黏菌素耐药基因mcr-1 TaqMan-MGB荧光定量PCR检测方法的建立
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摘要
【目的】建立针对黏菌素耐药基因mcr-1的Taq Man-MGB荧光定量PCR检测方法,为监控mcr-1基因携带细菌提供技术支持。【方法】针对mcr-1基因保守序列设计特异性引物和MGB探针,构建重组质粒作为阳性标准品,优化引物、探针浓度及Rox参比染料用量、退火温度等反应条件,建立针对mcr-1基因的Taq Man-MGB荧光定量PCR检测方法,并通过特异性、敏感性、重复性试验及临床应用验证其实用性。【结果】Taq Man-MGB荧光定量PCR最佳反应体系20.00μL:Premix Ex Taq~(TM)10.00μL,Rox参比染料(50×)0.25μL,引物MCR-1F/MCR-1R(10μmol/L)各0.50μL,MCR-1-P探针(10μmol/L)0.50μL,重组质粒pMCR-1(模板)2.00μL,灭菌双蒸水6.25μL。扩增程序:95℃预变性20 s;95℃10 s,60℃20 s,进行40个循环。该检测方法能特异性扩增出mcr-1基因,其检测敏感性为2.85拷贝/μL,是常规PCR的100倍,组内和组间重复性试验的变异系数为0.37%~1.18%,均小于1.20%。采用建立的Taq Man-MGB荧光定量PCR对82株广西鸡源致病性沙门氏菌分离株进行检测,结果未发现有携带mcr-1基因的菌株。【结论】针对mcr-1基因建立的Taq Man-MGB荧光定量PCR检测方法具有特异性强、敏感性高、重复性好的特点,可用于临床筛查mcr-1基因,为监控mcr-1基因携带细菌提供技术支持。
【Objective】TaqMan-MGB fluorescent quantitative PCR detection method targeting colistin resistant gene mcr-1 was established in order to supply an effective technique for surveillance of bacteria harboring gene mcr-1.【Method】Specific primers and MGB probe were designed basbased on mcr-1 gene conserved sequence,and recombinant plasmid was constructed as positive standard. After optimizing the reaction conditions including primer concentration,probe concentration,Rox reference dyeamount and annealing temperature,a Taq Man-MGB fluorescent quantitative PCR for detection of mcr-1 gene was established. The assay was further verified by specificity test,sensitivity test,repeatability test and clinical application.【Result】The optimal 20.00 μL reaction system of Taq Man-MGB fluorescent quantitative PCR included:Premix Ex TaqTM10.00 μL,Rox reference dye(50×)0.25 μL,MCR-1 F/MCR-1 R primers(10 μmol/L)0.50 μL each,MCR-1-P probe(10 μmol/L)0.50 μL,recombinant plasmid p MCR-1(template)2.00 μL and distilled water 6.25μL. The amplification procedure consisted of an initial predenaturation step for 20 s at 95 ℃,followed by 40 cycles of 10 s at 95 ℃ and 20 s at 60 ℃. The established assay could specifically amplify gene mcr-1. The detection limit was 2.85 copy/μL,which was as 100 times as that of conventional PCR. The coefficient of variation for intra-and inter-assay was from0.37 % to 1.18 %,which was less than 1.20%. Eighty-two pathogenic chicken Salmonella isolates from Guangxi were detected for mcr-1 gene by the assay and none was positive.【Conclusion】The established Taq Man-MGB fluorescent quantitative PCR detection method was highly specific,sensitive and reproducible,and it can be used for screening mcr-1 gene in clinical practice and provide technical support for surveillance of bacteria harboring mcr-1 gene.
【Objective】TaqMan-MGB fluorescent quantitative PCR detection method targeting colistin resistant gene mcr-1 was established in order to supply an effective technique for surveillance of bacteria harboring gene mcr-1.【Method】Specific primers and MGB probe were designed basbased on mcr-1 gene conserved sequence,and recombinant plasmid was constructed as positive standard. After optimizing the reaction conditions including primer concentration,probe concentration,Rox reference dyeamount and annealing temperature,a Taq Man-MGB fluorescent quantitative PCR for detection of mcr-1 gene was established. The assay was further verified by specificity test,sensitivity test,repeatability test and clinical application.【Result】The optimal 20.00 μL reaction system of Taq Man-MGB fluorescent quantitative PCR included:Premix Ex TaqTM10.00 μL,Rox reference dye(50×)0.25 μL,MCR-1 F/MCR-1 R primers(10 μmol/L)0.50 μL each,MCR-1-P probe(10 μmol/L)0.50 μL,recombinant plasmid p MCR-1(template)2.00 μL and distilled water 6.25μL. The amplification procedure consisted of an initial predenaturation step for 20 s at 95 ℃,followed by 40 cycles of 10 s at 95 ℃ and 20 s at 60 ℃. The established assay could specifically amplify gene mcr-1. The detection limit was 2.85 copy/μL,which was as 100 times as that of conventional PCR. The coefficient of variation for intra-and inter-assay was from0.37 % to 1.18 %,which was less than 1.20%. Eighty-two pathogenic chicken Salmonella isolates from Guangxi were detected for mcr-1 gene by the assay and none was positive.【Conclusion】The established Taq Man-MGB fluorescent quantitative PCR detection method was highly specific,sensitive and reproducible,and it can be used for screening mcr-1 gene in clinical practice and provide technical support for surveillance of bacteria harboring mcr-1 gene.
