应用多重PCR快速鉴定自然发酵肉制品中6种葡萄球菌
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摘要
肉葡萄球菌(Staphylococcuscarnosus)、木糖葡萄球菌(S.xylosus)、腐生葡萄球菌(S. saprophyticus)、表皮葡萄球菌(S. epidermidis)、马胃葡萄球菌(S. equorum)和松鼠葡萄球菌(S. sciuri)是发酵肉制品中常见的葡萄球菌,为准确、快速地检测和鉴定这些菌种,建立6种葡萄球菌的多重聚合酶链式反应(polymerase chain reaction,PCR)方法并进行验证。以上述菌种的gap、rpoB、SodA和SNc为靶基因,设计和筛选菌种特异性引物6对,优化PCR体系,建立6种葡萄球菌的多重PCR鉴定方法。实验结果显示多重PCR的基因组DNA检测灵敏度可达到4 pg,活菌检测灵敏度可达到2×10~2 CFU;采用建立的多重PCR方法对传统肉制品中分离的8株葡萄球菌进行鉴定,结果与16S rDNA序列分析、生理生化鉴定结果一致。建立的多重PCR方法具有较高的种间特异性,可快速、准确地用于发酵肉制品中肉葡萄球菌、木糖葡萄球菌、表皮葡萄球菌、马胃葡萄球菌、松鼠葡萄球菌和腐生葡萄球菌的检测和鉴定,具有较好的应用前景。
A multiplex polymerase chain reaction(mPCR) assay was developed and validated for simultaneous detection of Staphylococcus carnosus, S. xylosus, S. saprophyticus, S. epidermidis, S. equorum and S. sciuri, which are commonly found in fermented meat products. In this study, six pairs of species-specific primers were designed targeting the gap, rpoB, SodA and SNc genes, and the PCR reaction system was optimized. The results showed that The sensitivity of the mPCR system was 4 pg for bacterial genomic DNA and 2 × 10~2 CFU for living cultures. Meanwhile, eight Staphylococcus strainsfrom traditional fermented meat products were consistently identified by this mPCR assay and 16 S rDNA sequence and biochemical analysis. This mPCR method was species-specific and effective, and could be used in the rapid and accurate identification of S. carnosus, S. xylosus, S. epidermidis, S. equorum, S. sciuri and S. saprophyticus. Thus, it has great application prospects.
A multiplex polymerase chain reaction(mPCR) assay was developed and validated for simultaneous detection of Staphylococcus carnosus, S. xylosus, S. saprophyticus, S. epidermidis, S. equorum and S. sciuri, which are commonly found in fermented meat products. In this study, six pairs of species-specific primers were designed targeting the gap, rpoB, SodA and SNc genes, and the PCR reaction system was optimized. The results showed that The sensitivity of the mPCR system was 4 pg for bacterial genomic DNA and 2 × 10~2 CFU for living cultures. Meanwhile, eight Staphylococcus strainsfrom traditional fermented meat products were consistently identified by this mPCR assay and 16 S rDNA sequence and biochemical analysis. This mPCR method was species-specific and effective, and could be used in the rapid and accurate identification of S. carnosus, S. xylosus, S. epidermidis, S. equorum, S. sciuri and S. saprophyticus. Thus, it has great application prospects.
引文
[1]龙强,聂乾忠,刘成国.发酵肉制品功能性发酵剂研究现状[J].食品科学,2016,37(17):263-269.DOI:10.7506/spkx1002-6630-201617044.
[2]夏秀芳,张金铎,孔保华.木糖葡萄球菌添加量对发酵牛肉串品质特性的影响[J].食品科学,2013,34(7):47-50.DOI:10.7506/spkx1002-6630-201307011.
[3]张晓琼,桂萌,靳晓娇,等.不同发酵肉中优良葡萄球菌的筛选鉴定[J].肉类研究,2011,25(8):17-21.DOI:10.3969/j.issn.1001-8123.2011.08.004.
[4]COTON E,DESMONTS M-H,LEROY S,et al.Biodiversity of coagulase-negative staphylococci in French cheeses,dry fermented sausages,processing environments and clinical samples[J].International Journal of Food Microbiology,2010,137(1/2/3):221-229.DOI:10.1016/j.ijfoodmicro.2009.11.023.
