奶牛隐性乳房炎三种致病菌的多重PCR检测方法的建立
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摘要
为建立快速并能同时检测无乳链球菌、金黄色葡萄球菌、绿脓杆菌3种奶牛隐性乳房炎致病菌的方法,根据无乳链球菌16S r RNA基因、金黄色葡萄球菌NUC耐热基因、绿脓杆菌ETA基因序列并参考文献各合成一对特异性引物,通过对PCR反应各项参数的调整优化,建立多重PCR检测体系,并同时进行灵敏性试验和特异性试验。结果显示:该检测方法可以同时扩增出无乳链球菌270 bp、金黄色葡萄球菌153 bp和绿脓杆菌375 bp的特异性片段以及细菌16S rRNA 1500 bp的通用片段,而对于其他的6种病原菌只能扩增出1 500 bp的通用片段,具有较强的特异性。三种特异性引物扩增条带与Gen Bank上的细菌基因序列同源性为99%。增菌培养24h,该体系对无乳链球菌、金黄色葡萄球菌、绿脓杆菌的最低灵敏度分别为8×10~4、4×10~4、1. 5×10~5CFU·m L~(-1)。本研究建立的检测方法能够完成无乳链球菌、金黄色葡萄球菌、绿脓杆菌的快速检测,对于生产上奶牛隐性乳房炎的鉴别诊断和快速检测具有重大意义。
In order to establish a method for the rapid and simultaneous detection of three pathogenic bacteria of mastitis of Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa,three pairs of specific primers were synthesized based on the Streptococcus agalactiae 16 S rRNA gene,Staphylococcus aureus NUC heat-resistance gene,Pseudomonas aeruginosa ETA gene sequence and references. By adjusting and optimizing the parameters of the PCR reaction,a multiplex PCR detection system was established,and the sensitivity test and specificity test were simultaneously performed. The results showed that the detection method can simultaneously amplify specific fragments of 270 bp of Streptococcus agalactiae,153 bp of Staphylococcus aureus and 375 bp of Pseudomonas aeruginosa and a generic 1 500 bp fragment of bacterial 16 S rRNA. For other six kinds of pathogenic bacteria,only 1 500 bp universal fragments could be amplified,which had strong specificity. Three specific primer amplification bands were 99% homologous to the Gen Bank and bacterial gene sequences. After culture for 24 h,the minimum sensitivity of the system to Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa was 8 × 10~4,4 × 10~4 and 1. 5 × 10~5 CFU·m L~(-1),respectively. The detection method established in this study can complete the rapid detection of Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa,and is of great significance for the differential diagnosis and rapid detection of subclinical mastitis in dairy cows.
In order to establish a method for the rapid and simultaneous detection of three pathogenic bacteria of mastitis of Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa,three pairs of specific primers were synthesized based on the Streptococcus agalactiae 16 S rRNA gene,Staphylococcus aureus NUC heat-resistance gene,Pseudomonas aeruginosa ETA gene sequence and references. By adjusting and optimizing the parameters of the PCR reaction,a multiplex PCR detection system was established,and the sensitivity test and specificity test were simultaneously performed. The results showed that the detection method can simultaneously amplify specific fragments of 270 bp of Streptococcus agalactiae,153 bp of Staphylococcus aureus and 375 bp of Pseudomonas aeruginosa and a generic 1 500 bp fragment of bacterial 16 S rRNA. For other six kinds of pathogenic bacteria,only 1 500 bp universal fragments could be amplified,which had strong specificity. Three specific primer amplification bands were 99% homologous to the Gen Bank and bacterial gene sequences. After culture for 24 h,the minimum sensitivity of the system to Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa was 8 × 10~4,4 × 10~4 and 1. 5 × 10~5 CFU·m L~(-1),respectively. The detection method established in this study can complete the rapid detection of Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa,and is of great significance for the differential diagnosis and rapid detection of subclinical mastitis in dairy cows.
