紫外诱变筛选磺胺敏感性菌株及在牛奶磺胺抗生素检测中的应用
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摘要
本研究利用紫外诱变方法筛选到两株具有磺胺敏感性的菌株H1、H2。DNA鉴定为芽孢杆菌属嗜热脂肪地芽孢杆菌种。将H1、H2作为指示菌,进行牛奶磺胺残留总量检测,发现这两株菌对不同种类的磺胺药物具有不同的灵敏度。当H1、H2处理后芽孢悬液菌落总数比为3:4时,检测试剂盒有较好的灵敏度,分别为磺胺:30μg/L、磺胺嘧啶:30μg/L、磺胺甲嘧啶:30μg/L、磺胺二甲嘧啶:30μg/L、氨苯磺胺:60μg/L、磺胺噻唑:45μg/L、磺胺氯哒嗪:80μg/L、磺胺甲噁唑:75μg/L、磺胺胍:75μg/L。在此比例下对两株菌同时传代,探究菌株传代对检测试剂盒灵敏度的影响,结果表明以第四代菌为指示菌时,该检测试剂盒丧失对氨苯磺胺和磺胺胍的敏感性;第六代丧失对磺胺氯哒嗪、磺胺甲噁唑的敏感性;对于磺胺、磺胺嘧啶、磺胺甲嘧啶、磺胺噻唑的敏感性可稳定到第七代。通过与国内外商业试剂盒对比,本试剂盒灵敏度高于国内试剂盒,检测时间优于国外试剂盒。
In this study, two strains with sulfonamide-sensitive strains H1 and H2 were screened by UV mutagenesis. After DNA identification, the two strains were of the genus Bacillus, a species of Geobacillus stearothermophilus. Using H1 and H2 as the indicator bacteria,the total amount of sulfonamide residues in milk was detected, and the obtained results showed that these two strains exhibited different sensitivities to different kinds of sulfa drugs. When the total colony ratio of spore suspension for H1 and H2 was 3:4, the test kit had good sensitivity: sulfonamide, 30 μg/L; sulfadiazine, 30 μg/L; sulfamethazine, 30 μg/L; sulfamethazine, 30 μg/L; sulfonamide, 60 μg/L; sulfathiazole,45 μg/L; sulfachloropyridazine, 80 μg/L; sulfamethoxazole, 75 μg/L; sulfazone, 75 μg/L. Under these conditions, the two strains were simultaneously passaged, and the effect of strain passage on the sensitivity of the test kit was investigated. The results showed that the test kit lost sensitivities to sulfonamide and sulfaguanidine when the fourth generation bacteria was used as the indicator. The sensitivity to sulfachloropurazine or sulfamethoxazole also lost when the sixth generation was used. Whereas, the sensitivity to sulfonamides, sulfadiazine,sulfamethazine or sulfathiazole remained constant till the seventh generation. Compared with domestic and overseas commercial kits, this kit had a higher sensitivity than the domestic kits, and showed advantage in detection time over the overeas kits.
In this study, two strains with sulfonamide-sensitive strains H1 and H2 were screened by UV mutagenesis. After DNA identification, the two strains were of the genus Bacillus, a species of Geobacillus stearothermophilus. Using H1 and H2 as the indicator bacteria,the total amount of sulfonamide residues in milk was detected, and the obtained results showed that these two strains exhibited different sensitivities to different kinds of sulfa drugs. When the total colony ratio of spore suspension for H1 and H2 was 3:4, the test kit had good sensitivity: sulfonamide, 30 μg/L; sulfadiazine, 30 μg/L; sulfamethazine, 30 μg/L; sulfamethazine, 30 μg/L; sulfonamide, 60 μg/L; sulfathiazole,45 μg/L; sulfachloropyridazine, 80 μg/L; sulfamethoxazole, 75 μg/L; sulfazone, 75 μg/L. Under these conditions, the two strains were simultaneously passaged, and the effect of strain passage on the sensitivity of the test kit was investigated. The results showed that the test kit lost sensitivities to sulfonamide and sulfaguanidine when the fourth generation bacteria was used as the indicator. The sensitivity to sulfachloropurazine or sulfamethoxazole also lost when the sixth generation was used. Whereas, the sensitivity to sulfonamides, sulfadiazine,sulfamethazine or sulfathiazole remained constant till the seventh generation. Compared with domestic and overseas commercial kits, this kit had a higher sensitivity than the domestic kits, and showed advantage in detection time over the overeas kits.