[5]孙二娜,陈历水,刘松玲,等.传统发酵肉制品中葡萄球菌的分离筛选及鉴定[C]//第9届乳酸菌与健康国际研讨会.天津:中国食品科学技术学会,2014:80.
[6]SEMEDO-LEMSADDEK T,CARVALHO L,TEMPERA C,et al.Characterization and technological features of autochthonous coagulase-negative staphylococci as potential starters for portuguese dry fermented sausages[J].Journal of Food Science,2016,81(5):M1197-M1202.DOI:10.1111/1750-3841.13311.
[7]马宇霞,卢士玲,李开雄,等.熏马肠中生物胺氧化酶菌株的筛选与鉴定[J].现代食品科技,2014,30(5):49-55.
[8]LANDETA G,CURIEL J A,CARRASCOSA A V,et al.Characterization of coagulase-negative staphylococci isolated from Spanish dry cured meat products[J].Meat Science,2013,93(3):387-396.DOI:10.1016/j.meatsci.2012.09.019.
[9]SEITTER M.Safety assessment of coagulase-negative staphylococci used in food production[D].Stuttgart:Universit?t Hohenheim,2015.
[10]MAINAR M S,LEROY F.Process-driven bacterial community dynamics are key to cured meat colour formation by coagulasenegative staphylococci via nitrate reductase ornitric oxide synthase activities[J].International Journal of Food Microbiology,2015,212(6):60-66.DOI:10.1016/j.ijfoodmicro.2015.03.009.
[11]SANCHEZ M M,STAVROPOULOU D A,LEROY F.Exploring the metabolic heterogeneity of coagulase-negative staphylococci to improve the quality and safety of fermented meats:a review[J].International Journal of Food Microbiology,2017,247(1):24-37.DOI:10.1016/j.ijfoodmicro.2016.05.021.
[12]MARTY E,BODENMANN C,BUCHS J,et al.Prevalence of antibiotic resistance in coagulase-negative staphylococci from spontaneously fermented meat products and safety assessment for new starters[J].International Journal of Food Microbiology,2012,159(2):74-83.DOI:10.1016/j.ijfoodmicro.2012.07.025.
[13]RANTSIOU K,COCOLIN L.New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods:a review[J].International Journal of Food Microbiology,2006,108(2):255-267.DOI:10.1016/j.ijfoodmicro.2005.11.013.
[14]COCOLIN L,MANZANO M,CANTONI C,et al.Denaturing gradient gel electrophoresis analysis of the 16S RNA gene v1 region to monitor dynamic changes in the bacterial population during fermentation of italian sausages[J].Applied and Environmental Microbiology,2001,67(11):5113-5121.DOI:10.1128/AEM.67.11.5113-5121.2001.
[15]FONTANA C,VIGNOLO G,COCCONCELLI P.PCR-DGGEanalysis for the identification of microbial populations from Argentinean dry fermented sausages[J].Journal of Microbiological Methods,2005,63(3):254-263.DOI:10.1016/j.mimet.2005.03.010.
[16]MARTY E,BUCHS J,EUGSTER-MEIER E,et al.Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP[J].Food Microbiology,2012,29(2):157-166.DOI:10.1016/j.fm.2011.09.011.
[17]SPIEGELMAN D,WHISSELL G,GREER C W.A survey of the methods for the characterization of microbial consortia and communities[J].Canadian Journal of Microbiology,2005,51(5):355-386.DOI:10.1139/w05-003.
[18]HAN H W,CHANG H C,HUNAG A H,et al.Optimization of the score cutoff value for routine identifcation of Staphylococcus species by matrix-assisted laser desorption ionization-timeoffight mass spectrometry[J].Diagnostic Microbiology&Infectious Disease,2015,83(4):349-354.DOI:10.1016/j.diagmicrobio.2015.08.003.
[19]TOMAZI T,GONCALVES J L,BARREIRO J R.Identifcation of coagulase-negative staphylococci from bovine intramammary infection by matrix-assisted laser desorption ionization-time of fight mass spectrometry[J].Journal of Clinical Microbiology,2014,52(5):1658-1663.DOI:10.1128/JCM.03032-13.