引文
曹阳,遇晓杰,韩营营,李杰,阚飙,闫梅英.2017.我国非伤寒沙门菌对多粘菌素的耐药现况及mcr-1基因携带概况[J].疾病监测,32(5):365-371.[Cao Y,Yu X J,Han YY,Li J,Kan B,Yan M Y.2017.Polymyxin resistance and mcr-1 prevalence in non-typhoid Salmonella isolates in China[J].Disease Surveillancs,32(5):365-371.]
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毛福超,郁川,韩璐,韩于佩,刘文慧,刘静.2016.豫西地区禽源大肠杆菌的分离鉴定与耐药性分析[J].河南农业科学,45(1):127-130.[Mao F C,Yu C,Han L,Han Y P,Liu W H,Liu J.2016.Isolation,identification and drug resistance analysis of Escherichia coli from chickens in western Henan Province[J].Journal of Henan Agricultural Sciences,45(1):127-130.]
田亚凯.2018.犬源大肠杆菌质粒介导的喹诺酮类药物耐药基因的检测及药物敏感性分析[J].江西农业学报,30(4):79-82.[Tian Y K.2018.Detection of plasmid-mediated quinolone-resistant genes in Escherichia coli isolated from dogs and analysis of their drug sensitivity[J].Acta Agriculturae Jiangxi,30(4):79-82.]
王秀娜,张会敏,孙坚,刘雅红,冯友军.2017.多黏菌素耐药MCR-1:公共卫生领域的新挑战[J].科学通报,62(10):1018-1029.[Wang X N,Zhang H M,Sun J,Liu Y H,Feng Y J.2017.The MCR-1 colistin resistance:A new challenge to global public health[J].Chinese Science Bulletin,62(10):1018-1029.]
王影,李艳然,韩镌竹,高铎,李欣南.2017.多粘菌素耐药性的研究进展[J].微生物学通报,44(1):200-206.[Wang Y,Li Y R,Han J Z,Gao D,Li X N.2017.Progress in drug resistance of polymyxin[J].Microbiology China,44(1):200-206.]
易灵娴,刘艺云,吴仁杰,梁梓森,刘健华.2017.质粒介导的黏菌素耐药基因mcr-1研究进展[J].遗传,39(2):110-125.[Yi L X,Liu Y Y,Wu R J,Liang Z S,Liu J H.2017.Research progress on the plasmid-mediated colistin resistance gene mcr-1[J].Hereditas,39(2):110-125.]
Campos J,Cristino L,Peixe L,Antunes P.2016.MCR-1 in multidrug-resistant and copper-tolerant clinically relevant Salmonella 1,4[,5],12:i:-and S.Rissen clones in Portugal,2011 to 2015[J].Euro Surveillance,21(26).doi:10.2807/1560-7917.
Cannatelli A,Giani T,Antonelli A,Principe L,Luzzaro F,Rossolini G M.2016.First detection of the mcr-1 colistin resistance gene in Escherichia coli in Italy[J].Antimicrobial Agents and Chemotherapy,60(5):3257-3258.
Carnevali C,Morganti M,Scaltriti E,Bolzoni L,Pongolini S,Casadei G.2016.Occurrence of mcr-1 in colistin-resistant Salmonella enteric isolates recovered from human and animals in Italy,2012 to 2015[J].Antimicrobial Agents and Chemotherapy,60(12):7532-7534.
Chabou S,Leangapichart T,Okdah L,Le Page S,Hadjadj L,Rolain J M.2016.Real-time quantitative PCR assay with Taqman?probe for rapid detection of MCR-1 plasmid-mediated colistin resistance[J].New Microbes and New Infections,13:71-74.
