LAMP技术用于幽门螺旋杆菌检测与分型的研究
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摘要
目的建立环介导等温扩增(LAMP)体系用于检测唾液和胃液中的幽门螺旋杆菌(Hp)及基因分型。方法收集临床Hp阳性标本,应用荧光定量PCR进行分型检测,即分为Ⅰ型和Ⅱ型。分别根据Hp,HpⅠ型设计LAMP引物并优化反应条件,通过LAMP检测验证这3套引物的特异性,并通过检测稀释后模板验证引物的灵敏度,同时加入钙黄绿素使产物显色,使Hp分型检测可视化。用LAMP方法检测收集的临床标本,计算Hp阳性率,并与荧光定量PCR法相比较。结果建立的LAMP体系特异性良好,对Hp、HpⅠ和HpⅡ型检测的灵敏度均为102 IU/ml。利用钙黄绿素进行产物检测的效果和电泳结果相一致。对临床标本进行Hp分型检测,HpⅠ、HpⅡ型LAMP结果与荧光定量PCR法比较差异均有统计学意义(P<0.05)。结论 LAMP对Hp的检测与分型均具有较高的特异性和灵敏度,且操作简便,在基层医院具有良好的应用前景。
Objective A loop-mediated isothermal amplification(LAMP)system was created to detect Helicobacter pylori and genotype H.pylori from saliva and gastric juice. Methods Clinical specimens positive for H.pylori were collected and typed using fluorescence quantitative PCR.H.pylori was divided into type I and typeⅡ.LAMP primers were designed in accordance with H.pylori and H.pylori I,and reaction conditions were optimized.The specificity of the three primers was verified using a LAMP assay,and the sensitivity of the primers was verified by detecting the diluted template.Calcein was added to color the products in order to allow visualization of H.pylori typing.The visual typing of H.pylori can also be achieved by staining with hydroxynaphthol blue.The clinical specimens collected were tested using LAMP,and the rate of H.pylori detection was calculated.The results were compared to those of fluorescent quantitative PCR.The advantages and disadvantages of the two detection methods were compared. Results The LAMP system had a high level of specificity,and its sensitivity to H.pylori,H.pylori I,and H.pyloriⅡ was 102 IU/ml.Detection of products using calcein was consistent with the results of electrophoresis.H.pylori typing was performed on clinical specimens.There were significant differences between H.pylori I and H.pylori II LAMP results and fluorescence quantitative PCR(P<0.05).H.pylori type I was detected with LAMP at a rate of 96.07%and type II was detected at a rate of 93.33%.Compared to fluorescence quantitative PCR,LAMP had a higher Kappa value and greater consistency.Conclusion LAMP has a high level of specificity and sensitivity in the detection and typing of H.pylori,and it is easy to perform.LAMP has promise for use in primary hospitals.
Objective A loop-mediated isothermal amplification(LAMP)system was created to detect Helicobacter pylori and genotype H.pylori from saliva and gastric juice. Methods Clinical specimens positive for H.pylori were collected and typed using fluorescence quantitative PCR.H.pylori was divided into type I and typeⅡ.LAMP primers were designed in accordance with H.pylori and H.pylori I,and reaction conditions were optimized.The specificity of the three primers was verified using a LAMP assay,and the sensitivity of the primers was verified by detecting the diluted template.Calcein was added to color the products in order to allow visualization of H.pylori typing.The visual typing of H.pylori can also be achieved by staining with hydroxynaphthol blue.The clinical specimens collected were tested using LAMP,and the rate of H.pylori detection was calculated.The results were compared to those of fluorescent quantitative PCR.The advantages and disadvantages of the two detection methods were compared. Results The LAMP system had a high level of specificity,and its sensitivity to H.pylori,H.pylori I,and H.pyloriⅡ was 102 IU/ml.Detection of products using calcein was consistent with the results of electrophoresis.H.pylori typing was performed on clinical specimens.There were significant differences between H.pylori I and H.pylori II LAMP results and fluorescence quantitative PCR(P<0.05).H.pylori type I was detected with LAMP at a rate of 96.07%and type II was detected at a rate of 93.33%.Compared to fluorescence quantitative PCR,LAMP had a higher Kappa value and greater consistency.Conclusion LAMP has a high level of specificity and sensitivity in the detection and typing of H.pylori,and it is easy to perform.LAMP has promise for use in primary hospitals.
引文
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[12] Bakhtiari S,Alvandi A,Pajavand H,et al.Development and di-agnostic evaluation of loop-mediated isothermal amplification u-sing a new gene target for rapid detection of Helicobacter pylori[J].Jundishapur J Microbio,2016,9(5):e28831.
[13] Yari F,Abiri R,Aryan E,et al.Loop-mediated isothermal am-plification as a fast noninvasive method of Helicobacter pylori Di-agnosis[J].J Clin Lab Anal,2016,30(5):464-70.
[14] 韩慧,胡子有,姚芳,等.环介导等温扩增技术检测粪便幽门螺杆菌方法的建立及应用[J].实用医学杂志,2011,27(19):3485-7.
[15] Martineau RL,Murray SA,Ci S,et al.Improved Performanceof loop-mediated isothermal amplification assays via swarm prim-ing[J].Anal Chem,2017,89(1):625-32.
