3种病原菌多重IMS-荧光RPA检测体系的建立及初步应用
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摘要
建立一种同时检测沙门氏菌、大肠杆菌O157:H7和布氏杆菌的快速、灵敏、高通量的检测方法。利用特异性免疫磁珠,在37℃条件下从200 mL样液体系中循环捕获目标致病菌。磁珠液提取DNA后,对3种病原菌进行多重IMS-荧光RPA检测。结果表明:针对沙门氏菌、大肠杆菌O157:H7和布氏杆菌的检测限分别达到3.0、4.5和8.7 CFU/mL。使用新建多重IMS-荧光RPA方法对人工感染60份皮张、毛皮纺织品DNA样本进行扩增,结果显示与预期结果一致;对60份公共场所收集的皮张、毛皮、纺织品DNA样本进行扩增,结果显示有2个样本沙门氏菌阳性、1个样本大肠杆菌O157:H7阳性,3份阳性样品送测序,测序结果与GenBank检索沙门氏菌、大肠杆菌O157:H7 DNA序列一致。得出结论:建立的多重IMS-荧光RPA检测体系灵敏度、特异性、准确度符合要求,能够在2 h内完成对3种病原菌检测,可以作为快速应对此三类病原菌安全突发事件的检测手段。
This study was to establish a multiplex IMS real-time RPA method for simultaneous detection of Salmonella sp.,E coli O157 and Brucella.Target pathogens were isolated from a 200 ml sample system with specific an immunomagnetic bead based technique at 37 ℃.The three pathogens were tested using the multiplex IMS real-time RPA after the magnetic beads had been extracted.The results were that the limits of detection of the immunomagnetic separation(IMS)-multiplex real-time RPA(RT-RPA) method were 3.0 CFU/g for Salmonella sp.,4.5 CFU/g for E coli O157 and 8.7 CFU/g for Brucella.The new multiplex IMS real-time RPA method was used to test the textile DNA of 60 textile samples artificially infected,and the results were consistent with the expected ones.Sixty textile samples collected in public places were tested by DNA amplification;and the results showed that two samples were Salmonella positive and one was Escherichia coli O157:H7 positive.The three positive samples were then sent for sequencing with a result consistent with the DNA sequences of Salmonella and Escherichia coli O157:H7 in Gen Bank.In conclusion,the multiplex IMS real-time RPA method was established with satisfactory sensitivity,specificity and accuracy,being able to detect three pathogenic bacteria in two hours.The method may be used for rapid detection of these bacteria in emergent cases.
This study was to establish a multiplex IMS real-time RPA method for simultaneous detection of Salmonella sp.,E coli O157 and Brucella.Target pathogens were isolated from a 200 ml sample system with specific an immunomagnetic bead based technique at 37 ℃.The three pathogens were tested using the multiplex IMS real-time RPA after the magnetic beads had been extracted.The results were that the limits of detection of the immunomagnetic separation(IMS)-multiplex real-time RPA(RT-RPA) method were 3.0 CFU/g for Salmonella sp.,4.5 CFU/g for E coli O157 and 8.7 CFU/g for Brucella.The new multiplex IMS real-time RPA method was used to test the textile DNA of 60 textile samples artificially infected,and the results were consistent with the expected ones.Sixty textile samples collected in public places were tested by DNA amplification;and the results showed that two samples were Salmonella positive and one was Escherichia coli O157:H7 positive.The three positive samples were then sent for sequencing with a result consistent with the DNA sequences of Salmonella and Escherichia coli O157:H7 in Gen Bank.In conclusion,the multiplex IMS real-time RPA method was established with satisfactory sensitivity,specificity and accuracy,being able to detect three pathogenic bacteria in two hours.The method may be used for rapid detection of these bacteria in emergent cases.
引文
[1]邓清贵,张明辉,殷海平.肉品检疫中常见的几种人畜共患传染病[J].农业技术与装备.2010.11(22):39-41.
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[9]徐潮,李亮,金芜军,等.荧光RPA技术检测转基因水稻科丰6号[J].分子植物育种.2014,12(5):875-880.