引文
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[2]汪善良,程茹,钟新敏,等.化学发光微粒子免疫法检测虾肉中磺胺类药物的残留[J].安徽农业科学,2018,46(8):170-173WANG Shan-liang,CHENG Ru,ZHONG Xin-min,et al.Detection of sulfonamides residues in shrimp by chemiluminescence microparticleimmuno assay(CMIA)[J].Anhui Agriculture Science,2018,46(8):170-173
[3]张元,李伟青,周伟娥,等.食品中磺胺类药物前处理及检测方法研究进展[J].食品科学,2015,36(23):340-346ZHANG Yuan,LI Wei-qing,ZHOU Wei-e,et al.Progress in sample pretreatment and analytical techniques for the determination of sulfonamide residues in foods[J].Food Science,2015,36(23):340-346
[4]Li F L,Guo Y M,Wang X Y,et al.Multiplexed aptasensor based on metal ions labels for simultaneous detection of multiple antibiotic residues in milk[J].Biosensors and Bioelectronics,2018,115:7-13
[5]Wang H X,Ren L H,Yu X,et al.Antibiotic residues in meat,milk and aquatic products in Shanghai and human exposure assessment[J].Food Control,2017,80:217-225
[6]黄晓蓉,于慈,郑晶,等.牛奶中的β-内酰胺类抗生素残留的快速检测方法[J].中国乳品工业,2004,32(2):44-47HUANG Xiao-rong,YU Ci,ZHENG Jing,et al.The study of rapid impedence method for detecting beta-lactam antibiotic residues in milk[J].China Dairy Industry,2004,32(2):44-47
[7]Orlando N,María P M,Rafael A.Microbiological system in microtitre plates for detection and classification of antibiotic residues in milk[J].International Dairy Journal,2013,32(10):150-155
[8]Melisa T,Orlando N,Maria P M,et al.Microbiological assay with Bacillus licheniformis for the easy detection of quinolones in milk[J].International Dairy Journal,2017,64(8):9-13
[9]农业部781号公告-12-2006,牛奶中磺胺类药物残留量的测定液相色谱-串联质谱法[S]Announcement No.781 of the ministry of agriculture-12-2006,determination of sulfonamide residues in milk by liquid chromatography-tandem mass spectrometry[S]
[10]GB/T 22966-2008,牛奶和奶粉中16种磺胺类药物残留量的测定液相色谱-串联质谱法[S]GB/T 22966-2008,Determination of 16 sulfonamide residues in milk and milk powder by liquid chromatography-tandem mass spectrometry[S]
[11]J M K J K Premarathne,D A Satharasinghe,A R C Gunasena.Establishment of a method to detect sulfonamide residues in chicken meat and eggs by high-performance liquid chromatography[J].Food Control,2017,72:276-282
[12]Jiang W X,Natalia V.Beloglazova,Wang Z H,et al.Development of a multiplex flow-through immunoaffinity chromatography test for the on-site screening of 14sulfonamide and 13 quinolone residues in milk[J].Biosensors and Bioelectronics,2015,66:124-128
[13]Muhammad S,Na N,Muhammad S,et al.Rapid trace level determination of sulfonamide residues in honey with online extraction using short C-18 column by high-performance liquid chromatography with fluorescence detection[J].Journal of Chromatography A,2013,1314:173-179
[14]顾欣.牛奶中青霉素残留的微生物学检测方法研究进展[J].中国兽药杂志,2017,1:40-45GU Xin.Research progress of microbiological methods for the detection of penicillin residues in milk[J].Chinese Journal of Veterinary Drug,2017,1:40-45
[15]朱建恩.牛奶中抗生素残留的微生物检测方法应用分析[J].食品安全导刊,2018,21:66-67ZHU Jian-en.Application analysis of microbial detection methods for antibiotic residues in milk[J].China Food Safety Magazine,2018,21:66-67
[16]刘雪杰.牛奶中抗生素残留检测指示菌的筛选鉴定及检测检测试剂盒研发[D].福州:福州大学,2013LIU Xue-jie.Screening and identification of indicator bacteria for antibiotic detection in milk and the production of test kit[D].Fuzhou:Fuzhou University,2013
[17]尹明浩,徐慧,程殿林,等.紫外诱变选育高产3-羟基丁酮枯草芽孢杆菌[J].中国酿造,2010,4:76-79YIN Ming-hao,XU Hui,CHENG Dian-lin,et al.Selection of high acetoin-producing strain of Bacillus subtilis by UVmutation[J].China Brewing,2010,4:76-79