Chen K,Chan E W,Xie M,Ye L,Dong N,Chen S.2017.Widespread distribution of mcr-1-bearing bacteria in the ecosystem,2015 to 2016[J].Euro Surveillance,22(39).doi:10.2807/1560-7917.ES.2017.22.39.17-00206.
Cui M,Zhang J,Gu Z,Li R,Chan E W,Yan M,Wu C,Xu X,Chen S.2017.Prevalence and molecular characterization of mcr-1-positive Salmonella strains recovered from clinical specimens in China[J].Antimicrobial Agents and Chemotherapy,61(5).pii:e02471-16.
EI Garch F,de Jong A,Bertrand X,Hocguet D,Sauget M.2018.mcr-1-like detection in commensal Escherichia coli and Salmonella spp.from food-producing animals at slaughter in Europe[J].Veterinary Microbiology,213:42-46.
Gay N,Belmonte O,Collard J M,Halifa M,Issack M I,Mindiae S,Palmyre P,Ibrahim A A,Rasamoelina H,Flachet L,Filleul L,Cardinale E.2017.Review of antibiotic resistance in the Indian Ocean Commission:A human and animal health issue[J].Front iers in Public Health,5:162.
Huang X,Yu L,Chen X,Zhi C,Yao X,Liu Y,Wu S,Guo Z,Yi L,Zeng Z,Liu J H.2017.High prevalence of colistin resistance and mcr-1 gene in Escherichia coli isolated from food animals in China[J].Frontiers in Microbiology,8:562.
Imirzalioglu C,Falgenhauer L,Schmiedel J,Waezsada S E,Gwozdzinski K,Roschanski N,Roesler U,Kreienbrock L,Schiffmann A P,Irrgang A,K?sbohrer A,Bauerfeind R,Domann E,Chakraborty T.2017.Evaluation of a loopmediated isothermal amplification-based assay for the rapid detection of plasmid-encoded colistin resistance gene mcr-1 in Enterobacteriaceae isolates[J].Antimicrobial Agents and Chemotherapy,61(4).pii:e02326-16.
Irrgang A,Roschanski N,Tenhagen B A,Grobbel M,Skladnikjewicz-Zjemer T,Thomas K,Roesler U,K?sbohrer A.2016.Prevalence of mcr-1 in E.coli from livestock and food in Germany,2010-2015[J].PLo S One,11(7):e0159863.
Kempf I,Jouy E,Chauvin C.2016.Colistin use and colistin resistance in bacteria from animals[J].International Journal of Antimicrobial Agents,48(6):598-606.
Li J,Shi X,Yin W,Wang Y,Shen Z,Ding S,Wang S.2017.A multiplex SYBR Green real-time PCR assay for the detection of three colistin resistance genes from cultured bacteria,feces,and environment samples[J].Frontiers in Microbiology,8:2078.
Liu Y Y,Wang Y,Walsh T R,Yi L X,Zhang R,Spencer J,Doi Y,Tian G,Dong B,Huang X,Yu L F,Gu D,Ren H,Chen X,Lv L,He D,Zhou H,Liang Z,Liu J H,Shen J.2016.Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China:A microbiological and molecular biological study[J].The Lancet Infectious Diseases,16(2):161-168.
Mc Gann P,Snesrud E,Maybank R,Corey B,Ong A C,Clifford R,Hinkle M,Whitman T,Lesho E,Schaecher K E.2016.Escherichia coli harboring mcr-1 and bla CTX-Mon a novel Inc F plasmid:First report of mcr-1 in the United States[J].Antimicrobial Agents and Chemotherapy,60(8):5107.
von Wintersdorff C J,Wolffs P F,van Niekerk J M,Beuken E,van Alphen L B,Stobberingh E E,Oude Lashof A M,Hoebe C J,Saveelkoul P H,Penders J.2016.Detection of the plasmid-mediated colistin-resistance gene mcr-1 in faecal metagenomes of Dutch travelers[J].The Journal of Antimicrobial Chemotherapy,71(12):3416-3419.
Yi L,Wang J,Gao Y,Liu Y,Doi Y,Wu R,Zeng Z,Liang Z,Liu J H.2017.mcr-1-harboring Salmonella enterica serovar typhimurium sequence type 34 in pigs,China[J].Emerging Infectious Diseases,23(2):291-295.
Zou D,Huang S,Lei H,Yang Z,Su Y,He X,Zhao Q,Wang Y,Liu W,Huang L.2017.Sensitive and rapid detection of the plasmid-encoded colistin-resistance gene mcr-1 in Enterobacteriaceae isolates by loop-mediated isothermal amplification[J].Frontiers in Microbiology,8:2356.
