贵州省畜禽源葡萄球菌和链球菌快速鉴定双重PCR方法的建立及应用
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摘要
为建立一种可准确、快速鉴定畜禽临床病例常见病原葡萄球菌和链球菌的双重PCR方法,本试验选取葡萄球菌的nuc基因和链球菌的EF-TU基因保守片段分别设计合成了1对特异性引物,构建可同时快速鉴别葡萄球菌和链球菌的双重PCR体系,并进行反应条件优化,筛选出最佳引物浓度和退火温度;应用该方法对其他73株革兰氏阳性临床分离细菌进行检测,评价该方法的特异性;将培养的葡萄球菌和链球菌倍比稀释计数后鉴定检测方法的敏感性;应用该检测方法对贵州省部分畜禽葡萄球菌和链球菌临床分离样本进行检测。结果显示,所建立的方法最佳引物添加量均为1μL,最佳退火温度为56℃;其他73株供试菌株检测结果均无扩增条带出现,所建双重PCR方法具有较好的特异性;敏感性试验结果显示,葡萄球菌和链球菌敏感度分别达1.50 ng/μL和1.44 pg/μL;临床样本复检结果显示,73株临床分离细菌中葡萄球菌40株(54.79%)、链球菌33株(45.21%),与传统细菌分离鉴定方法的符合率为97.26%。本试验建立了一种特异、敏感和快速鉴定贵州省畜禽葡萄球菌和链球菌病原的双重PCR方法,为临床病例快速诊断及流行病学调查提供了有效技术。
In order to establish a duplex PCR method for accurately and quickly identifying common pathogens Staphylococcus and Streptococcus in clinical cases of livestock and poultry,this experiment selected nuc gene of Staphylococcus and EF-TU gene conserved fragments of Streptococcus to design a pair of specific primers,construct a duplex PCR system which could simultaneously identify Staphylococcus and Streptococcus,optimize the reaction conditions,screen out the optimal primer concentration and annealing temperature;Using this method to detect 73 other gram-positive clinical isolates,the specificity of the method was evaluated;The sensitivity of the detection method was determined by diluting and counting the cultured Staphylococcus and Streptococcus;The detection method was used to detect the clinical isolates of Staphylococcus and Streptococcus of livestock and poultry in Guizhou province.The results showed that the optimal primer addition amount was 1 μL,and the optimal annealing temperature was 56 ℃.The other 73 strains showed no amplification bands,and the duplex PCR method was better.The specificity of the sensitivity test showed that the sensitivity of Staphylococcus and Streptococcus were 1.50 ng/μL and 1.44 pg/μL,respectively.The retest results of clinical samples showed that there were 40 strains of Staphylococcus(54.79%) and 33 strains of Streptococcus(45.21%) in 73 clinical isolates,the coincidence rate with traditional bacteria separation and identification method was 97.26%.This experiment established a duplex PCR method for specific,sensitive and rapid identification of Staphylococcus and Streptococcus pathogens in Guizhou province,providing effective techniques for rapid diagnosis and epidemiological investigation of clinical cases.
In order to establish a duplex PCR method for accurately and quickly identifying common pathogens Staphylococcus and Streptococcus in clinical cases of livestock and poultry,this experiment selected nuc gene of Staphylococcus and EF-TU gene conserved fragments of Streptococcus to design a pair of specific primers,construct a duplex PCR system which could simultaneously identify Staphylococcus and Streptococcus,optimize the reaction conditions,screen out the optimal primer concentration and annealing temperature;Using this method to detect 73 other gram-positive clinical isolates,the specificity of the method was evaluated;The sensitivity of the detection method was determined by diluting and counting the cultured Staphylococcus and Streptococcus;The detection method was used to detect the clinical isolates of Staphylococcus and Streptococcus of livestock and poultry in Guizhou province.The results showed that the optimal primer addition amount was 1 μL,and the optimal annealing temperature was 56 ℃.The other 73 strains showed no amplification bands,and the duplex PCR method was better.The specificity of the sensitivity test showed that the sensitivity of Staphylococcus and Streptococcus were 1.50 ng/μL and 1.44 pg/μL,respectively.The retest results of clinical samples showed that there were 40 strains of Staphylococcus(54.79%) and 33 strains of Streptococcus(45.21%) in 73 clinical isolates,the coincidence rate with traditional bacteria separation and identification method was 97.26%.This experiment established a duplex PCR method for specific,sensitive and rapid identification of Staphylococcus and Streptococcus pathogens in Guizhou province,providing effective techniques for rapid diagnosis and epidemiological investigation of clinical cases.
