Abstract
Syphilis is a sexually transmitted disease caused by the spirochaete bacterium Treponema pallidum. This study has developed a multiplex High-Resolution Melt-curve Loop-mediated isothermal amplification (multiplex HRM-LAMP) assay targeting the marker genes polA and tprL to detect T. pallidum. The multiplex HRM-LAMP assay conditions were optimized at 65 °C for 45 min. Real-time melt-curve analysis of multiplex HRM-LAMP shows two melt-curve peaks corresponding to polA and tprL with a Tm value of 80 ± 0.5 °C and 87 ± 0.5 °C, respectively. The detection limit of multiplex HRM-LAMP was found to be 6.4 × 10–4 ng/μL (3.79 copies/μL) of T. pallidum. The specificity was evaluated using seven different bacterial species, and the developed method was 100% specific in detecting T. pallidum. A total of 64 blood samples of T. pallidum suspected cases were used to validate the assay method. The clinical validation showed that the assay was 96.43% sensitive and 100% specific in detecting syphilis. Thus, the developed method was more rapid and sensitive than other available methods and provides a multigene-based diagnostic approach to detect T. pallidum.
Similar content being viewed by others
References
Augkarawaritsawong S, Srisurapanon S, Wachiralurpan S et al (2019) Comparatively rapid screening tests for diagnosis of hepatitis b virus infection using loop-mediated isothermal amplification (LAMP) paired with lateral flow dipstick (LFD), gold nanoparticles (AuNPs) and real-time turbidimetry. Sci Technol Asia 23:45–47
Becherer L, Bakheit M, Frischmann S et al (2018) Simplified real-time multiplex detection of loop-mediated isothermal amplification using novel mediator displacement probes with universal reporters. Anal Chem. https://doi.org/10.1021/acs.analchem.7b05371
Chi KH, Danavall D, Taleo F et al (2015) Molecular differentiation of Treponema pallidum subspecies in skin ulceration clinically suspected as yaws in vanuatu using real-time multiplex PCR and serological methods. Am J Trop Med Hyg. https://doi.org/10.4269/ajtmh.14-0459
Couturier E, Michel A, Janier M et al (2004) Syphilis surveillance in France 2000–2003. Euro Surveill Bull Transm Eur Commun Dis Bull. https://doi.org/10.2807/esm.09.12.00493-en
Curtis KA, Morrison D, Rudolph DL et al (2018) A multiplexed RT-LAMP assay for detection of group M HIV-1 in plasma or whole blood. J Virol Methods. https://doi.org/10.1016/j.jviromet.2018.02.012
Donà V, Kasraian S, Lupo A et al (2016) Multiplex real-time PCR assay with high-resolution melting analysis for characterization of antimicrobial resistance in Neisseria gonorrhoeae. J Clin Microbiol. https://doi.org/10.1128/JCM.03354-15
Dong J, Xu Q, Li CC, Zhang CY (2019) Single-color multiplexing by the integration of high-resolution melting pattern recognition with loop-mediated isothermal amplification. Chem Commun. https://doi.org/10.1039/c8cc09741k
Gayet-Ageron A, Sednaoui P, Lautenschlager S et al (2015) Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis. Emerg Infect Dis. https://doi.org/10.3201/eid2101.140790
Goto M, Honda E, Ogura A et al (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques. https://doi.org/10.2144/000113072
Grange PA, Gressier L, Dion PL et al (2012) Evaluation of a PCR test for detection of Treponema pallidum in swabs and blood. J Clin Microbiol. https://doi.org/10.1128/JCM.00702-11
Heymans R, Van Der Helm JJ, De Vries HJC et al (2010) Clinical value of Treponema pallidum real-time PCR for diagnosis of syphilis. J Clin Microbiol. https://doi.org/10.1128/JCM.00720-09
Hulse LS, McDonald S, Johnston SD, Beagley KW (2019) Rapid point-of-care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop-mediated isothermal amplification without nucleic acid purification. MicrobiologyOpen. https://doi.org/10.1002/mbo3.916
Kim JH, Kang M, Park E et al (2019) A simple and multiplex loop-mediated isothermal amplification (LAMP) assay for rapid detection of SARS-CoV. BioChip J. https://doi.org/10.1007/s13206-019-3404-3
Knauf S, Lüert S, Šmajs D et al (2018) Gene target selection for loop-mediated isothermal amplification for rapid discrimination of Treponema pallidum subspecies. PLoS Negl Trop Dis. https://doi.org/10.1371/journal.pntd.0006396