[18]魏云阶.紫外线诱变苏云金芽孢杆菌最佳条件探索[J].生物技术世界,2016,4:10-12WEI Yun-jie.Exploration on the optimum conditions of uv mutagenesis of Bacillus thuringiensis[J].Biotech World,2016,4:10-12
[19]艾冰花,李秉钧,冯俊荣.一株产蛋白酶芽孢杆菌紫外诱变育种的初步研究[J].渔业现代化,2015,42(4):39-43AI Bing-hua,LI Bing-jun,FENG Jun-rong.Preliminary study on the ultraviolet mutagenesis of a protease producing strain Bacillus amyloliquef[J].Fishery Modernization,2015,42(4):39-43
[20]GB 4789.2-2010,食品微生物学检验菌落总数测定[S]GB 4789.2-2010,Food microbiology test Total number of colonies[S]
[21]R E布坎南,N E吉本斯.《伯杰细菌鉴定手册》(第八版)[M].北京:科学出版社,1995Buchanan R E,Gibbons N E.Bergey’s Manual of Systemaic Bacteriology[M].Beijing:Science Press,1995
[22]Wilson K,Blitchington R.Human colonic biota studied by ribsomal DNA sequence analysis[J].Appl Environ Microbiol,1996,62(4):2273-2278
[23]Enren Z,Wenjing Z,Yue L,et al.Acclimatization of microbial consortia to alkaline conditions and enhanced electricity generation[J].Bioresource Technology,2016,211(7):736-742
[24]关颖,吴泳标,林奕云,等.一种基于多种微生物的急性毒性测定方法[J].生态科学,2016,35(6):6-13GUAN Ying,WU Yong-biao,LIN Yi-yun,et al.A method for testing acute toxicity based on 11 microbial species[J].Ecological Science,2016,35(6):6-13
[25]Yuancai Lv,Yuancai Chen,Shiying Sun,et al.Interaction among multiple microorganisms and effects of nitrogen and carbon supplementations on lignin degradation[J].Bioresource Technology,2013,155:144-151
[26]朱强.牛奶、尿液中主要抗菌药残留微生物学筛选方法及其检测试剂盒研究[D].武汉:华中农业大学,2008ZHU Qiang.Development of microbiological methods and their kits for screening the antimicrobial residues in milk and urine[D].Wuhan:Huazhong Agriculture University,2008
[27]范维,高晓月,李贺楠,等.高通量微生物显色法快速检测动物源性食品中抗生素残留[J].食品科学,2017,38(16):239-244FAN Wei,GAO Xiao-yue,LI He-nan,et al.Rapid and high-throughput detection of antibiotic residues in animal-derived food samples by microbial chromotest[J].Food Science,2017,38(16):239-244
[2]汪善良,程茹,钟新敏,等.化学发光微粒子免疫法检测虾肉中磺胺类药物的残留[J].安徽农业科学,2018,46(8):170-173WANG Shan-liang,CHENG Ru,ZHONG Xin-min,et al.Detection of sulfonamides residues in shrimp by chemiluminescence microparticleimmuno assay(CMIA)[J].Anhui Agriculture Science,2018,46(8):170-173
[3]张元,李伟青,周伟娥,等.食品中磺胺类药物前处理及检测方法研究进展[J].食品科学,2015,36(23):340-346ZHANG Yuan,LI Wei-qing,ZHOU Wei-e,et al.Progress in sample pretreatment and analytical techniques for the determination of sulfonamide residues in foods[J].Food Science,2015,36(23):340-346
[4]Li F L,Guo Y M,Wang X Y,et al.Multiplexed aptasensor based on metal ions labels for simultaneous detection of multiple antibiotic residues in milk[J].Biosensors and Bioelectronics,2018,115:7-13
[5]Wang H X,Ren L H,Yu X,et al.Antibiotic residues in meat,milk and aquatic products in Shanghai and human exposure assessment[J].Food Control,2017,80:217-225
[6]黄晓蓉,于慈,郑晶,等.牛奶中的β-内酰胺类抗生素残留的快速检测方法[J].中国乳品工业,2004,32(2):44-47HUANG Xiao-rong,YU Ci,ZHENG Jing,et al.The study of rapid impedence method for detecting beta-lactam antibiotic residues in milk[J].China Dairy Industry,2004,32(2):44-47
[7]Orlando N,María P M,Rafael A.Microbiological system in microtitre plates for detection and classification of antibiotic residues in milk[J].International Dairy Journal,2013,32(10):150-155
[8]Melisa T,Orlando N,Maria P M,et al.Microbiological assay with Bacillus licheniformis for the easy detection of quinolones in milk[J].International Dairy Journal,2017,64(8):9-13
[9]农业部781号公告-12-2006,牛奶中磺胺类药物残留量的测定液相色谱-串联质谱法[S]Announcement No.781 of the ministry of agriculture-12-2006,determination of sulfonamide residues in milk by liquid chromatography-tandem mass spectrometry[S]