引文
[1] JANS C,MEILE L,LACROIX C,et al.Genomics,evolution,and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC)[J].Infection Genetics & Evolution,2015,33:419-436.
[2] MORACH M,STEPHAN R,SCHMITT S,et al.Population structure and virulence gene profiles of Streptococcus agalactiae,collected from different hosts worldwide[J]. European Journal of Clinical Microbiology & Infectious Diseases,2018,37(3):527-536.
[3] XIA X J,WANG L,CHENG L K,et al.Expression and immunological evaluation of elongation factor Tu of Streptococcus suis serotype 2[J]. Polish Journal of Veterinary Sciences,2017,20(2):277.
[4] STEWARD K F,ROBINSON C,HOIDEN M T G,et al.Diversity of Streptococcus equi,subsp.zooepidemicus,strains isolated from the Spanish sheep and goat population and the identification,function and prevalence of a novel arbutin utilisation system[J]. Veterinary Microbiology,2017,207:231-238.
[5] 李书光,李天芝,李峰,等.快速鉴定猪链球菌和马链球菌兽疫亚种双重PCR方法的建立[J].中国畜牧兽医,2013,40(7):43-46. LI S G,LI T Z,LI F,et al.Establishment of double PCR for rapid identification of Streptococcus suis and Streptococcus equi subspecies[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):43-46.(in Chinese)
[6] 冯洁,谢建云,胡建华,等.3种条件性致病菌三重PCR检测方法的建立及初步应用[J].中国畜牧兽医,2015,42(6):1389-1395. FENG J,XIE J Y,HU J H,et al.Establishment and preliminary application of three PCR detection methods for 3 conditional pathogenic bacteria[J].China Animal Husbandry & Veterinary Medicine,2015,42(6):1389-1395.(in Chinese)
[7] 程龙飞,傅光华,林群群,等.同时检测水禽3种常见病原菌的环介导间接PCR方法的建立[J].中国兽医学报,2018,1:96-99. CHENG L F,FU G H,LIN Q Q,et al.Development of loop-mediated indirect PCR for simultaneously detecting 3 pathogenic bacteria of waterfowl[J].Chinese Journal of Veterinary Science,2018,1:96-99.(in Chinese)
[8] BOGESTAM K,VONDRACEK M,KARLSSON M,et al.Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus[J]. PLoS One,2018,13(2):e0192782.
[9] SHEET O H,GRABOWSKI N T,KLEIN G,et al.Development and validation of a loop mediated isothermal amplification (LAMP) assay for the detection of Staphylococcus aureus in bovine mastitis milk samples[J].Molecular and Cellular Probes,2016,30(5):320-325.
[10] 杜华茂,丁玉春,王丽丽,等.4株猪链球菌的病原生物学特性分析[J].中国兽医学报,2010,30(1):24-28. DU H M,DING Y C,WANG L L,et al.Pathogenic characteristics of four Streptococcus suis strains[J].Chinese Journal of Veterinary Science,2010,30(1):24-28.(in Chinese)
[11] CHRISTOPH J,LEO M,CHRISTOPHE L.Genomics,evolution,and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC)[J].Infection,Genetics and Evolution,2015,33:419-436.
[12] XU Y,HUO B,SUN X,et al.Rapid detection of staphylococcal enterotoxin B in milk samples based on fluorescence hybridization chain reaction amplifica-tion[J].RSC Advances,2018,8(29):16024-16031.
