羊梭菌病快速鉴别PCR方法的建立及初步应用
|
摘要
根据GenBank已公布的产气荚膜梭菌和腐败梭菌的α毒素基因序列,分别设计并合成针对2种α毒素基因的特异性引物,通过扩增条件的优化,建立快速鉴别产气荚膜梭菌和腐败梭菌的双重PCR方法。特异性试验显示,产气荚膜梭菌和腐败梭菌参考菌株均扩增出了相应的预期目的条带,而大肠埃希菌、沙门菌、金黄色葡萄球菌和链球菌则均未能扩增出相应条带。灵敏度试验结果显示,产气荚膜梭菌和腐败梭菌基因组DNA最低检测量分别为17.95 pg/μL和1.261 pg/μL。应用该方法对样品进行检测,其中5份为产气荚膜梭菌阳性。成功建立了特异性强、敏感性高的双重PCR方法,可以有效进行产气荚膜梭菌和腐败梭菌的快速鉴别,对羊梭菌病病原快速鉴定及流行病学调查具有重要意义。
According to the sequences published in GenBank,two pairs of specific primers were designed for the specific gene sequences of alpha toxin from C.perfringens and C.septium.A rapid distinguishing PCR for Clostridium perfringens and Clostridium septium was established by optimization of amplification conditions.Specificity test showed the strains of C.perfringens and C.septium both amplified the corresponding expected bands,while no amplifications were detected from Escherichia coli,Salmonella,Staphylococcus aureus and Streptococcus.The sensitivity analysis showed minimum detection amounts of C.perfringens genomic DNA and C.septium genomic DNA were 17.95 pg/μL and 1.261 pg/μL,respectively.Applying this method to detecting samples,the results showed that 5 of them were positive for C.perfringens. This study successfully established a duplex PCR method with high specificity and high sensitivity,which can effectively distinguish C.perfringens and C.septium.And it has a great significance for rapidly distinguishing and epidemiological investigation of the pathogens of clostridial diseases.
According to the sequences published in GenBank,two pairs of specific primers were designed for the specific gene sequences of alpha toxin from C.perfringens and C.septium.A rapid distinguishing PCR for Clostridium perfringens and Clostridium septium was established by optimization of amplification conditions.Specificity test showed the strains of C.perfringens and C.septium both amplified the corresponding expected bands,while no amplifications were detected from Escherichia coli,Salmonella,Staphylococcus aureus and Streptococcus.The sensitivity analysis showed minimum detection amounts of C.perfringens genomic DNA and C.septium genomic DNA were 17.95 pg/μL and 1.261 pg/μL,respectively.Applying this method to detecting samples,the results showed that 5 of them were positive for C.perfringens. This study successfully established a duplex PCR method with high specificity and high sensitivity,which can effectively distinguish C.perfringens and C.septium.And it has a great significance for rapidly distinguishing and epidemiological investigation of the pathogens of clostridial diseases.
引文
[1] Low L Y,Harrison P F,Lin Y H,et al.RNA-seq analysis of virR and revR mutants of Clostridium perfringens[J].BMC Genomics,2016,17:391.
[2] Rood J I,Adams V,Lacey J,et al.Expansion of the Clostridium perfringens toxin-based typing scheme[J].Anaerobe,2018,53:5-10.
[3] 王全龙.羊快疫的发病机制及防治措施[J].甘肃畜牧兽医,2018,48(7):53-54.
[4] 陈发魁.浅谈羊快疫的诊断和防控措施[J].中国畜牧兽医文摘,2018,34(2):203.
[5] 叶力克·阿勒泰别克.羊梭菌病的流行病学、临床症状、剖检变化及其防控[J].现代畜牧科技,2018(3):102.
[6] 扎西措.默勒镇羊快疫类病流行情况的调查[J].畜牧兽医科技信息,2010(8):67.
[7] 孟林明,贺奋义.张掖市羊梭菌病的流行情况及防控措施[J].中兽医学杂志,2015(8):64.
[8] 徐超.羊梭菌病的流行调查及防控策略[J].中兽医学杂志,2018(1):79.
[9] 张喜国.山羊快疫和肠毒血症混合感染的综合防治[J].畜牧兽医杂志,2013,32(2):135.