Zurfuh K,Poirel L,Nordmann P,Nüesch-inderbinen M,H?chler H,Stephan R.2016.Occurrence of the plasmidborne mcr-1 colistin resistance gene in extended-spectrum-β-lactamase-producing Enterobacteriaceae in river water and imported vegetable samples in Switzerland[J].Antimicrobial Agents and Chemotherapy,60(4):2594-2595.
黎宗强,施开创,李凤梅,王海清,张珍,尹彦文.2016.鸡源致病性沙门氏菌耐药表型与耐药基因、血清群的相关性研究[J].动物医学进展,37(3):25-32.[Li Z Q,Shi K C,Li F M,Wang H Q,Zhang Z,Yin Y W.2016.Correlation analysis of antimicrobial resistance,resistance genes and serogroup of pathogenic Salmonella isolates from chickens[J].Progress in Veterinary Medicine,37(3):25-32.]
毛福超,郁川,韩璐,韩于佩,刘文慧,刘静.2016.豫西地区禽源大肠杆菌的分离鉴定与耐药性分析[J].河南农业科学,45(1):127-130.[Mao F C,Yu C,Han L,Han Y P,Liu W H,Liu J.2016.Isolation,identification and drug resistance analysis of Escherichia coli from chickens in western Henan Province[J].Journal of Henan Agricultural Sciences,45(1):127-130.]
田亚凯.2018.犬源大肠杆菌质粒介导的喹诺酮类药物耐药基因的检测及药物敏感性分析[J].江西农业学报,30(4):79-82.[Tian Y K.2018.Detection of plasmid-mediated quinolone-resistant genes in Escherichia coli isolated from dogs and analysis of their drug sensitivity[J].Acta Agriculturae Jiangxi,30(4):79-82.]
王秀娜,张会敏,孙坚,刘雅红,冯友军.2017.多黏菌素耐药MCR-1:公共卫生领域的新挑战[J].科学通报,62(10):1018-1029.[Wang X N,Zhang H M,Sun J,Liu Y H,Feng Y J.2017.The MCR-1 colistin resistance:A new challenge to global public health[J].Chinese Science Bulletin,62(10):1018-1029.]
王影,李艳然,韩镌竹,高铎,李欣南.2017.多粘菌素耐药性的研究进展[J].微生物学通报,44(1):200-206.[Wang Y,Li Y R,Han J Z,Gao D,Li X N.2017.Progress in drug resistance of polymyxin[J].Microbiology China,44(1):200-206.]
易灵娴,刘艺云,吴仁杰,梁梓森,刘健华.2017.质粒介导的黏菌素耐药基因mcr-1研究进展[J].遗传,39(2):110-125.[Yi L X,Liu Y Y,Wu R J,Liang Z S,Liu J H.2017.Research progress on the plasmid-mediated colistin resistance gene mcr-1[J].Hereditas,39(2):110-125.]
Campos J,Cristino L,Peixe L,Antunes P.2016.MCR-1 in multidrug-resistant and copper-tolerant clinically relevant Salmonella 1,4[,5],12:i:-and S.Rissen clones in Portugal,2011 to 2015[J].Euro Surveillance,21(26).doi:10.2807/1560-7917.
Cannatelli A,Giani T,Antonelli A,Principe L,Luzzaro F,Rossolini G M.2016.First detection of the mcr-1 colistin resistance gene in Escherichia coli in Italy[J].Antimicrobial Agents and Chemotherapy,60(5):3257-3258.
Carnevali C,Morganti M,Scaltriti E,Bolzoni L,Pongolini S,Casadei G.2016.Occurrence of mcr-1 in colistin-resistant Salmonella enteric isolates recovered from human and animals in Italy,2012 to 2015[J].Antimicrobial Agents and Chemotherapy,60(12):7532-7534.
Chabou S,Leangapichart T,Okdah L,Le Page S,Hadjadj L,Rolain J M.2016.Real-time quantitative PCR assay with Taqman?probe for rapid detection of MCR-1 plasmid-mediated colistin resistance[J].New Microbes and New Infections,13:71-74.
Chen K,Chan E W,Xie M,Ye L,Dong N,Chen S.2017.Widespread distribution of mcr-1-bearing bacteria in the ecosystem,2015 to 2016[J].Euro Surveillance,22(39).doi:10.2807/1560-7917.ES.2017.22.39.17-00206.
Cui M,Zhang J,Gu Z,Li R,Chan E W,Yan M,Wu C,Xu X,Chen S.2017.Prevalence and molecular characterization of mcr-1-positive Salmonella strains recovered from clinical specimens in China[J].Antimicrobial Agents and Chemotherapy,61(5).pii:e02471-16.