[10]GB/T 22966-2008,牛奶和奶粉中16种磺胺类药物残留量的测定液相色谱-串联质谱法[S]GB/T 22966-2008,Determination of 16 sulfonamide residues in milk and milk powder by liquid chromatography-tandem mass spectrometry[S]
[11]J M K J K Premarathne,D A Satharasinghe,A R C Gunasena.Establishment of a method to detect sulfonamide residues in chicken meat and eggs by high-performance liquid chromatography[J].Food Control,2017,72:276-282
[12]Jiang W X,Natalia V.Beloglazova,Wang Z H,et al.Development of a multiplex flow-through immunoaffinity chromatography test for the on-site screening of 14sulfonamide and 13 quinolone residues in milk[J].Biosensors and Bioelectronics,2015,66:124-128
[13]Muhammad S,Na N,Muhammad S,et al.Rapid trace level determination of sulfonamide residues in honey with online extraction using short C-18 column by high-performance liquid chromatography with fluorescence detection[J].Journal of Chromatography A,2013,1314:173-179
[14]顾欣.牛奶中青霉素残留的微生物学检测方法研究进展[J].中国兽药杂志,2017,1:40-45GU Xin.Research progress of microbiological methods for the detection of penicillin residues in milk[J].Chinese Journal of Veterinary Drug,2017,1:40-45
[15]朱建恩.牛奶中抗生素残留的微生物检测方法应用分析[J].食品安全导刊,2018,21:66-67ZHU Jian-en.Application analysis of microbial detection methods for antibiotic residues in milk[J].China Food Safety Magazine,2018,21:66-67
[16]刘雪杰.牛奶中抗生素残留检测指示菌的筛选鉴定及检测检测试剂盒研发[D].福州:福州大学,2013LIU Xue-jie.Screening and identification of indicator bacteria for antibiotic detection in milk and the production of test kit[D].Fuzhou:Fuzhou University,2013
[17]尹明浩,徐慧,程殿林,等.紫外诱变选育高产3-羟基丁酮枯草芽孢杆菌[J].中国酿造,2010,4:76-79YIN Ming-hao,XU Hui,CHENG Dian-lin,et al.Selection of high acetoin-producing strain of Bacillus subtilis by UVmutation[J].China Brewing,2010,4:76-79
[18]魏云阶.紫外线诱变苏云金芽孢杆菌最佳条件探索[J].生物技术世界,2016,4:10-12WEI Yun-jie.Exploration on the optimum conditions of uv mutagenesis of Bacillus thuringiensis[J].Biotech World,2016,4:10-12
[19]艾冰花,李秉钧,冯俊荣.一株产蛋白酶芽孢杆菌紫外诱变育种的初步研究[J].渔业现代化,2015,42(4):39-43AI Bing-hua,LI Bing-jun,FENG Jun-rong.Preliminary study on the ultraviolet mutagenesis of a protease producing strain Bacillus amyloliquef[J].Fishery Modernization,2015,42(4):39-43
[20]GB 4789.2-2010,食品微生物学检验菌落总数测定[S]GB 4789.2-2010,Food microbiology test Total number of colonies[S]
[21]R E布坎南,N E吉本斯.《伯杰细菌鉴定手册》(第八版)[M].北京:科学出版社,1995Buchanan R E,Gibbons N E.Bergey’s Manual of Systemaic Bacteriology[M].Beijing:Science Press,1995
[22]Wilson K,Blitchington R.Human colonic biota studied by ribsomal DNA sequence analysis[J].Appl Environ Microbiol,1996,62(4):2273-2278
[23]Enren Z,Wenjing Z,Yue L,et al.Acclimatization of microbial consortia to alkaline conditions and enhanced electricity generation[J].Bioresource Technology,2016,211(7):736-742
[24]关颖,吴泳标,林奕云,等.一种基于多种微生物的急性毒性测定方法[J].生态科学,2016,35(6):6-13GUAN Ying,WU Yong-biao,LIN Yi-yun,et al.A method for testing acute toxicity based on 11 microbial species[J].Ecological Science,2016,35(6):6-13
[25]Yuancai Lv,Yuancai Chen,Shiying Sun,et al.Interaction among multiple microorganisms and effects of nitrogen and carbon supplementations on lignin degradation[J].Bioresource Technology,2013,155:144-151
[26]朱强.牛奶、尿液中主要抗菌药残留微生物学筛选方法及其检测试剂盒研究[D].武汉:华中农业大学,2008ZHU Qiang.Development of microbiological methods and their kits for screening the antimicrobial residues in milk and urine[D].Wuhan:Huazhong Agriculture University,2008
[27]范维,高晓月,李贺楠,等.高通量微生物显色法快速检测动物源性食品中抗生素残留[J].食品科学,2017,38(16):239-244FAN Wei,GAO Xiao-yue,LI He-nan,et al.Rapid and high-throughput detection of antibiotic residues in animal-derived food samples by microbial chromotest[J].Food Science,2017,38(16):239-244