[10] 焦振东,李春东,王妍妍,等.羊快疫和羊肠毒血症混合感染的诊治[J].兽医导刊,2015(3):69.
[11] Neumann A P,Dunham S M,Rehberger T G,et al.Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples[J].Mol Cell Probes,2010,24(4):211-218.
[12] 石玉玲,曾兰兰,陈丽丹,等.产气荚膜梭菌实时荧光PCR方法的建立[J].生物技术通讯,2010,21(3):389-392.
[13] 刘立兵,李睿文,陈志敏,等.产气荚膜梭菌荧光PCR和RPA检测方法的建立和比较[J].食品科学,2019(1):1-8.
[14] 刘哲,马晓燕,张会彦,等.环介导等温扩增技术快速检测产气荚膜梭菌的研究[J].中国食品学报,2012,12(4):168-174.
[15] Vázquez-Iglesias L,Estefanell-Ucha B,Barcia-Castro L,et al.A simple electroelution method for rapid protein purification:isolation and antibody production of alpha toxin from Clostridium septicum[J].Peer J,2017,5(11):e3407.
[16] 孙佳芝,王新桐,刘雪慧,等.产气荚膜梭菌α毒素双抗体夹心ELISA检测方法的建立及初步应用[J].中国兽医学报,2014,34(6):930-935.
[17] 董洁,杨晓静,苗承霞,等.产气荚膜梭菌菌落多重PCR方法的建立及初步应用[J].中国兽医学报,2013,33(12):1842-1847.
[2] Rood J I,Adams V,Lacey J,et al.Expansion of the Clostridium perfringens toxin-based typing scheme[J].Anaerobe,2018,53:5-10.
[3] 王全龙.羊快疫的发病机制及防治措施[J].甘肃畜牧兽医,2018,48(7):53-54.
[4] 陈发魁.浅谈羊快疫的诊断和防控措施[J].中国畜牧兽医文摘,2018,34(2):203.
[5] 叶力克·阿勒泰别克.羊梭菌病的流行病学、临床症状、剖检变化及其防控[J].现代畜牧科技,2018(3):102.
[6] 扎西措.默勒镇羊快疫类病流行情况的调查[J].畜牧兽医科技信息,2010(8):67.
[7] 孟林明,贺奋义.张掖市羊梭菌病的流行情况及防控措施[J].中兽医学杂志,2015(8):64.
[8] 徐超.羊梭菌病的流行调查及防控策略[J].中兽医学杂志,2018(1):79.
[9] 张喜国.山羊快疫和肠毒血症混合感染的综合防治[J].畜牧兽医杂志,2013,32(2):135.
[10] 焦振东,李春东,王妍妍,等.羊快疫和羊肠毒血症混合感染的诊治[J].兽医导刊,2015(3):69.
[11] Neumann A P,Dunham S M,Rehberger T G,et al.Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples[J].Mol Cell Probes,2010,24(4):211-218.
[12] 石玉玲,曾兰兰,陈丽丹,等.产气荚膜梭菌实时荧光PCR方法的建立[J].生物技术通讯,2010,21(3):389-392.
[13] 刘立兵,李睿文,陈志敏,等.产气荚膜梭菌荧光PCR和RPA检测方法的建立和比较[J].食品科学,2019(1):1-8.
[14] 刘哲,马晓燕,张会彦,等.环介导等温扩增技术快速检测产气荚膜梭菌的研究[J].中国食品学报,2012,12(4):168-174.
[15] Vázquez-Iglesias L,Estefanell-Ucha B,Barcia-Castro L,et al.A simple electroelution method for rapid protein purification:isolation and antibody production of alpha toxin from Clostridium septicum[J].Peer J,2017,5(11):e3407.
[16] 孙佳芝,王新桐,刘雪慧,等.产气荚膜梭菌α毒素双抗体夹心ELISA检测方法的建立及初步应用[J].中国兽医学报,2014,34(6):930-935.
[17] 董洁,杨晓静,苗承霞,等.产气荚膜梭菌菌落多重PCR方法的建立及初步应用[J].中国兽医学报,2013,33(12):1842-1847.