[20]王稳航,徐倩倩,滕安国,等.发酵肉制品菌群的非培养鉴定技术研究进展[J].肉类研究,2014,28(9):17-21.
[21]KIM J,HONG J,LIM J A,et al.Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci[J].Archives of Microbiology,2017,200(1):73-83.DOI:10.1007/s00203-017-1415-9.
[22]LANDETA G,REVERON I,CARRASCOSA A V,et al.Use of recA gene sequence analysis for the identification of Staphylococcus equorum strains predominant on dry-cured hams[J].Food Microbiology,2011,28(6):1205-1210.DOI:10.1016/j.fm.2011.04.006.
[23]MOROT-BIZOT S,TALON R,LEROY-SETRINB S.Development of specific PCR primers for a rapid and accurate identification of Staphylococcus xylosus,a species used in food fermentation[J].Journal of Microbiological Methods,2003,55(1):279-286.DOI:10.1016/S0167-7012(03)00159-3.
[24]MOROT-BIZOT C S,TALON R,LEROY S.Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food[J].Journal of Applied Microbiology,2004,97(5):1087-1094.DOI:10.1111/j.1365-2672.2004.02399.x.
[25]AYMERICH T,MARTIN B,GARRIGA M,et al.Microbial quality and direct PCR identification of lactic acid bacteria and nonpathogenic staphylococci from artisanal low-acid sausages[J].Applied and Environmental Microbiology,2003,69(8):4583-4594.DOI:10.1128/AEM.69.8.4583-4594.2003.
[26]GANDRA E A,FERNANDEZ M A,SILVA J A,et al.Detection by multiplex PCR of Staphylococcus aureus,S.intermedius and S.hyicus in artificially contaminated milk[J].Ciencia Rural,2016,46(1):1418-1423.DOI:10.1590/0103-8478cr20151391.
[27]BLAIOTTA G,ERCOLINI D,MAURIELLO G,et al.Rapid and reliable identification of Staphylococcus equorum by aspecies specific PCR assay targeting the sod Agene[J].Systematic&Applied Microbiology,2004,27(6):696-702.DOI:10.1078/0723202042369901.
[28]HIROTAKI S,SASAKI T,KUWAHARA-ARAI K,et al.Rapid and accurate identification of human-associated Staphylococci by use of multiplex PCR[J].Journal of Clinical Microbiology,2011,49(10):3627-3631.DOI:10.1128/JCM.00488-11.
[29]SHOME B R,MITRAL S D,BHUVANAL M,et al.Multiplex PCRassay for species identification of bovine mastitis pathogens[J].Journal of Applied Microbiology,2011,111(6):1349-1356.DOI:10.1111/j.1365-2672.2011.05169.x.
[30]孙鸿燕.四种食源性致病菌多重PCR检测方法的建立[D].长春:吉林大学,2011:37.
[31]周明东,张臣伟,张海滨,等.食源性致病菌快速检测方法的建立与应用[J].中国食品学报,2012,12(10):169-176.
[32]姜彦君,都启晶,范颖华,等.食源性致病菌快速检测试剂盒的研制与应用[J].中国食品学报,2014,14(1):224-231.
[33]刘红玉,李岩,崔洪斌.肉中4种致病菌的PCR快速检测方法的建立[J].食品科学,2011,32(6):213-216.
[34]钱志伟,孙新城.食品中3种致病菌多重PCR检测体系的建立及初步应用[J].食品科学,2011,32(16):236-239.
[35]胡冰雪,舒沿沿,潘道东,等.荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立[J].食品科学,2016,37(20):209-214.DOI:10.7506/spkx1002-6630-201620036.
[36]张莎莎,郭新梅,赵美爱,等.微芯片电泳与聚丙烯酰胺凝胶电泳的比较分析[J].中国农业科技导报,2013,15(2):173-178.
[2]夏秀芳,张金铎,孔保华.木糖葡萄球菌添加量对发酵牛肉串品质特性的影响[J].食品科学,2013,34(7):47-50.DOI:10.7506/spkx1002-6630-201307011.
[3]张晓琼,桂萌,靳晓娇,等.不同发酵肉中优良葡萄球菌的筛选鉴定[J].肉类研究,2011,25(8):17-21.DOI:10.3969/j.issn.1001-8123.2011.08.004.