Lin Q, Ye X, Yang B et al (2019) Real-time fluorescence loop-mediated isothermal amplification assay for rapid and sensitive detection of Streptococcus gallolyticus subsp. Gallolyticus associated with colorectal cancer. Anal Bioanal Chem. https://doi.org/10.1007/s00216-019-02059-8
Liu H, Rodes B, Chen CY, Steiner B (2001) New tests for syphilis: rational design of a PCR method for detection of Treponema pallidum in clinical specimens using unique regions of the DNA polymerase I gene. J Clin Microbiol. https://doi.org/10.1128/JCM.39.5.1941-1946.2001
Liu ML, Xia Y, Wu XZ et al (2017) Loop-mediated isothermal amplification of Neisseria gonorrhoeae porA pseudogene: a rapid and reliable method to detect gonorrhea. AMB Express. https://doi.org/10.1186/s13568-017-0349-6
Liu Y, Singh P, Mustapha A (2018) Multiplex high resolution melt-curve real-time PCR assay for reliable detection of Salmonella. Food Control. https://doi.org/10.1016/j.foodcont.2018.03.043
Manali KM, Arunraj R, Kumar T, Ramya M (2017) Detection of microcystin producing cyanobacteria in Spirulina dietary supplements using multiplex HRM quantitative PCR. J Appl Phycol. https://doi.org/10.1007/s10811-016-1011-4
Martin IE, Gu W, Yang TRSW (2009) Macrolide resistance and molecular types of Treponema pallidum causing primary syphilis in Shanghai, China. Clin Infect Dis. https://doi.org/10.1086/600878
Monica NI, Rathinasabapathi P, Ramya M (2019) Development of real-time loop-mediated isothermal amplification (RealAmp) method for sensitive and rapid detection of pathogenic and nonpathogenic Leptospira. Lett Appl Microbiol. https://doi.org/10.1111/lam.13108
Naidu NK, Bharucha ZS, Sonawane V, Ahmed I (2012) Comparative study of treponemal and non-treponemal test for screening of blood donated at a blood center. Asian J Transfus Sci. https://doi.org/10.4103/0973-6247.95048
Park IU, Fakile YF, Chow JM et al (2019) Performance of treponemal tests for the diagnosis of syphilis. Clin Infect Dis 68:913–918
Ramya M, Kayalvizhi M, Haripriya G, Rathinasabapathi P (2018) Detection of microcystin-producing cyanobacteria in water samples using loop-mediated isothermal amplification targeting mcyB gene. 3 Biotech. https://doi.org/10.1007/s13205-018-1402-0
Sutton MY, Liu H, Steiner B et al (2001) Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: use of specimens from ulcers and blood. J Infect Dis. https://doi.org/10.1086/320698
Tipple C, Hanna MOF, Hill S et al (2011) Getting the measure of syphilis: qPCR to better understand early infection. Sex Transm Infect. https://doi.org/10.1136/sti.2011.049494
Wang C, Cheng Y, Liu B et al (2018) Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay article. Emerg Microbe Infect. https://doi.org/10.1038/s41426-018-0085-2
Xiao Y, Xie Y, Xu M et al (2017) Development and evaluation of a loop-mediated isothermal amplification assay for the detection of Treponema pallidum DNA in the peripheral blood of secondary syphilis patients. Am J Trop Med Hyg. https://doi.org/10.4269/ajtmh.17-0051
Xiao long, Zhang, Wu, XLH et al (2014) Simultaneous detection of salmonella, listeria monocytogenes, and Staphylococcus aureus by multiplex real-time PCR assays using high-resolution melting. Food Anal Method. https://doi.org/10.1007/s12161-014-9875-x
Xu G, Gunson RN, Cooper JM, Reboud J (2015) Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis-applications in the clinical diagnosis of sexually transmitted diseases. Chem Commun. https://doi.org/10.1039/c4cc08389j
Zhang J, Cao J, Zhu M et al (2019) Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae. World J Microbiol Biotechnol. https://doi.org/10.1007/s11274-019-2601-5
Acknowledgements
The authors acknowledge the SRM Institute of Science and Technology, Kanchipuram, Tamil Nadu, for providing the research facilities.
Funding
SRM Institute of Science and Technology, Kanchipuram, provided financial support for this project.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Erko Stackebrandt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Priya, K., Rathinasabapathi, P., Arunraj, R. et al. Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum. Arch Microbiol 204, 355 (2022). https://doi.org/10.1007/s00203-022-02973-z
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00203-022-02973-z