引文
[1]阚威,王华,马友记,等.牛源性无乳链球菌分离鉴定及cfb基因的克隆和真核表达载体的构建[J].华北农学报,2014,29(5):39-44.KAN W,WANG H,MA Y J,et al.Construction of eukaryotic expression vector of cfb gene from bovine Streptococcus agalactiaee[J].Acta Agriculturae Boreali-Sinica,2014,29(5):39-44.(in Chinese with English abstract)
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[9]刘玉梅,古丽扎尔·阿不都纳斯尔,丁剑冰,等.环介导等温扩增与实时荧光定量PCR检测布鲁氏菌的比较[J].中国病原生物学杂志,2016,11(12):1070-1073.LIU Y M,GULIZHAER A,DING J B,et al.Comparison of the detection of Brucella by loop-mediated isothermal amplification and real-time PCR[J].Journal of Pathogen Biology,2016,11(12):1070-1073.(in Chinese with English abstract)
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[11]王鹏飞.实验动物金黄色葡萄球菌、绿脓杆菌和肺支原体多重PCR检测方法的建立与初步应用[D].石家庄:河北医科大学,2016.WANG P F.The establishment and preliminary application of multiple PCR for detecting Staphylococcus aureus,Pseudomonas aeruginosa,and Mycoplasma Pulmonis in laboratory animals[D].Shijiazhuang:Hebei Medical University,2016.(in Chinese with English abstract)
[12]WANG W,XI H,HUANG M,et al.Performance of mass spectrometric identification of bacteria and yeasts routinely isolated in a clinical microbiology laboratory using MALDI-TOF MS[J].Journal of Thoracic Disease,2014,6(5):524-533.
[13]GUNAWARDANA S,THILAKARATHNE D,ABEGUNAWAR-DANA I S,et al.Risk factors for bovine mastitis in the Central Province of Sri Lanka[J].Tropical Animal Health&Production,2014,46(7):1105-1112.
[14]WANG X,WANG X,WANG Y,et al.Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk[J].Letters in Applied Microbiology,2014,58(6):527-534.
[15]解绍栋,唐金茹.奶牛乳房炎的预防和治疗[J].山东畜牧兽医,2012(12):97.XIE S D,TANG J R.Dairy cow mastitis prevention and treatment[J].Shandong Journal of Animal Science and Veterinary Medicine,2012(12):97.(in Chinese)
[16]丁天翊,潘阳阳,何翃闳,等.快速检测奶牛乳房炎四种病原菌的多重PCR方法的建立及应用[J].中国兽医科学,2018,48(1):19-26.DING T Y,PAN Y Y,HE H H,et al.Development and applications of a multiplex-PCR for quick detection of four pathogens resulting in bovine mastitis[J].Chinese Veterinary Science,2018,48(1):19-26.(in Chinese with English abstract)
[17]李栋梁,寇亚楠,宋月,等.利用16S r DNA检测奶牛乳房炎5种病原菌PCR方法的建立及应用[J].河南农业科学,2014,43(6):132-136.LI D L,KOU Y N,SONG Y,et al.Detection of five bacterial species in bovine mastitis by PCR based on 16S r DNA sequence[J].Journal of Henan Agricultural Sciences,2014,43(6):132-136.(in Chinese with English abstract)
[18]赵弥,丁玉林,毕艳楠,等.无乳链球菌对体外培养的奶牛乳腺成纤维细胞瘦素及其受体表达的影响[J].中国预防兽医学报,2017,39(9):750-754.ZHAO M,DING Y L,BI Y N,et al.Effects of Streptococcus agalactiae on the expression of leptin and OB-Rb in bovine mammary fibroblasts cultured in vitro[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(9):750-754.(in Chinese with English abstract)
[19]罗素,马可,王兆飞,等.无乳链球菌透明质酸酶的可溶性表达及其对小鼠的致病作用[J].中国兽医科学,2016(8):997-1002.LUO S,MA K,WANG Z F,et al.Prokaryotic expression of hyaluronidase from Streptococcus agalactiae and its pathogenicity in mice[J].Chinese Veterinary Science,2016(8):997-1002.(in Chinese with English abstract)
[20]MAISEY H C,HENSLER M,NIZET V,et al.Group BStreptococcal pilus proteins contribute to adherence to and invasion of brain microvascular endothelial cells[J].Journal of Bacteriology,2007,189(4):1464-1467.