EI Garch F,de Jong A,Bertrand X,Hocguet D,Sauget M.2018.mcr-1-like detection in commensal Escherichia coli and Salmonella spp.from food-producing animals at slaughter in Europe[J].Veterinary Microbiology,213:42-46.
Gay N,Belmonte O,Collard J M,Halifa M,Issack M I,Mindiae S,Palmyre P,Ibrahim A A,Rasamoelina H,Flachet L,Filleul L,Cardinale E.2017.Review of antibiotic resistance in the Indian Ocean Commission:A human and animal health issue[J].Front iers in Public Health,5:162.
Huang X,Yu L,Chen X,Zhi C,Yao X,Liu Y,Wu S,Guo Z,Yi L,Zeng Z,Liu J H.2017.High prevalence of colistin resistance and mcr-1 gene in Escherichia coli isolated from food animals in China[J].Frontiers in Microbiology,8:562.
Imirzalioglu C,Falgenhauer L,Schmiedel J,Waezsada S E,Gwozdzinski K,Roschanski N,Roesler U,Kreienbrock L,Schiffmann A P,Irrgang A,K?sbohrer A,Bauerfeind R,Domann E,Chakraborty T.2017.Evaluation of a loopmediated isothermal amplification-based assay for the rapid detection of plasmid-encoded colistin resistance gene mcr-1 in Enterobacteriaceae isolates[J].Antimicrobial Agents and Chemotherapy,61(4).pii:e02326-16.
Irrgang A,Roschanski N,Tenhagen B A,Grobbel M,Skladnikjewicz-Zjemer T,Thomas K,Roesler U,K?sbohrer A.2016.Prevalence of mcr-1 in E.coli from livestock and food in Germany,2010-2015[J].PLo S One,11(7):e0159863.
Kempf I,Jouy E,Chauvin C.2016.Colistin use and colistin resistance in bacteria from animals[J].International Journal of Antimicrobial Agents,48(6):598-606.
Li J,Shi X,Yin W,Wang Y,Shen Z,Ding S,Wang S.2017.A multiplex SYBR Green real-time PCR assay for the detection of three colistin resistance genes from cultured bacteria,feces,and environment samples[J].Frontiers in Microbiology,8:2078.
Liu Y Y,Wang Y,Walsh T R,Yi L X,Zhang R,Spencer J,Doi Y,Tian G,Dong B,Huang X,Yu L F,Gu D,Ren H,Chen X,Lv L,He D,Zhou H,Liang Z,Liu J H,Shen J.2016.Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China:A microbiological and molecular biological study[J].The Lancet Infectious Diseases,16(2):161-168.
Mc Gann P,Snesrud E,Maybank R,Corey B,Ong A C,Clifford R,Hinkle M,Whitman T,Lesho E,Schaecher K E.2016.Escherichia coli harboring mcr-1 and bla CTX-Mon a novel Inc F plasmid:First report of mcr-1 in the United States[J].Antimicrobial Agents and Chemotherapy,60(8):5107.
von Wintersdorff C J,Wolffs P F,van Niekerk J M,Beuken E,van Alphen L B,Stobberingh E E,Oude Lashof A M,Hoebe C J,Saveelkoul P H,Penders J.2016.Detection of the plasmid-mediated colistin-resistance gene mcr-1 in faecal metagenomes of Dutch travelers[J].The Journal of Antimicrobial Chemotherapy,71(12):3416-3419.
Yi L,Wang J,Gao Y,Liu Y,Doi Y,Wu R,Zeng Z,Liang Z,Liu J H.2017.mcr-1-harboring Salmonella enterica serovar typhimurium sequence type 34 in pigs,China[J].Emerging Infectious Diseases,23(2):291-295.
Zou D,Huang S,Lei H,Yang Z,Su Y,He X,Zhao Q,Wang Y,Liu W,Huang L.2017.Sensitive and rapid detection of the plasmid-encoded colistin-resistance gene mcr-1 in Enterobacteriaceae isolates by loop-mediated isothermal amplification[J].Frontiers in Microbiology,8:2356.
Zurfuh K,Poirel L,Nordmann P,Nüesch-inderbinen M,H?chler H,Stephan R.2016.Occurrence of the plasmidborne mcr-1 colistin resistance gene in extended-spectrum-β-lactamase-producing Enterobacteriaceae in river water and imported vegetable samples in Switzerland[J].Antimicrobial Agents and Chemotherapy,60(4):2594-2595.