实时荧光环介导等温扩增技术检测土壤中的类鼻疽伯克霍尔德菌
|
摘要
目的结合荧光染料EvaGreen与环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)探索快速检测类鼻疽伯克霍尔德菌的可行性。方法根据类鼻疽伯克霍尔德菌filc基因片段设计了LAMP引物,利用荧光染料EvaGreen建立实时荧光环介导等温扩增技术(real-time fluorescence loop-mediated isothermal amplification,RealAmp),优化反应条件,扩增产物经电泳鉴定。通过类鼻疽伯克霍尔德菌和其他致病菌验证特异性,比较RealAmp与普通LAMP技术的敏感度,并测定人工污染土壤模拟样本的检出限。结果恒温63℃条件下,20～60 min即可完成RealAmp检测;利用该方法检测类鼻疽伯克霍尔德菌为阳性,其余致病菌如鼠疫杆菌、布鲁氏杆菌、大肠杆菌等13株参考菌株及蜱虫的DNA(含大量假单胞菌属细菌)呈扩增阴性;RealAmp技术检测类鼻疽伯克霍尔德菌核酸的灵敏度为10~2 CFU/mL,检测克隆株质粒的灵敏度可达10~1 copies/μL,比普通LAMP技术的灵敏度高10倍;人工污染土壤模拟样本RealAmp的检出限为4.4×10~1 CFU/g,且20 min左右即可判定结果;直接检测24份浓度为4.4×10~1 CFU/g～4.4×10~8 CFU/g的类鼻疽伯克霍尔德菌人工污染土壤样本,在检出限以上,检出率同荧光定量PCR一致。结论该方法特异性强、灵敏度高,且可实时监测类鼻疽伯克霍尔德菌,有望成为快速检测类鼻疽伯克霍尔德菌的有效方法,增强抵御生物恐怖战争的能力。
The purpose of this study is using fluorescent dye EvaGreen and loop-mediated isothermal amplification technology to explore the feasibility of the rapid detection of Burkholderia pseudomallei. The specific LAMP primers designed were based on the filc gene of Burkholderia pseudomallei, and the best reaction conditions with EvaGreen inside for the real-time fluorescence loop-mediated isothermal amplification were optimized,and the products of amplification were detected by electrophoresis. Comparison of the sensitivity between RealAmp and general LAMP technology was conducted, and the detection limits of artificial contaminated soil was evaluated. The results showed that the RealAmp detection could be completed in 20-60 min under the condition of isothermality of 63 ℃; RealAmp is positive to detect Burkholderia pseudomallei but negative for others pathogenic bacteria such as Yersinia pestis, Brucella, Escherichia coli and other 13 strains of standard strains and the DNA from tick(including a large number of pseudomonas bacteria); the detection limita of Burkholderia pseudomallei with RealAmp technology is 10~2 CFU/mL for nucleic acid, and 10~1 copies/μL for cloning plasmid, which is 10 times higher than those of general LAMP technology. The detection limit by RealAmp in the artificial contaminated soil is 44 CFU/g, and can be determined by the reation in 20 min. The efficiency by RealAmp is in accord with that by the fluorescent quantitative PCR based on the detection of 24 samples of soil contaminated by Burkholderia pseudomallei with concentration gradient 4.4×10~1 CFU/g to 4.4×10~8 CFU/g. It that due to the high specificity and sensibility on Burkholderia pseudomallei, RealAmp can be a potential method for the fast detection of Burkholderia Pseudomallei, and decreasing the risk of biological terror.