[21]FAUST S N,PATHAN N,LEVIN M.Bacterial meningitis and brain abscess[J].Medicine,2009,33(4):55-60.
[22]DOS SANTOS F F,REIS D R,et al.Presence of mec A-positive multidrug-resistant Staphylococcus epidermidis in bovine milk samples in Brazil[J].Journal of Dairy Science,2016,99(2):1374-1382.
[23]XU J,TENG D,JIANG F,et al.Mycoplasma gallisepticum MGA_0676 is a membrane-associated cytotoxic nuclease with a staphylococcal nuclease region essential for nuclear translocation and apoptosis induction in chicken cells[J].Applied Microbiology&Biotechnology,2015,99(4):1859-1871.
[24]叶芬,曹杰,魏学良.奶牛乳房炎防治研究进展[J].中国牛业科学,2007,33(5):53-55.YE F,CAO J,WEI X L.Research advance of prevention and treatment of mastitis in dairy cattle[J].China Cattle Science,2007,33(5):53-55.(in Chinese with English abstract)
[25]MEIRI-BENDEK I,LIPKIN E,FRIEDMANN A,et al.APCR-based method for the detection of Streptococcus agalactiae in milk[J].Journal of Dairy Science,2002,85(7):1717-1723.
[26]VOULHOUX R,TAUPIAC M P,CZJZEK M,et al.Influence of deletions within domain II of exotoxin A on its extracellular secretion from Pseudomonas aeruginosa[J].Journal of Bacteriology,2000,182(14):4051-4058.
[27]刘祥.重组铜绿假单胞菌外毒素A的原核表达和多克隆抗体制备及免疫保护功能分析[J].湖南农业大学学报(自然科学版),2016,42(1):33-38.LIU X.Prokaryotic expression,polyclonal antibody preparation and immunoprotection potential of exotoxin A from recombinant Pseudomonas aeruginosa[J].Journal of Hunan Agricultural University(Natural Science Edition),2016,42(1):33-38.(in Chinese with English abstract)
[28]韩青松,简永利,涂宜强,等.绿脓杆菌研究进展[J].畜牧与饲料科学,2012,33(1):122-124.HAN Q S,JIAN Y L,TU Y Q,et al.Research advances on Pseudomonas aeruginosa[J].Animal Husbandry and Feed Science,2012,33(1):122-124.(in Chinese with English abstract)
[29]唐俊妮.金黄色葡萄球菌耐热核酸酶相关基因的功能与特征分析[D].武汉:华中农业大学,2007.TANG J N.Characterization and function analysis of the thermostable nuclease genes in Staphylococcus aureus[D].Wuhan:Huazhong Agricultural University,2007.(in Chinese with English abstract)
[2]王剑,肖小东,耿建虎,等.奶牛临床型乳房炎的主要发病原因及防治措施[J].当代畜牧,2017(6):21-22.WANG J,XIAO X D,GENG J H,et al.The main causes and prevention measures of clinical mastitis in dairy cows[J].Contemporary Animal Husbandry,2017(6):21-22.(in Chinese)
[3]GAO G S,GAO G P,LI Z B,et al.The investigation of main pathogens causing dairy cattle recessive mastitis in Eastern Hebei[J].Animal Husbandry and Feed Science,2013(4):198-200.
[4]刘鸽,张培生,于会举,等.用测SCC法调查石河子某奶牛场隐性乳房炎的发病情况[J].中国乳业,2017(1):54-57.LIU G,ZHANG P S,YU H J,et al.Investigation on incidence mastitis of a dairy farm in Shihezi by SCC method[J].China Dairy,2017(1):54-57.(in Chinese)
[5]曲家华,周庆民.奶牛隐性乳房炎的几种检测方法[J].养殖技术顾问,2012(10):155.QU J H,ZHOU Q M.Several methods for detection of recessive mastitis in dairy cows[J].Technical Advisor for Animal Husbandry,2012(10):155.(in Chinese)
[6]CHAMBERLAIN J S,GIBBS R A,RANIER J E,et al.Deletion screening of the Duchenne muscular dystrophy locus via multiple DNA amplification[J].Nucleic Acids Research,1988,16(23):11141-11156.
