树鼩粪便中沙门氏菌LAMP检测方法建立及应用
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摘要
目的建立树鼩粪便沙门氏菌的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,并对此方法进行初步应用。方法根据沙门氏菌种属特异性基因inv A(侵袭蛋白基因A)的保守序列,设计合成特异性LAMP引物。分别设定十个反应温度即57℃~66℃和十个反应时间即24~42 min进行优化,并测定本方法的特异性和灵敏度;同时进行普通PCR实验,作为本方法的验证和比较。用建立的LAMP法对91份野生来源的树鼩稀便样品进行检测。结果优化反应条件选出反应温度为62℃,反应时间34 min;该方法对沙门氏菌的灵敏度可达3.36×101CFU/m L,比普通PCR方法高10~100倍;对10种树鼩来源的肠道细菌进行LAMP检测,只有沙门氏菌属的肠炎沙门氏菌和乙型副伤寒沙门氏菌检测为阳性,其余为阴性;对91份野生树鼩粪便样品进行LAMP检测,阳性检出率为20.88%;LAMP法的所有反应可在40 min内完成。结果可通过肉眼观察颜色变化直接判定。结论所建立的LAMP方法具有便捷、快速、灵敏、特异等特点,可用于树鼩粪便中沙门氏菌的大规模快速检测。
Objective To Establish a loop-mediated isothermal amplification( LAMP) assay for detection of Salmonella in fecal samples of tree shrews,and report the result of preliminary application of this method. Methods LAMP primers were designed and synthesized according to the conserved sequence of Salmonella specific gene inv A( invasive protein gene A). To optimize the reaction time and temperature by setting 10 reaction times( 24 to 42 min) and temperature( 57℃ to 66℃) and tested its specificity and sensitivity. At the same time,a conventional PCR test was performed to verify and compare with the LAMP assay. 91 fecal samples of wild-derived tree shrews were detected by theLAMP assay. Results The experimental condition was confirmed as 62℃ and 34 min. The sensitivity of Salmonella was3. 36 × 101 CFU/mL,which was 10 to 100 times higher than that of conventional PCR assay. In 10 kinds of intestinal bacteria for LAMP amplification,Salmonella enteritidis and Salmonella paratyphi B were positive,the others were negative.Among the 91 samples of tree shrew fecal samples detected by the LAMP assay,the positive detection rate was 20. 88%.The LAMP assay can be completed within 40 min,the result can be observed and judged visually by color changes.Conclusions The LAMP assay established in this study is convenient,rapid,sensitive and specific. It can be used as a rapid measure for large-scale detection of Salmonella in feces of tree shrews.
Objective To Establish a loop-mediated isothermal amplification( LAMP) assay for detection of Salmonella in fecal samples of tree shrews,and report the result of preliminary application of this method. Methods LAMP primers were designed and synthesized according to the conserved sequence of Salmonella specific gene inv A( invasive protein gene A). To optimize the reaction time and temperature by setting 10 reaction times( 24 to 42 min) and temperature( 57℃ to 66℃) and tested its specificity and sensitivity. At the same time,a conventional PCR test was performed to verify and compare with the LAMP assay. 91 fecal samples of wild-derived tree shrews were detected by theLAMP assay. Results The experimental condition was confirmed as 62℃ and 34 min. The sensitivity of Salmonella was3. 36 × 101 CFU/mL,which was 10 to 100 times higher than that of conventional PCR assay. In 10 kinds of intestinal bacteria for LAMP amplification,Salmonella enteritidis and Salmonella paratyphi B were positive,the others were negative.Among the 91 samples of tree shrew fecal samples detected by the LAMP assay,the positive detection rate was 20. 88%.The LAMP assay can be completed within 40 min,the result can be observed and judged visually by color changes.Conclusions The LAMP assay established in this study is convenient,rapid,sensitive and specific. It can be used as a rapid measure for large-scale detection of Salmonella in feces of tree shrews.
引文
[1]彭丽萍,陈博文,徐建超,等.食品沙门氏菌检测方法进展[J].中国人兽共患病杂志,1999,16(5):89-91.
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[8]Gao H,Lei Z,Jia J,et al.Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat[J].J Microbiol Methods,2009,77(2):198-201.
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[11]殷安国,匡德宣,李晓飞,等.树鼩模型在人类病毒性疾病研究中的应用进展[J].中国实验动物学报,2014,22(2):86-89.