The purpose of this study is using fluorescent dye EvaGreen and loop-mediated isothermal amplification technology to explore the feasibility of the rapid detection of Burkholderia pseudomallei. The specific LAMP primers designed were based on the filc gene of Burkholderia pseudomallei, and the best reaction conditions with EvaGreen inside for the real-time fluorescence loop-mediated isothermal amplification were optimized,and the products of amplification were detected by electrophoresis. Comparison of the sensitivity between RealAmp and general LAMP technology was conducted, and the detection limits of artificial contaminated soil was evaluated. The results showed that the RealAmp detection could be completed in 20-60 min under the condition of isothermality of 63 ℃; RealAmp is positive to detect Burkholderia pseudomallei but negative for others pathogenic bacteria such as Yersinia pestis, Brucella, Escherichia coli and other 13 strains of standard strains and the DNA from tick(including a large number of pseudomonas bacteria); the detection limita of Burkholderia pseudomallei with RealAmp technology is 10~2 CFU/mL for nucleic acid, and 10~1 copies/μL for cloning plasmid, which is 10 times higher than those of general LAMP technology. The detection limit by RealAmp in the artificial contaminated soil is 44 CFU/g, and can be determined by the reation in 20 min. The efficiency by RealAmp is in accord with that by the fluorescent quantitative PCR based on the detection of 24 samples of soil contaminated by Burkholderia pseudomallei with concentration gradient 4.4×10~1 CFU/g to 4.4×10~8 CFU/g. It that due to the high specificity and sensibility on Burkholderia pseudomallei, RealAmp can be a potential method for the fast detection of Burkholderia Pseudomallei, and decreasing the risk of biological terror.
引文
[1] 蒋忠军,宋阳,陆振豸.我国类鼻疽研究的历史与现状[J].热带医学杂志,2002,2(2):176-180. DOI:10.3969/j.issn.1672-3619.2002.02.026
[2] Pumpuang A,Chantratita N,Wikraiphat C, et al.Survival of Burkholderia pseudomallei in distilled water for 16 years[J].Trans R Soc Trop Med Hyg,2011,105(10):598-600. DOI;10.1016/j.trstmh.2011.06.004
[3] Karuppal R,Marthya A,Justus S,et al.Burkholderia pseudomallei- a rare cause for septic arthritis[J].Joint Bone Spine,2007,74(6):659-660. DOI:10.1016/j.jbspin.2007.01.040
[4] Cruz-Migoni A,Ruzheinikov SN,Sedelnikova SE,et al.Cloning, purification and crystallographic analysis of a hypothetical protein, BPSL1549, from Burkholderia pseudomallei[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2011,67(12):1623-1626. DOI: 10.1107/S1744309111028995
[5] 刘青芹,段雄波,李金钟.类鼻疽伯克霍尔德菌分离鉴定进展[J].中国人兽共患病学报,2008,24(10):970-973. DOI:10.3969/j.issn.1002-2694.2008.10.021
[6] Howard K,Inglis TJ.Novel selective medium for isolation of Burkholderia pseudomallei[J].J Clin Microbiol,2003,41(7):3312-3316. DOI:10.1128/JCM.41.7.3312-3316.2003
[7] Glass MB,Popovic T.Preliminary evaluation of the API 20NE and RapID NF Plus Systems for rapid identification of Burkholderia pseudomallei and B. mallei [J].J Clin Microbiol,2005,43(1):479-483. DOI:10.1128/JCM.43.1.479-483.2005
[8] Anuntagool N,Naigowit P,Petkanchanapong V,et al.Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia [J].J Med Microbiol,2000,49(12):1075-1078. DOI:10.1099/0022-1317-49-12-1075
[9] Cheng AC,Currie BJ.Melioidosis: epidemiology, pathophysiology, and management [J].Clin Microbiol Rev,2005,18(2):383-416. DOI:10.1128/CMR.18.2.383-416.2005
