黄君富,等.铜绿假单胞菌OprD2基因环介导等温扩增快速检测技术的研究[J].中华检验医学杂志,2013,36(6):543-547
[13]Xu YM,Zou DY,Tang F,et al.Design and analysis of LAMPprimers used in differentially detecting Yersinia enterocolitica and Brucella spp.[J].Chinese Journal of Laboratory Diagnosis,2013,17(1):19-22(in Chinese)徐云明,邹德颖,唐峰,等.区别检测小肠结肠炎耶尔森氏菌与布鲁氏菌LAMP引物的设计与分析[J].中国实验诊断学,2013,17(1):19-22
[14]Xiao LP.Application and study of LAMP technique in the detection of Salmonella in infectious diarrhea[J].Chinese Journal of Health Laboratory Technology,2015,25(24):4240-4242(in Chinese)肖丽萍.环介导技术在感染性腹泻沙门菌检测中的应用与研究[J].中国卫生检验杂志,2015,25(24):4240-4242
[15]Zhou Y,Wan Q,Cai ZH,et al.Loop-mediated isothermal amplification-based rapid detection for Shigella dysenteriae in food samples[J].Microbiology China,2017,44(9):2247-2254(in Chinese)周杨,万强,蔡芷荷,等.环介导等温扩增技术在食品中痢疾志贺氏菌快速检测[J].微生物学通报,2017,44(9):2247-2254
[16]Ding WC,Hu JR,Shi YH,et al.Establishment of loop-mediated isothermal amplification for detection of Vibrio alginolyticus[J].Journal of Molecular Cell Biology,2009,42(1):70-76(in Chinese)丁文超,胡健饶,史雨红,等.环介导恒温扩增技术快速检测溶藻弧菌[J].分子细胞生物学报,2009,42(1):70-76
[17]Dong DR,Zou DY,Liu H,et al.Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing,China[J].Frontiers in Microbiology,2015,6:1100
[18]Dong DR.The study of a new nucleic acid amplification technology termed Polymerase Spiral Reaction(PSR)and application in POCT detection[D].Beijing:Doctoral Dissertation of Academy of Military Medical Sciences,2016(in Chinese)董德荣.一种新型核酸恒温扩增方法的研究及其在现场检测中的应用[D].北京:中国人民解放军军事医学科学院博士学位论文,2016
[19]Liu W,Dong DR,Yang Z,et al.Polymerase Spiral Reaction(PSR):a novel isothermal nucleic acid amplification method[J].Scientific Reports,2015,5:12723
[20]Dong DR,Liu W,Li H,et al.Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing,China[J].Frontiers in Microbiology,2015,6:519
[21]Liu W,Xu YH,Dong DR,et al.Survey and rapid detection of Bordetella pertussis in clinical samples targeting the BP485 in China[J].Frontiers in Public Health,2015,3:39
[22]Kidd TJ,Grimwood K,Ramsay KA,et al.Comparison of three molecular techniques for typing Pseudomonas aeruginosa isolates in sputum samples from patients with cystic fibrosis[J].Journal of Clinical Microbiology,2011,49(1):263-268.
[23]Wang P,Zhang ZL,Qiao YS.Differentiation and identification of Pseudomonas aeruginosa and its interference strains by Fourier transform-infrared spectroscopy[J].Food Science and Technology,2017,42(4):302-305(in Chinese)王萍,张占林,乔勇升.傅立叶变换红外光谱技术对铜绿假单胞菌及其干扰菌的分类鉴定[J].食品科技,2017,42(4):302-305
[24]Goto M,Honda E,Ogura A,et al.Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue[J].Biotechniques,2009,46(3):167-172
[25]Iglewski BH,Kabat D.NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin[J].Proceedings of the National Academy of Sciences of the United States of America,1975,72(6):2284-2288
[26]Gray GL,Smith DH,Baldridge JS,et al.Cloning,nucleotide sequence,and expression in Escherichia coli of the exotoxin Astructural gene of Pseudomonas aeruginosa[J].Proceedings of the National Academy of Sciences of the United States of America,1984,81(9):2645-2649
[27]Zhang W,Li W,Zhang WW,et al.The methodological study on rapid identification of Pseudomonas aeruginosa based on PCRtechnique[J].Chinese Journal of Health Laboratory Technology,2005,15(9):1065-1067(in Chinese)张伟,李闻,张伟尉,等.基于PCR技术的绿脓杆菌快速检测方法研究[J].中国卫生检验杂志,2005,15(9):1065-1067
[28]Khan AA,Cerniglia CE.Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR[J].Applied and Environmental Microbiology,1994,60(10):3739-3745.
[29]Qin X,Emerson J,Stapp J,et al.Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis[J].Journal of Clinical Microbiology,2003,41(9):4312-4317
[30]Xu JR,Moore JE,Murphy PG,et al.Early detection of Pseudomonas aeruginosa-comparison of conventional versus molecular(PCR)detection directly from adult patients with cystic fibrosis(CF)[J].Annals of Clinical Microbiology and Antimicrobials,2004,3:21