[13] GREISEN K,LOEFFELHOLZ M,PUROHIT A,et al.PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria,including bacteria found in cerebrospinal fluid[J].Journal of Clinical Microbiology,1994,32(2):335-351.
[14] FENG L,NIU X,MEI W,et al.Immunogenicity and protective capacity of EF-Tu and FtsZ of Streptococcus suis serotype 2 against lethal infection[J].Vaccine,2018,36(19):2581-2588.
[15] 吴静波,南文金,黄健强,等.猪链球菌通用型和2型双重荧光定量PCR快速检测技术的建立和应用[J].畜牧兽医学报,2018,49(2):368-377. WU J B,NAN W J,HUANG J Q,et al.Development of a rapid duplex Real-time PCR assay for Streptococcus suis and S.suis serotype 2[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):368-377.(in Chinese)
[16] 戈胜强.快速鉴别奶牛乳房炎细菌或真菌的双重PCR方法的建立与应用[J].山东农业大学学报,2008,39(11):1554-1561. GE S Q.Establishment and application of dual-PCR method to rapid detection of bacteria and yeast from bovine mastitis[D].Journal of Shandong Agricultural University,2008,39(11):1554-1561.(in Chinese)
[17] WEI C,ZHONG J,HU T,et al.Simultaneous detection of Escherichia coli O157∶H7,Staphylococcus aureus and Salmonella by multiplex PCR in milk[J]. Biotech,2018,8(1):76.
[18] YU Y,MA X,ZHANG W.Detection of Staphylococcus aureus in milk using Real-time fluorescence loop-mediated isothermal amplification[J].Advance Journal of Food Science & Technology,2015,8(9):678-684.
[19] ARUNRUT N,KIATPATHOMCHAI W,ANANCHAIPATTANA C.Multiplex PCR assay and lyophilization for detection of Salmonella,spp.Staphylococcus aureus,and Bacillus cereus,in pork products[J].Food Science & Biotechnology,2018,27(3):867-875.
[20] 刘继超,姜铁民,陈历俊,等.多重PCR检测原料乳中沙门氏菌、无乳链球菌和黄色葡萄球菌的研究[J].中国食品学报,2010,10(6):173-179. LIU J C,JIANG T M,CHEN L J,et al.Study on the detection of Salmonella,Streptococcus agalactiae and Staphylococcus aureus in raw milk by multiplex PCR[J].Journal of Chinese Institute of Food Science and Technology,2010,10(6):173-179.(in Chinese)
[21] YAMADA R,LE H T T,ARAI S,et al.Development of PCR for identifying Streptococcus parasuis,a close relative of Streptococcus suis[J].Journal of Veterinary Medical Science,2018,80(7):1101-1107.
[22] 孙学飞,倪艳秀,茅爱华,等.猪链球菌、胸膜肺炎放线杆菌和副猪嗜血杆菌多重PCR检测方法的建立和应用[J].江苏农业学报,2016,32(5):1094-1099. SUN X F,NI Y X,MAO A H,et al.Development and application of a multiplex PCR for detection of Streptococcus suis,Actinobacillus pleuropneumoniae and Haemophilus parasuis[J].Jiangsu Journal of Agricultural Sciences,2016,32(5):1094-1099.(in Chinese)
[23] 邓海平,蒲万霞,梁剑平,等.贵州地区牛源黄色葡萄球菌分离株16S rRNA分子鉴定及RAPD分型研究[J].中国畜牧兽医,2011,38(1):112-115. DENG H P,PU W X,LIANG J P,et al.Research of identification by 16S rRNA and RAPD typing on Staphylococcus aureus isolated from dairy cattle in Guizhou[J].China Animal Husbandry & Veterinary Medicine,2011,38(1):112-115.(in Chinese)
[24] 覃娟娟,顾凡,蔡雨涵,等.副猪嗜血杆菌毒力菌株和猪支气管败血波氏杆菌毒力菌株双重PCR检测方法的建立及应用[J].中国兽医学报,2015,35(8):1217-1222. QIN J J,GU F,CAI Y H,et al.Establishment and application of duplex PCR for detecting virulent strains of Haemophilus parasuis and Bordetella bronchiseptica[J].Chinese Journal of Veterinary Science,2015,35(8):1217-1222.(in Chinese)
[2] MORACH M,STEPHAN R,SCHMITT S,et al.Population structure and virulence gene profiles of Streptococcus agalactiae,collected from different hosts worldwide[J]. European Journal of Clinical Microbiology & Infectious Diseases,2018,37(3):527-536.
