Development and application of loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for rapid detection of Leptospira interrogans serogroup icterohaemorrhagiae
-
摘要:
目的 基于环介导恒温扩增(LAMP)结合纳米生物传感条(LFB)技术,建立并评价黄疸出血群钩端螺旋体(钩体)的LAMP-LFB快速检测方法。 方法 以黄疸出血群钩体O抗原基因簇中的糖基转移酶基因(gtf)为检测靶标并设计特异性LAMP引物。 通过对LAMP引物的特定标记(FIP-FAM和LF-Biotin)和优化试验,建立LAMP-LFB快速检测方法,并分析其灵敏度和特异性。 应用建立的LAMP-LFB、普通PCR方法和显微凝集试验(MAT),分别对53株钩体分离菌株进行鉴定,比较分析鉴定结果并评价LAMP-LFB方法的实用性。 结果 灵敏度和特异性试验显示,LAMP-LFB方法可检出黄疸出血群赖型56601基因组DNA的最低浓度为100 fg/µL,检测特异性为100%,与其余血清群和非钩体菌株核酸无交叉反应。 菌株鉴定结果显示,LAMP-LFB与MAT鉴定结果完全一致,符合率为100%,其敏感性高于PCR方法。 此外,LAMP扩增产物经LFB验证,可直接通过观察检测线(TL)和质控线(CL)进行结果判定。 结论 基于gtf基因建立的LAMP-LFB检测技术方便、快捷且重复性好,可快速、灵敏、特异地鉴定黄疸出血群钩体菌株,能够作为有价值的黄疸出血群钩体菌株快速鉴别或诊断方法。 Abstract:Objective To develop and evaluate a rapid detection assay for Leptospira interrogans serogroup icterohaemorrhagiae based on loop-mediated isothermal amplification (LAMP) combined with nanoparticle-based lateral flow biosensor (LFB). Methods The LAMP primers targeting the glycotransferase gene (gtf) in the O-antigen gene cluster of L.serogoup icterohaemorrhagiae were designed. After specific label of primers (FIP-FAM and LF-Biotin) and condition optimization, we evaluated the sensitivity, specificity and feasibility of gtf-LAMP-LFB assay. A total of 53 strains of Leptospira were identified by using LAMP-LFB assay, PCR and microscopic agglutination test (MAT), and the results were analyzed to evaluate the practicality of LAMP-LFB assay. Results Our data showed that the detection limit of the assay was 100 fg/μL for genomic DNA of reference strain of L. icterohaemorrhagiae (56601), and the specificity was 100% because there were no cross reactions with nucleic acids of other Leptospira serogroups and non-Leptospira strains. For the application examination, LAMP-LFB and MAT identification results were completely consistent, while the sensitivity of LAMP-LFB assay was higher than PCR. In addition, the color of CL and TL bands could be observed directly to determine the results by using LFB to detect LAMP amplicons. Conclusion The LAMP-LFB assay developed based on LAMP technique has high repeatability, sensitivity and specificity, which can be used for the rapid and accurate identification of the strains of L.interrogans serogroup icterohaemorrhagiae, and can be used as a potential screening and diagnosis tool for L.interrogans serogroup icterohaemorrhagiae. -
图 1 环介导恒温扩增结合纳米生物传感条技术检测黄疸出血群钩端螺旋体的灵敏度
注:A. LFB生物传感条验证;B. MG 可视化反应;C. 1.5% 琼脂糖凝胶电泳;D. 实时浊度仪监测;A/B/C/D中1~8. 10 ng/µL、1 ng/µL、100 pg/µL、10 pg/µL、1 pg/µL、100 fg/µL、10 fg/µL、1 fg/µL;9. 空白对照(水);M. 100 bp ladder
Figure 1. Sensitivity of LAMP-LFB assay for detection of Leptospira interrogans serogroup icterohaemorrhagiae
图 2 环介导恒温扩增结合纳米生物传感条技术检测黄疸出血群钩端螺旋体的特异性
注:1. 黄疸出血群赖型56601;2~5. 黄疸出血群分离菌株;6. 爪哇群56602;7. 犬群56603;8. 拜伦群56604;9. 致热群56605;10. 秋季群56606;11. 澳洲群56607;12. 波摩那群56608;13. 流感伤寒群56609;14. 七日热群56610;15. 赛罗群56612;16. 明尼群56613;17. 巴达维亚群56615;18. 塔拉索夫群56635;19. 曼耗群56655;20. 双曲钩体57601;21~26. 结核分枝杆菌(H37Rv)、牛分枝杆菌、沙门菌、肺炎克雷伯菌、单核细胞增生李斯特菌、大肠埃希菌;27. 阴性对照
Figure 2. Specificity of LAMP-LFB assay for detection of Leptospira interrogans serogroup icterohaemorrhagiae
表 1 用于扩增gtf基因的引物
Table 1. Primers used for amplification of gene gtf