[12]周广龙,朱勤,李振宇,等.树鼩在眼科学的基础研究进展[J].中国实验动物学报,2015,23(6):652-655.
[13]徐新平,陈红波,贲昆龙.树鼩在医学生物学中的应用[J].中国实验动物学报,2005,13(3):187-190.
[14]刘丽君,余柄廷,胡凝珠,等.树鼩粪便细菌分离培养与鉴定[J].中国比较医学杂志.2015,25(210):64-68.
[15]LaM ontagne MG,Michel FC Jr,Holden PA,et al.Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis[J].J Microbiol Methods,2002,49(3):255-264.
[16]骞宇,赵欣.大鼠粪便中细菌基因组DNA提取方法的比较[J].食品工业科技,2014,35(4):166-169.
[17]Rahn K,De Grandis SA,Clarke RC,et al.Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J].Mol Cell Probes,1992,6(4):271-279.
[18]Yang Q,Domesle KJ,Wang F,et al.Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time[J].BMC Microbiol,2016,16(1):112.
[19]GB4789.4-2010,食品微生物学检验沙门氏菌检验[S].
[20]Eriksson E,Aspan A.Comparison of culture,ELISA and PCR techniques for Salmonella detection in faecal samples for cattle,pig and poultry[J].BMC Vet Res,2007,3:21.
[21]张凤玉,胡丹,吕恒,等.2型猪链球菌89K毒力岛Ⅳ型分泌系统LAMP检测方法的建立[J].中国病原生物学杂志,2014,9(2):113-116,121.
[22]熊春蓉,殷旭仁,宋丽君,等.环介导同温DNA扩增法(LAMP)与解剖-显微镜检法检测血吸虫感染性钉螺效果的比较[J].中国病原生物学杂志,2014,9(12):1084-1087.
[23]Ashida H,Toyotome T,Nagai T,et al.Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors[J].Mol Microbiol,2007,63(3):680-693.
[24]Galán JE,Curtiss R.Distribution of the invA,-B,-C,and-D genes of Salmonella typhimurium among other Salmonella serovars:invA mutants of Salmonella typhi are deficient for entry into mammalian cells[J].Infect Immun,1991,59(9),2901-2908.
[25]Zhuang L,Gong J,Li Q,et al.Detection of Salmonella spp.by a loop-mediated isothermal amplification(LAMP)method targeting bcfD gene[J].Lett Appl Microbiol.2014,59(6):658-664.
[26]Boyd EF,Hartl DL.Analysis of the type 1 Pilin gene cluster fim in Salmonella:its distinct evolutionary histories in the 5’and 3’regions[J].J Bacteriol,1999,181(4):1301-1308.
[27]吴家林,沙丹,马广源,等.沙门氏菌LAMP检测方法的建立[J].中国病原生物学杂志,2015,10(7):611-614.
[28]邱索平,林志雄,游勇来,等.实验猴粪便样品中沙门氏菌LAMP检测方法的建立及应用[J].中国畜牧兽医,2014,41(1):51-56.
[29]马晨,陈雪华,李建国.国标法与快速法检测沙门氏菌的结果比较[J].食品科技,2015,40(9):276-282.
[30]邢进,冯育芳,付瑞,等.野生树鼩可培养细菌和真菌携带情况的调查[J]实验动物科学,2012,29(3):34-38.
[31]高家红,江勤芳,罗志武,等.树鼩正常肠道细菌的培养分离鉴定及其药敏试验研究[J].中国比较医学杂志,2009,19(12):24-26,34.
[2]徐桂云,樊世杰.家禽沙门氏菌感染现状及不同国家的防治策略[J].中国家禽,2012,34(9):7-12.
[3]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):e63.
[4]Nagamine K,Hase T,Notomi T,et al.Accelerated reaction by loop mediated isothermal amplification using loop primers[J].Mol Cell Probes,2002,16(3):223-229.
[5]Fukuta S,Ohishi K,Yoshida K,et al.Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum[J].J Virol Methods,2004,121(1):49-55.
[6]Mori Y,Nagamine K,Tomita N,et al.Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J].Biochem Biophys Res Commun,2001,289(1):150-154.
[7]Tomita N,Mori Y,Kanda H,et al.Loop-mediated isothermal amplification(LAMP)of gene sequences and simple visual detection of products[J].Nat Protoc,2008,3(5):877-882.