[16] 张亚云,徐智渊,赵大鹏,等.幽门螺杆菌Caga通过B-Raf和JNK2调节原癌蛋白CIP2A表达及其细胞生物学作用研究[J].中国病原生物学杂志,2017,12(12):1148-51.
[17] Aryan E,Makvandi M,Farajzadeh A,et al.Clinical value ofIS6110-based loop-mediated isothermal amplification for detectionof Mycobacterium tuberculosis complex in respiratory specimens[J].J Infect,2013,66(6):487-93.
[18] Minami M,Ohta M,Ohkura T,et al.Use of a combination ofbrushing technique and the loop-mediated isothermal amplificationmethod as a novel,rapid,and safe system for detection of Heli-cobacter pylori[J].J Clin Microbiol,2006,44(11):4032-7.
[19] Yano A,Ishimaru R,Hujikata R.Rapid and sensitive detectionof heat-labile I and heat-stable I enterotoxin genes of enterotoxi-genic Escherichia coli by loop-mediated isothermal amplification[J].J Microbiol Methods,2007,68(2):414-20.
[2] Roy RK,Hoppe MM,Srivastava S,et al.CEACAM6is upregu-lated by Helicobacter pylori Caga and is a biomarker for earlygastric cancer[J].Oncotarget,2016,7(34):55290-301.
[3] Agudo S,Perez-Perez G,Alarcon T,et al.High prevalence ofclarithromycin-resistant Helicobacter pylori strains and risk fac-tors associated with resistance in Madrid,Spain[J].J Clin Micro-biol,2010,48(10):3703-7.
[4] Palframan SL,Kwok T,Gabriel K.Vacuolating cytotoxin A(Vaca),a key toxin for Helicobacter pylori pathogenesis[J].Front Cell Infect Microbiol,2012,2:92.
[5] Atherton JC.The pathogenesis of Helicobacter pylori-inducedgastro-duodenal diseases[J].Annu Rev Pathol,2006(1):63-96.
[6] Odenbreit S,Puls J,Sedlmaier B,et al.Translocation of Helico-bacter pylori Caga into gastric epithelial cells by type IV secretion[J].Science,2000,287(5457):1497-500.
[7] Kwok T,Zabler D,Urman S,et al.Helicobacter exploits inte-grin for type IV secretion and kinase activation.[J]Nature,2007,449(7164):862-6.
[8] Caliendo AM,Valsamakis A,Bremer JW,et al.Multilaboratoryevaluation of real-time PCR tests for hepatitis B virus DNA quan-tification[J].J Clin Microbio,2011,49(8):2854-8.
[9] 赵娜,刘金霞,孙殿兴.环介导等温扩增技术在临床检测中的优势与展望[J].中国病原生物学杂志,2016,11(4):381-4.
[10] 杨德全,鞠厚斌,葛菲菲,等.环介导等温扩增技术及其在动物疫病诊断中的应用[J].中国动物传染病学报,2011(2):75-81.
[11] Amiri N,Abiri R,Eyvazi M,et al.The frequency of Helico-bacter pylori in dental plaque is possibly underestimated[J].ArchOral Biol,2015,60(5):782-8.
[12] Bakhtiari S,Alvandi A,Pajavand H,et al.Development and di-agnostic evaluation of loop-mediated isothermal amplification u-sing a new gene target for rapid detection of Helicobacter pylori[J].Jundishapur J Microbio,2016,9(5):e28831.
[13] Yari F,Abiri R,Aryan E,et al.Loop-mediated isothermal am-plification as a fast noninvasive method of Helicobacter pylori Di-agnosis[J].J Clin Lab Anal,2016,30(5):464-70.
[14] 韩慧,胡子有,姚芳,等.环介导等温扩增技术检测粪便幽门螺杆菌方法的建立及应用[J].实用医学杂志,2011,27(19):3485-7.
[15] Martineau RL,Murray SA,Ci S,et al.Improved Performanceof loop-mediated isothermal amplification assays via swarm prim-ing[J].Anal Chem,2017,89(1):625-32.
[16] 张亚云,徐智渊,赵大鹏,等.幽门螺杆菌Caga通过B-Raf和JNK2调节原癌蛋白CIP2A表达及其细胞生物学作用研究[J].中国病原生物学杂志,2017,12(12):1148-51.
[17] Aryan E,Makvandi M,Farajzadeh A,et al.Clinical value ofIS6110-based loop-mediated isothermal amplification for detectionof Mycobacterium tuberculosis complex in respiratory specimens[J].J Infect,2013,66(6):487-93.
[18] Minami M,Ohta M,Ohkura T,et al.Use of a combination ofbrushing technique and the loop-mediated isothermal amplificationmethod as a novel,rapid,and safe system for detection of Heli-cobacter pylori[J].J Clin Microbiol,2006,44(11):4032-7.
[19] Yano A,Ishimaru R,Hujikata R.Rapid and sensitive detectionof heat-labile I and heat-stable I enterotoxin genes of enterotoxi-genic Escherichia coli by loop-mediated isothermal amplification[J].J Microbiol Methods,2007,68(2):414-20.