用途 名称 序列(5′~3′) 长度(bp) LAMP F3 GAAAACAAAATATTCTATTGGGC 23 B3 CTCAGAGGAATTTAAATGGTT 21 FIP* FAM-CCTCGAGTACATTCCATTGATGTTT-AAAAAACGACCGATAGAATTGC 47 BIP GAGTGATTTAAATTGTGCAGGGATT-GAAAAGTTTATTTACGGAAGCT 47 LF* Biotin-ATATGCACCAATAAATGAAACA 22 LB GCTTGGTTGGTTCTCATAA 19 PCR F CAGGATTTATCGGTGGTACT 20 R TTACGAAAAGAAGTTCCTTGAA 22 注:FAM为6-羧基荧光素 表 2 53株钩端螺旋体分离株的鉴定结果
Table 2. Identification results of 53 isolates of Leptospira
检测方法 阳性 阴性 PCR 49 4 LAMP-LFB 53 0 MAT 53 0 -
[1] 严杰, 戴保民, 于恩庶. 钩端螺旋体病学[M]. 3版. 北京: 人民卫生出版社, 2006: 1–3.Yan J, Dai BM, Yu ES. Leptospirosis[M]. 3rd ed. Beijing: People's Medical Publishing House, 2006: 1–3. [2] Han HJ, Wen HL, Liu JW, et al. Pathogenic Leptospira species in insectivorous bats, China, 2015[J]. Emerg Infect Dis, 2018, 24(6): 1123–1126. DOI: 10.3201/eid2406.171585. [3] Zhang CC, Xu JM, Zhang TL, et al. Genetic characteristics of pathogenic Leptospira in wild small animals and livestock in Jiangxi province, China, 2002–2015[J]. PLoS Negl Trop Dis, 2019, 13(6): e0007513. DOI: 10.1371/journal.pntd.0007513. [4] Zhang CL, Wang H, Yan J. Leptospirosis prevalence in Chinese populations in the last two decades[J]. Microbes Infect, 2012, 14(4): 317–323. DOI: 10.1016/j.micinf.2011.11.007. [5] Erol E, Jackson CB, Steinman M, et al. A diagnostic evaluation of real-time PCR, fluorescent antibody and microscopic agglutination tests in cases of equine leptospiral abortion[J]. Equine Vet J, 2015, 47(2): 171–174. DOI: 10.1111/evj.12281. [6] Stoddard RA, Gee JE, Wilkins PP, et al. Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene[J]. Diagn Microbiol Infect Dis, 2009, 64(3): 247–255. DOI: 10.1016/j.diagmicrobio.2009.03.014. [7] Nurul Najian AB, Engku Nur Syafirah EAR, Ismail N, et al. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira[J]. Anal Chim Acta, 2016, 903: 142–148. DOI: 10.1016/j.aca.2015.11.015. [8] Suwancharoen D, Kulchim C, Chirathaworn C, et al. Development of a novel primer combination to detect pathogenic Leptospira by loop-mediated isothermal amplification[J]. J Microbiol Methods, 2012, 91(1): 171–173. DOI: 10.1016/j.mimet.2012.08.008. [9] Li SJ, Jiang WJ, Huang JF, et al. Highly sensitive and specific diagnosis of COVID-19 by reverse transcription multiple cross-displacement amplification-labelled nanoparticles biosensor[J]. Eur Respir J, 2020, 56(6): 2002060. DOI: 10.1183/13993003.02060−2020. [10] Li SJ, Liu Y, Chen X, et al. Visual and rapid detection of Leptospira interrogans using multiple cross-displacement amplification coupled with nanoparticle-based lateral flow biosensor[J]. Vector Borne Zoonotic Dis, 2019, 19(8): 604–612. DOI: 10.1089/vbz.2018.2395. [11] Cai CS, Zhu YZ, Zhong Y, et al. Development of O-antigen gene cluster-specific PCRs for rapid typing six epidemic serogroups of Leptospira in China[J]. BMC Microbiol, 2010, 10(1): 67. DOI: 10.1186/1471−2180−10−67. [12] Chen HW, Weissenberger G, Atkins E, et al. Highly sensitive loop-mediated isothermal amplification for the detection of Leptospira[J]. Int J Bacteriol, 2015, 2015: 147173. DOI: 10.1155/2015/147173. [13] 刘英, 陈红, 李沛丽, 等. 贵州省钩端螺旋体血清群特异性PCR鉴定结果与分析[J]. 