[3] XIA X J,WANG L,CHENG L K,et al.Expression and immunological evaluation of elongation factor Tu of Streptococcus suis serotype 2[J]. Polish Journal of Veterinary Sciences,2017,20(2):277.
[4] STEWARD K F,ROBINSON C,HOIDEN M T G,et al.Diversity of Streptococcus equi,subsp.zooepidemicus,strains isolated from the Spanish sheep and goat population and the identification,function and prevalence of a novel arbutin utilisation system[J]. Veterinary Microbiology,2017,207:231-238.
[5] 李书光,李天芝,李峰,等.快速鉴定猪链球菌和马链球菌兽疫亚种双重PCR方法的建立[J].中国畜牧兽医,2013,40(7):43-46. LI S G,LI T Z,LI F,et al.Establishment of double PCR for rapid identification of Streptococcus suis and Streptococcus equi subspecies[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):43-46.(in Chinese)
[6] 冯洁,谢建云,胡建华,等.3种条件性致病菌三重PCR检测方法的建立及初步应用[J].中国畜牧兽医,2015,42(6):1389-1395. FENG J,XIE J Y,HU J H,et al.Establishment and preliminary application of three PCR detection methods for 3 conditional pathogenic bacteria[J].China Animal Husbandry & Veterinary Medicine,2015,42(6):1389-1395.(in Chinese)
[7] 程龙飞,傅光华,林群群,等.同时检测水禽3种常见病原菌的环介导间接PCR方法的建立[J].中国兽医学报,2018,1:96-99. CHENG L F,FU G H,LIN Q Q,et al.Development of loop-mediated indirect PCR for simultaneously detecting 3 pathogenic bacteria of waterfowl[J].Chinese Journal of Veterinary Science,2018,1:96-99.(in Chinese)
[8] BOGESTAM K,VONDRACEK M,KARLSSON M,et al.Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus[J]. PLoS One,2018,13(2):e0192782.
[9] SHEET O H,GRABOWSKI N T,KLEIN G,et al.Development and validation of a loop mediated isothermal amplification (LAMP) assay for the detection of Staphylococcus aureus in bovine mastitis milk samples[J].Molecular and Cellular Probes,2016,30(5):320-325.
[10] 杜华茂,丁玉春,王丽丽,等.4株猪链球菌的病原生物学特性分析[J].中国兽医学报,2010,30(1):24-28. DU H M,DING Y C,WANG L L,et al.Pathogenic characteristics of four Streptococcus suis strains[J].Chinese Journal of Veterinary Science,2010,30(1):24-28.(in Chinese)
[11] CHRISTOPH J,LEO M,CHRISTOPHE L.Genomics,evolution,and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC)[J].Infection,Genetics and Evolution,2015,33:419-436.
[12] XU Y,HUO B,SUN X,et al.Rapid detection of staphylococcal enterotoxin B in milk samples based on fluorescence hybridization chain reaction amplifica-tion[J].RSC Advances,2018,8(29):16024-16031.
[13] GREISEN K,LOEFFELHOLZ M,PUROHIT A,et al.PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria,including bacteria found in cerebrospinal fluid[J].Journal of Clinical Microbiology,1994,32(2):335-351.