[4]COTON E,DESMONTS M-H,LEROY S,et al.Biodiversity of coagulase-negative staphylococci in French cheeses,dry fermented sausages,processing environments and clinical samples[J].International Journal of Food Microbiology,2010,137(1/2/3):221-229.DOI:10.1016/j.ijfoodmicro.2009.11.023.
[5]孙二娜,陈历水,刘松玲,等.传统发酵肉制品中葡萄球菌的分离筛选及鉴定[C]//第9届乳酸菌与健康国际研讨会.天津:中国食品科学技术学会,2014:80.
[6]SEMEDO-LEMSADDEK T,CARVALHO L,TEMPERA C,et al.Characterization and technological features of autochthonous coagulase-negative staphylococci as potential starters for portuguese dry fermented sausages[J].Journal of Food Science,2016,81(5):M1197-M1202.DOI:10.1111/1750-3841.13311.
[7]马宇霞,卢士玲,李开雄,等.熏马肠中生物胺氧化酶菌株的筛选与鉴定[J].现代食品科技,2014,30(5):49-55.
[8]LANDETA G,CURIEL J A,CARRASCOSA A V,et al.Characterization of coagulase-negative staphylococci isolated from Spanish dry cured meat products[J].Meat Science,2013,93(3):387-396.DOI:10.1016/j.meatsci.2012.09.019.
[9]SEITTER M.Safety assessment of coagulase-negative staphylococci used in food production[D].Stuttgart:Universit?t Hohenheim,2015.
[10]MAINAR M S,LEROY F.Process-driven bacterial community dynamics are key to cured meat colour formation by coagulasenegative staphylococci via nitrate reductase ornitric oxide synthase activities[J].International Journal of Food Microbiology,2015,212(6):60-66.DOI:10.1016/j.ijfoodmicro.2015.03.009.
[11]SANCHEZ M M,STAVROPOULOU D A,LEROY F.Exploring the metabolic heterogeneity of coagulase-negative staphylococci to improve the quality and safety of fermented meats:a review[J].International Journal of Food Microbiology,2017,247(1):24-37.DOI:10.1016/j.ijfoodmicro.2016.05.021.
[12]MARTY E,BODENMANN C,BUCHS J,et al.Prevalence of antibiotic resistance in coagulase-negative staphylococci from spontaneously fermented meat products and safety assessment for new starters[J].International Journal of Food Microbiology,2012,159(2):74-83.DOI:10.1016/j.ijfoodmicro.2012.07.025.
[13]RANTSIOU K,COCOLIN L.New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods:a review[J].International Journal of Food Microbiology,2006,108(2):255-267.DOI:10.1016/j.ijfoodmicro.2005.11.013.
[14]COCOLIN L,MANZANO M,CANTONI C,et al.Denaturing gradient gel electrophoresis analysis of the 16S RNA gene v1 region to monitor dynamic changes in the bacterial population during fermentation of italian sausages[J].Applied and Environmental Microbiology,2001,67(11):5113-5121.DOI:10.1128/AEM.67.11.5113-5121.2001.
[15]FONTANA C,VIGNOLO G,COCCONCELLI P.PCR-DGGEanalysis for the identification of microbial populations from Argentinean dry fermented sausages[J].Journal of Microbiological Methods,2005,63(3):254-263.DOI:10.1016/j.mimet.2005.03.010.
[16]MARTY E,BUCHS J,EUGSTER-MEIER E,et al.Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP[J].Food Microbiology,2012,29(2):157-166.DOI:10.1016/j.fm.2011.09.011.
[17]SPIEGELMAN D,WHISSELL G,GREER C W.A survey of the methods for the characterization of microbial consortia and communities[J].Canadian Journal of Microbiology,2005,51(5):355-386.DOI:10.1139/w05-003.
[18]HAN H W,CHANG H C,HUNAG A H,et al.Optimization of the score cutoff value for routine identifcation of Staphylococcus species by matrix-assisted laser desorption ionization-timeoffight mass spectrometry[J].Diagnostic Microbiology&Infectious Disease,2015,83(4):349-354.DOI:10.1016/j.diagmicrobio.2015.08.003.