[8]Gao H,Lei Z,Jia J,et al.Application of loop-mediated isothermal amplification for detection of Yersinia enterocolitica in pork meat[J].J Microbiol Methods,2009,77(2):198-201.
[9]Lu CC,Dai TT,Zhang HF,et al.Development of a loop-mediated isothermal amplification assay to detect Fusarium oxysporum[J].J Phytopathol,2015,163(1):63-66.
[10]戴婷婷,陆辰晨,郑小波,等.环介导等温扩增技术在病原物检测上的应用研究进展[J].南京农业大学学报,2015,38(5):695-703.
[11]殷安国,匡德宣,李晓飞,等.树鼩模型在人类病毒性疾病研究中的应用进展[J].中国实验动物学报,2014,22(2):86-89.
[12]周广龙,朱勤,李振宇,等.树鼩在眼科学的基础研究进展[J].中国实验动物学报,2015,23(6):652-655.
[13]徐新平,陈红波,贲昆龙.树鼩在医学生物学中的应用[J].中国实验动物学报,2005,13(3):187-190.
[14]刘丽君,余柄廷,胡凝珠,等.树鼩粪便细菌分离培养与鉴定[J].中国比较医学杂志.2015,25(210):64-68.
[15]LaM ontagne MG,Michel FC Jr,Holden PA,et al.Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis[J].J Microbiol Methods,2002,49(3):255-264.
[16]骞宇,赵欣.大鼠粪便中细菌基因组DNA提取方法的比较[J].食品工业科技,2014,35(4):166-169.
[17]Rahn K,De Grandis SA,Clarke RC,et al.Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J].Mol Cell Probes,1992,6(4):271-279.
[18]Yang Q,Domesle KJ,Wang F,et al.Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time[J].BMC Microbiol,2016,16(1):112.
[19]GB4789.4-2010,食品微生物学检验沙门氏菌检验[S].
[20]Eriksson E,Aspan A.Comparison of culture,ELISA and PCR techniques for Salmonella detection in faecal samples for cattle,pig and poultry[J].BMC Vet Res,2007,3:21.
[21]张凤玉,胡丹,吕恒,等.2型猪链球菌89K毒力岛Ⅳ型分泌系统LAMP检测方法的建立[J].中国病原生物学杂志,2014,9(2):113-116,121.
[22]熊春蓉,殷旭仁,宋丽君,等.环介导同温DNA扩增法(LAMP)与解剖-显微镜检法检测血吸虫感染性钉螺效果的比较[J].中国病原生物学杂志,2014,9(12):1084-1087.
[23]Ashida H,Toyotome T,Nagai T,et al.Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors[J].Mol Microbiol,2007,63(3):680-693.
[24]Galán JE,Curtiss R.Distribution of the invA,-B,-C,and-D genes of Salmonella typhimurium among other Salmonella serovars:invA mutants of Salmonella typhi are deficient for entry into mammalian cells[J].Infect Immun,1991,59(9),2901-2908.
[25]Zhuang L,Gong J,Li Q,et al.Detection of Salmonella spp.by a loop-mediated isothermal amplification(LAMP)method targeting bcfD gene[J].Lett Appl Microbiol.2014,59(6):658-664.
[26]Boyd EF,Hartl DL.Analysis of the type 1 Pilin gene cluster fim in Salmonella:its distinct evolutionary histories in the 5’and 3’regions[J].J Bacteriol,1999,181(4):1301-1308.
[27]吴家林,沙丹,马广源,等.沙门氏菌LAMP检测方法的建立[J].中国病原生物学杂志,2015,10(7):611-614.
[28]邱索平,林志雄,游勇来,等.实验猴粪便样品中沙门氏菌LAMP检测方法的建立及应用[J].中国畜牧兽医,2014,41(1):51-56.
[29]马晨,陈雪华,李建国.国标法与快速法检测沙门氏菌的结果比较[J].食品科技,2015,40(9):276-282.
[30]邢进,冯育芳,付瑞,等.野生树鼩可培养细菌和真菌携带情况的调查[J]实验动物科学,2012,29(3):34-38.
[31]高家红,江勤芳,罗志武,等.树鼩正常肠道细菌的培养分离鉴定及其药敏试验研究[J].中国比较医学杂志,2009,19(12):24-26,34.