[10]Shen F,Davydova E K,Du W,et al.Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on slipchip[J].Anal Chem,2011,83(9):3533-3540.
[11]吴耀东,徐民俊,,郑文斌,冯胜勇等.重组酶聚合酶扩增技术及其在动物病原快速检测中的应用[J].中国兽医学报,2016,36(10):1797-1801.
[12]中华人民共和国农业部.动物病原微生物分类名录:2005年5月24日农业部令第53号公布.http://www.360doc.com/document/15/0710/18/26295974_484069737.shtml.
[13]SUN Z Y,NING B A,SU P,et al.High-throughput suspension array for detecting four pathogens[J].Anal Methods,2012,8(4):2528-2536.
[14]Abdullah J,Saffie N,Sjasri F A,et al.Rapid detection of Salmonella typhi by loop-mediated isothermal amplification(LAMP)method[J].Braz J Microbiol,2015,45(4):1385-1391.
[15]SN/T 1088-2010.布氏杆菌检疫技术规范[S].
[16]苗玉强.布鲁氏菌免疫磁珠及免疫荧光检测方法的研究[D].长春:吉林农业大学,2016.
[2]中华人民共和国农业部.人畜共患传染病名录:农业部公告第1149号.https://wenku.baidu.com/view/6a783fe90975f46527d3e1f6.html
[3]Tang T,Cheng A,Wang M,et al.Development and clinical verifycation of a loop-medeated isothermal amplification method for detection of Salmonella species in suspect infected ducks[J].Poultry Science,2012,91:979-986.
[4]Masanori W,Junko I,Keiko K,et al.Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan[J].Journal of Clinical Microbiology,2014,52(8):2757-2763.
[5]Simborio H L,Lee J J,Bernardo Reyes A W,et al.Evaluation of the combined use of the recombinant Brucella abortus Omp10,Omp19 and Omp28 proteins for the clinical diagnosis of bovine brucellosis[J].Microb Pathog,2015,83-84C:41-46.
[6]Elizabeth Trembath-Reichert;Abigail Green-Saxena;Victoria J.Orphan.Whole Cell Immunomagnetic Enrichment of Environmental Microbial Consortia Using rRNA-Targeted Magneto-FISH[J].Methods in Enzymology,2013,531(9)21-44.
[7]Fedio W M,Jinneman K C,Yoshitomi K J,et al.Detection of E.coli O157:H7 in raw ground beef by Pathatrix?immunomagneticseparation,real-time PCR and cultural methods[J].International Journal of Food Microbiology,2011,148(2):87-92.
[8]Corchero JL,Villaverde A.Biomedical applications of distally controlled magnetic nanoparticles.Trends Biotechnol,2009,27(8):468-476.
[9]徐潮,李亮,金芜军,等.荧光RPA技术检测转基因水稻科丰6号[J].分子植物育种.2014,12(5):875-880.
[10]Shen F,Davydova E K,Du W,et al.Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on slipchip[J].Anal Chem,2011,83(9):3533-3540.
[11]吴耀东,徐民俊,,郑文斌,冯胜勇等.重组酶聚合酶扩增技术及其在动物病原快速检测中的应用[J].中国兽医学报,2016,36(10):1797-1801.
[12]中华人民共和国农业部.动物病原微生物分类名录:2005年5月24日农业部令第53号公布.http://www.360doc.com/document/15/0710/18/26295974_484069737.shtml.
[13]SUN Z Y,NING B A,SU P,et al.High-throughput suspension array for detecting four pathogens[J].Anal Methods,2012,8(4):2528-2536.
[14]Abdullah J,Saffie N,Sjasri F A,et al.Rapid detection of Salmonella typhi by loop-mediated isothermal amplification(LAMP)method[J].Braz J Microbiol,2015,45(4):1385-1391.
[15]SN/T 1088-2010.布氏杆菌检疫技术规范[S].
[16]苗玉强.布鲁氏菌免疫磁珠及免疫荧光检测方法的研究[D].长春:吉林农业大学,2016.