[19]TOMAZI T,GONCALVES J L,BARREIRO J R.Identifcation of coagulase-negative staphylococci from bovine intramammary infection by matrix-assisted laser desorption ionization-time of fight mass spectrometry[J].Journal of Clinical Microbiology,2014,52(5):1658-1663.DOI:10.1128/JCM.03032-13.
[20]王稳航,徐倩倩,滕安国,等.发酵肉制品菌群的非培养鉴定技术研究进展[J].肉类研究,2014,28(9):17-21.
[21]KIM J,HONG J,LIM J A,et al.Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci[J].Archives of Microbiology,2017,200(1):73-83.DOI:10.1007/s00203-017-1415-9.
[22]LANDETA G,REVERON I,CARRASCOSA A V,et al.Use of recA gene sequence analysis for the identification of Staphylococcus equorum strains predominant on dry-cured hams[J].Food Microbiology,2011,28(6):1205-1210.DOI:10.1016/j.fm.2011.04.006.
[23]MOROT-BIZOT S,TALON R,LEROY-SETRINB S.Development of specific PCR primers for a rapid and accurate identification of Staphylococcus xylosus,a species used in food fermentation[J].Journal of Microbiological Methods,2003,55(1):279-286.DOI:10.1016/S0167-7012(03)00159-3.
[24]MOROT-BIZOT C S,TALON R,LEROY S.Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food[J].Journal of Applied Microbiology,2004,97(5):1087-1094.DOI:10.1111/j.1365-2672.2004.02399.x.
[25]AYMERICH T,MARTIN B,GARRIGA M,et al.Microbial quality and direct PCR identification of lactic acid bacteria and nonpathogenic staphylococci from artisanal low-acid sausages[J].Applied and Environmental Microbiology,2003,69(8):4583-4594.DOI:10.1128/AEM.69.8.4583-4594.2003.
[26]GANDRA E A,FERNANDEZ M A,SILVA J A,et al.Detection by multiplex PCR of Staphylococcus aureus,S.intermedius and S.hyicus in artificially contaminated milk[J].Ciencia Rural,2016,46(1):1418-1423.DOI:10.1590/0103-8478cr20151391.
[27]BLAIOTTA G,ERCOLINI D,MAURIELLO G,et al.Rapid and reliable identification of Staphylococcus equorum by aspecies specific PCR assay targeting the sod Agene[J].Systematic&Applied Microbiology,2004,27(6):696-702.DOI:10.1078/0723202042369901.
[28]HIROTAKI S,SASAKI T,KUWAHARA-ARAI K,et al.Rapid and accurate identification of human-associated Staphylococci by use of multiplex PCR[J].Journal of Clinical Microbiology,2011,49(10):3627-3631.DOI:10.1128/JCM.00488-11.
[29]SHOME B R,MITRAL S D,BHUVANAL M,et al.Multiplex PCRassay for species identification of bovine mastitis pathogens[J].Journal of Applied Microbiology,2011,111(6):1349-1356.DOI:10.1111/j.1365-2672.2011.05169.x.
[30]孙鸿燕.四种食源性致病菌多重PCR检测方法的建立[D].长春:吉林大学,2011:37.
[31]周明东,张臣伟,张海滨,等.食源性致病菌快速检测方法的建立与应用[J].中国食品学报,2012,12(10):169-176.
[32]姜彦君,都启晶,范颖华,等.食源性致病菌快速检测试剂盒的研制与应用[J].中国食品学报,2014,14(1):224-231.
[33]刘红玉,李岩,崔洪斌.肉中4种致病菌的PCR快速检测方法的建立[J].食品科学,2011,32(6):213-216.
[34]钱志伟,孙新城.食品中3种致病菌多重PCR检测体系的建立及初步应用[J].食品科学,2011,32(16):236-239.
[35]胡冰雪,舒沿沿,潘道东,等.荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立[J].食品科学,2016,37(20):209-214.DOI:10.7506/spkx1002-6630-201620036.
[36]张莎莎,郭新梅,赵美爱,等.微芯片电泳与聚丙烯酰胺凝胶电泳的比较分析[J].中国农业科技导报,2013,15(2):173-178.