[7]覃芳芸,熊毅,盛宇明,等.猪链球菌2型多重PCR检测方法的建立及应用[J].畜牧与兽医,2007,39(9):4-6.QIN F Y,XIONG Y,SHENG Y M,et al.Establishment and preliminary application of a multiplex PCR for rapid detection of Streptococcus suis type 2[J].Animal Husbandry&Veterinary Medicine,2007,39(9):4-6.(in Chinese with English abstract)
[8]马保臣,秦卓明,蔡玉梅,等.多重PCR检测奶牛乳腺炎金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母菌方法的建立与应用[J].畜牧兽医学报,2006,37(11):1202-1208.MA B C,QIN Z M,CAI Y M,et al.Development of a multiplex PCR for simultaneous detection of Staphylococcus aureus,Streptococcus agalactiae,Streptococcus dysgalactiae and yeasts in Bovine mastitis milk samples[J].Acta Veterinaria et Zootechnica Sinica,2006,37(11):1202-1208.(in Chinese with English abstract)
[9]刘玉梅,古丽扎尔·阿不都纳斯尔,丁剑冰,等.环介导等温扩增与实时荧光定量PCR检测布鲁氏菌的比较[J].中国病原生物学杂志,2016,11(12):1070-1073.LIU Y M,GULIZHAER A,DING J B,et al.Comparison of the detection of Brucella by loop-mediated isothermal amplification and real-time PCR[J].Journal of Pathogen Biology,2016,11(12):1070-1073.(in Chinese with English abstract)
[10]PHUEKTES P,MANSELL P D,BROWNING G F.Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and streptococcal causes of bovine mastitis[J].Journal of Dairy Science,2001,84(5):1140-1148.
[11]王鹏飞.实验动物金黄色葡萄球菌、绿脓杆菌和肺支原体多重PCR检测方法的建立与初步应用[D].石家庄:河北医科大学,2016.WANG P F.The establishment and preliminary application of multiple PCR for detecting Staphylococcus aureus,Pseudomonas aeruginosa,and Mycoplasma Pulmonis in laboratory animals[D].Shijiazhuang:Hebei Medical University,2016.(in Chinese with English abstract)
[12]WANG W,XI H,HUANG M,et al.Performance of mass spectrometric identification of bacteria and yeasts routinely isolated in a clinical microbiology laboratory using MALDI-TOF MS[J].Journal of Thoracic Disease,2014,6(5):524-533.
[13]GUNAWARDANA S,THILAKARATHNE D,ABEGUNAWAR-DANA I S,et al.Risk factors for bovine mastitis in the Central Province of Sri Lanka[J].Tropical Animal Health&Production,2014,46(7):1105-1112.
[14]WANG X,WANG X,WANG Y,et al.Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk[J].Letters in Applied Microbiology,2014,58(6):527-534.