中国人兽共患病学报,2015,31(12):1146–1150,1156. DOI:10.3969/j.issn.1002−2694.2015.12.012.Liu Y, Chen H, Li PL, et al. Serogroup identification and analysis of Leptospira isolates using serogroup specific PCR in Guizhou province, China[J]. Chin J Zoonoses, 2015, 31(12): 1146–1150,1156. DOI: 10.3969/j.issn.1002−2694.2015.12.012. [14] Shimabukuro FH, da Costa VM, da Silva RC, et al. Prozone effects in microscopic agglutination tests for leptospirosis in the sera of mice infected with the pathogenic Leptospira interrogans serovar Canicola[J]. Mem Inst Oswaldo Cruz, 2013, 108(5): 668–670. DOI: 10.1590/0074−0276108052013022. [15] Latifah I, Halim AA, Rahmat MS, et al. Isolation by culture and PCR identification of LipL32 gene of pathogenic Leptospira spp. in wild rats of Kuala Lumpur[J]. Malays J Pathol, 2017, 39(2): 161–166. [16] Riediger IN, Stoddard RA, Ribeiro GS, et al. Rapid, actionable diagnosis of urban epidemic leptospirosis using a pathogenic Leptospira lipL32-based real-time PCR assay[J]. PLoS Negl Trop Dis, 2017, 11(9): e0005940. DOI: 10.1371/journal.pntd.0005940. [17] Koizumi N, Izumiya H, Mu JJ, et al. Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia[J]. Infect Genet Evol, 2015, 36: 434–440. DOI: 10.1016/j.meegid.2015.08.013. [18] Xu YH, Zhang JL, Cui SH, et al. Genetic stability of vaccine strains by multilocus sequence typing and pulsed-field gel electrophoresis analysis: Implications for quality control of the leptospiral vaccine[J]. Hum Vaccin Immunother, 2015, 11(5): 1272–1276. DOI: 10.1080/21645515.2015.1020266. [19] Karunanayake L, Gamage CD, Gunasekara CP, et al. Multilocus sequence typing reveals diverse known and novel genotypes of Leptospira spp. circulating in Sri Lanka[J]. PLoS Negl Trop Dis, 2020, 14(8): e0008573. DOI: 10.1371/journal.pntd.0008573. [20] Wang Y, Li H, Wang Y, et al. Loop-mediated isothermal amplification label-based gold nanoparticles lateral flow biosensor for detection of Enterococcus faecalis and Staphylococcus aureus[J]. Front Microbiol, 2017, 8: 192. DOI: 10.3389/fmicb.2017.00192. [21] Hsu YH, Chou SJ, Chang CC, et al. Development and validation of a new loop-mediated isothermal amplification for detection of pathogenic Leptospira species in clinical materials[J]. J Microbiol Methods, 2017, 141: 55–59. DOI: 10.1016/j.mimet.2017.07.010. [22] Wang YC, Wang Y, Jiao WW, et al. Development of loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay for Mycoplasma pneumoniae detection[J]. AMB Express, 2019, 9(1): 196. DOI: 10.1186/s13568−019−0921−3. [23] 杨幸贵, 黄俊飞, 陈依江, 等. 结核分枝杆菌双靶标环介导恒温扩增检测方法的建立与应用[J]. 中国病原生物学杂志,2021,16(5):513–519. DOI: 10.13350/j.cjpb.210504.Yang XG, Huang JF, Chen YJ, et al. Development and use of loop-mediated isothermal amplification (LAMP) based on 2 target genes for rapid detection of Mycobacterium tuberculosis[J]. J Pathog Biol, 2021, 16(5): 513–519. DOI: 10.13350/j.cjpb.210504. [24] Chen X, Ma K, Yi X, et al. A novel detection of Enterococcus faecalis using multiple cross displacement amplification linked with gold nanoparticle lateral flow biosensor[J]. Infect Drug Resist, 2019, 12: 3771–3781. DOI: 10.2147/IDR.S235325. [25] Benacer D, Zain SNM, Lewis JW, et al. A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains[J]. Rev Soc Bras Med Trop, 2017, 50(2): 239–242. DOI: 10.1590/0037−8682−0364−2016. -