[14] FENG L,NIU X,MEI W,et al.Immunogenicity and protective capacity of EF-Tu and FtsZ of Streptococcus suis serotype 2 against lethal infection[J].Vaccine,2018,36(19):2581-2588.
[15] 吴静波,南文金,黄健强,等.猪链球菌通用型和2型双重荧光定量PCR快速检测技术的建立和应用[J].畜牧兽医学报,2018,49(2):368-377. WU J B,NAN W J,HUANG J Q,et al.Development of a rapid duplex Real-time PCR assay for Streptococcus suis and S.suis serotype 2[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):368-377.(in Chinese)
[16] 戈胜强.快速鉴别奶牛乳房炎细菌或真菌的双重PCR方法的建立与应用[J].山东农业大学学报,2008,39(11):1554-1561. GE S Q.Establishment and application of dual-PCR method to rapid detection of bacteria and yeast from bovine mastitis[D].Journal of Shandong Agricultural University,2008,39(11):1554-1561.(in Chinese)
[17] WEI C,ZHONG J,HU T,et al.Simultaneous detection of Escherichia coli O157∶H7,Staphylococcus aureus and Salmonella by multiplex PCR in milk[J]. Biotech,2018,8(1):76.
[18] YU Y,MA X,ZHANG W.Detection of Staphylococcus aureus in milk using Real-time fluorescence loop-mediated isothermal amplification[J].Advance Journal of Food Science & Technology,2015,8(9):678-684.
[19] ARUNRUT N,KIATPATHOMCHAI W,ANANCHAIPATTANA C.Multiplex PCR assay and lyophilization for detection of Salmonella,spp.Staphylococcus aureus,and Bacillus cereus,in pork products[J].Food Science & Biotechnology,2018,27(3):867-875.
[20] 刘继超,姜铁民,陈历俊,等.多重PCR检测原料乳中沙门氏菌、无乳链球菌和黄色葡萄球菌的研究[J].中国食品学报,2010,10(6):173-179. LIU J C,JIANG T M,CHEN L J,et al.Study on the detection of Salmonella,Streptococcus agalactiae and Staphylococcus aureus in raw milk by multiplex PCR[J].Journal of Chinese Institute of Food Science and Technology,2010,10(6):173-179.(in Chinese)
[21] YAMADA R,LE H T T,ARAI S,et al.Development of PCR for identifying Streptococcus parasuis,a close relative of Streptococcus suis[J].Journal of Veterinary Medical Science,2018,80(7):1101-1107.
[22] 孙学飞,倪艳秀,茅爱华,等.猪链球菌、胸膜肺炎放线杆菌和副猪嗜血杆菌多重PCR检测方法的建立和应用[J].江苏农业学报,2016,32(5):1094-1099. SUN X F,NI Y X,MAO A H,et al.Development and application of a multiplex PCR for detection of Streptococcus suis,Actinobacillus pleuropneumoniae and Haemophilus parasuis[J].Jiangsu Journal of Agricultural Sciences,2016,32(5):1094-1099.(in Chinese)
[23] 邓海平,蒲万霞,梁剑平,等.贵州地区牛源黄色葡萄球菌分离株16S rRNA分子鉴定及RAPD分型研究[J].中国畜牧兽医,2011,38(1):112-115. DENG H P,PU W X,LIANG J P,et al.Research of identification by 16S rRNA and RAPD typing on Staphylococcus aureus isolated from dairy cattle in Guizhou[J].China Animal Husbandry & Veterinary Medicine,2011,38(1):112-115.(in Chinese)
[24] 覃娟娟,顾凡,蔡雨涵,等.副猪嗜血杆菌毒力菌株和猪支气管败血波氏杆菌毒力菌株双重PCR检测方法的建立及应用[J].中国兽医学报,2015,35(8):1217-1222. QIN J J,GU F,CAI Y H,et al.Establishment and application of duplex PCR for detecting virulent strains of Haemophilus parasuis and Bordetella bronchiseptica[J].Chinese Journal of Veterinary Science,2015,35(8):1217-1222.(in Chinese)