[15]解绍栋,唐金茹.奶牛乳房炎的预防和治疗[J].山东畜牧兽医,2012(12):97.XIE S D,TANG J R.Dairy cow mastitis prevention and treatment[J].Shandong Journal of Animal Science and Veterinary Medicine,2012(12):97.(in Chinese)
[16]丁天翊,潘阳阳,何翃闳,等.快速检测奶牛乳房炎四种病原菌的多重PCR方法的建立及应用[J].中国兽医科学,2018,48(1):19-26.DING T Y,PAN Y Y,HE H H,et al.Development and applications of a multiplex-PCR for quick detection of four pathogens resulting in bovine mastitis[J].Chinese Veterinary Science,2018,48(1):19-26.(in Chinese with English abstract)
[17]李栋梁,寇亚楠,宋月,等.利用16S r DNA检测奶牛乳房炎5种病原菌PCR方法的建立及应用[J].河南农业科学,2014,43(6):132-136.LI D L,KOU Y N,SONG Y,et al.Detection of five bacterial species in bovine mastitis by PCR based on 16S r DNA sequence[J].Journal of Henan Agricultural Sciences,2014,43(6):132-136.(in Chinese with English abstract)
[18]赵弥,丁玉林,毕艳楠,等.无乳链球菌对体外培养的奶牛乳腺成纤维细胞瘦素及其受体表达的影响[J].中国预防兽医学报,2017,39(9):750-754.ZHAO M,DING Y L,BI Y N,et al.Effects of Streptococcus agalactiae on the expression of leptin and OB-Rb in bovine mammary fibroblasts cultured in vitro[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(9):750-754.(in Chinese with English abstract)
[19]罗素,马可,王兆飞,等.无乳链球菌透明质酸酶的可溶性表达及其对小鼠的致病作用[J].中国兽医科学,2016(8):997-1002.LUO S,MA K,WANG Z F,et al.Prokaryotic expression of hyaluronidase from Streptococcus agalactiae and its pathogenicity in mice[J].Chinese Veterinary Science,2016(8):997-1002.(in Chinese with English abstract)
[20]MAISEY H C,HENSLER M,NIZET V,et al.Group BStreptococcal pilus proteins contribute to adherence to and invasion of brain microvascular endothelial cells[J].Journal of Bacteriology,2007,189(4):1464-1467.
[21]FAUST S N,PATHAN N,LEVIN M.Bacterial meningitis and brain abscess[J].Medicine,2009,33(4):55-60.
[22]DOS SANTOS F F,REIS D R,et al.Presence of mec A-positive multidrug-resistant Staphylococcus epidermidis in bovine milk samples in Brazil[J].Journal of Dairy Science,2016,99(2):1374-1382.
[23]XU J,TENG D,JIANG F,et al.Mycoplasma gallisepticum MGA_0676 is a membrane-associated cytotoxic nuclease with a staphylococcal nuclease region essential for nuclear translocation and apoptosis induction in chicken cells[J].Applied Microbiology&Biotechnology,2015,99(4):1859-1871.
[24]叶芬,曹杰,魏学良.奶牛乳房炎防治研究进展[J].中国牛业科学,2007,33(5):53-55.YE F,CAO J,WEI X L.Research advance of prevention and treatment of mastitis in dairy cattle[J].China Cattle Science,2007,33(5):53-55.(in Chinese with English abstract)
[25]MEIRI-BENDEK I,LIPKIN E,FRIEDMANN A,et al.APCR-based method for the detection of Streptococcus agalactiae in milk[J].Journal of Dairy Science,2002,85(7):1717-1723.
[26]VOULHOUX R,TAUPIAC M P,CZJZEK M,et al.Influence of deletions within domain II of exotoxin A on its extracellular secretion from Pseudomonas aeruginosa[J].Journal of Bacteriology,2000,182(14):4051-4058.
[27]刘祥.重组铜绿假单胞菌外毒素A的原核表达和多克隆抗体制备及免疫保护功能分析[J].湖南农业大学学报(自然科学版),2016,42(1):33-38.LIU X.Prokaryotic expression,polyclonal antibody preparation and immunoprotection potential of exotoxin A from recombinant Pseudomonas aeruginosa[J].Journal of Hunan Agricultural University(Natural Science Edition),2016,42(1):33-38.(in Chinese with English abstract)
[28]韩青松,简永利,涂宜强,等.绿脓杆菌研究进展[J].畜牧与饲料科学,2012,33(1):122-124.HAN Q S,JIAN Y L,TU Y Q,et al.Research advances on Pseudomonas aeruginosa[J].Animal Husbandry and Feed Science,2012,33(1):122-124.(in Chinese with English abstract)
[29]唐俊妮.金黄色葡萄球菌耐热核酸酶相关基因的功能与特征分析[D].武汉:华中农业大学,2007.TANG J N.Characterization and function analysis of the thermostable nuclease genes in Staphylococcus aureus[D].Wuhan:Huazhong Agricultural University,2007.(in Chinese with English abstract)
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