[10] Inglis TJ,Aravena-Roman M,Ching S, et al.Cellular fatty acid profile distinguishes Burkholderia pseudomallei from avirulent Burkholderia thailandensis [J].J Clin Microbiol,2003,41(10):4812-4814. DOI:10.1128/JCM.41.10.4812-4814.2003
[11] Gee JE,Sacchi CT,Glass MB, et al.Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei [J].J Clin Microbiol,2003,41(10):4647-4654. DOI:10.1128/JCM.41.10.4647-4654.2003
[12] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA [J].Nucleic Acids Res,2000,28(12):E63. DOI:10.1093/nar/28.12.e63
[13] 李志强.LAMP技术在微生物检测中的应用[J].生命科学仪器,2009,07(7):7-10. DOI:10.3969/j.issn.1671-7929.2009.08.001
[14] 于波,方瑶,顾江,等.类鼻疽杆菌鞭毛蛋白FliC与EHEC O157∶H7 EspA蛋白的融合表达及其免疫学性质研究[J].免疫学杂志, 2013,29(3):251-255. DOI:10.13431/j.cnki.immunol.j.20130056
[15] 傅琼瑶,陈春羽,邬娇,等.一种快速定量检测类鼻疽伯克霍尔德菌的方法建立及评价[J].第三军医大学学报,2015,37(17):1734-1738. DOI:10.16016/j.1000-5404.201412143
[16] Suputtamongkol Y,Chaowagul W,Chetchotisakd P,et al.Risk factors for melioidosis and bacteremic melioidosis[J].Clin Infect Dis,1999,29(2):408-413. DOI:10.1086/520223
[2] Pumpuang A,Chantratita N,Wikraiphat C, et al.Survival of Burkholderia pseudomallei in distilled water for 16 years[J].Trans R Soc Trop Med Hyg,2011,105(10):598-600. DOI;10.1016/j.trstmh.2011.06.004
[3] Karuppal R,Marthya A,Justus S,et al.Burkholderia pseudomallei- a rare cause for septic arthritis[J].Joint Bone Spine,2007,74(6):659-660. DOI:10.1016/j.jbspin.2007.01.040
[4] Cruz-Migoni A,Ruzheinikov SN,Sedelnikova SE,et al.Cloning, purification and crystallographic analysis of a hypothetical protein, BPSL1549, from Burkholderia pseudomallei[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2011,67(12):1623-1626. DOI: 10.1107/S1744309111028995
[5] 刘青芹,段雄波,李金钟.类鼻疽伯克霍尔德菌分离鉴定进展[J].中国人兽共患病学报,2008,24(10):970-973. DOI:10.3969/j.issn.1002-2694.2008.10.021
[6] Howard K,Inglis TJ.Novel selective medium for isolation of Burkholderia pseudomallei[J].J Clin Microbiol,2003,41(7):3312-3316. DOI:10.1128/JCM.41.7.3312-3316.2003
[7] Glass MB,Popovic T.Preliminary evaluation of the API 20NE and RapID NF Plus Systems for rapid identification of Burkholderia pseudomallei and B. mallei [J].J Clin Microbiol,2005,43(1):479-483. DOI:10.1128/JCM.43.1.479-483.2005
[8] Anuntagool N,Naigowit P,Petkanchanapong V,et al.Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia [J].J Med Microbiol,2000,49(12):1075-1078. DOI:10.1099/0022-1317-49-12-1075
[9] Cheng AC,Currie BJ.Melioidosis: epidemiology, pathophysiology, and management [J].Clin Microbiol Rev,2005,18(2):383-416. DOI:10.1128/CMR.18.2.383-416.2005
[10] Inglis TJ,Aravena-Roman M,Ching S, et al.Cellular fatty acid profile distinguishes Burkholderia pseudomallei from avirulent Burkholderia thailandensis [J].J Clin Microbiol,2003,41(10):4812-4814. DOI:10.1128/JCM.41.10.4812-4814.2003
[11] Gee JE,Sacchi CT,Glass MB, et al.Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei [J].J Clin Microbiol,2003,41(10):4647-4654. DOI:10.1128/JCM.41.10.4647-4654.2003
[12] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA [J].Nucleic Acids Res,2000,28(12):E63. DOI:10.1093/nar/28.12.e63
[13] 李志强.LAMP技术在微生物检测中的应用[J].生命科学仪器,2009,07(7):7-10. DOI:10.3969/j.issn.1671-7929.2009.08.001
[14] 于波,方瑶,顾江,等.类鼻疽杆菌鞭毛蛋白FliC与EHEC O157∶H7 EspA蛋白的融合表达及其免疫学性质研究[J].免疫学杂志, 2013,29(3):251-255. DOI:10.13431/j.cnki.immunol.j.20130056
[15] 傅琼瑶,陈春羽,邬娇,等.一种快速定量检测类鼻疽伯克霍尔德菌的方法建立及评价[J].第三军医大学学报,2015,37(17):1734-1738. DOI:10.16016/j.1000-5404.201412143
[16] Suputtamongkol Y,Chaowagul W,Chetchotisakd P,et al.Risk factors for melioidosis and bacteremic melioidosis[J].Clin Infect Dis,1999,29(2):408-413. DOI:10.1086/520223