Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum

doi:10.1007/s00203-022-02973-z

. 2022 Jun 1;204(6):355.

doi: 10.1007/s00203-022-02973-z.

Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum

Krishnamoorthy Priya¹, Pasupathi Rathinasabapathi¹, Rex Arunraj¹, Dhanasekaran Sugapriya², Mohandass Ramya³

Affiliations

¹ Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Kanchipuram District, Chennai, Tamil Nadu, 603203, India.
² Department of Medical Laboratory (Pathology), College of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Wadi-Al Dawaser, 11451, Riyadh, Saudi Arabia.
³ Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Kanchipuram District, Chennai, Tamil Nadu, 603203, India. ramyam@srmist.edu.in.

PMID: 35648234
DOI: 10.1007/s00203-022-02973-z

Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum

Krishnamoorthy Priya et al. Arch Microbiol. 2022.

. 2022 Jun 1;204(6):355.

doi: 10.1007/s00203-022-02973-z.

Authors

Krishnamoorthy Priya¹, Pasupathi Rathinasabapathi¹, Rex Arunraj¹, Dhanasekaran Sugapriya², Mohandass Ramya³

Affiliations

¹ Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Kanchipuram District, Chennai, Tamil Nadu, 603203, India.
² Department of Medical Laboratory (Pathology), College of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Wadi-Al Dawaser, 11451, Riyadh, Saudi Arabia.
³ Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur, Kanchipuram District, Chennai, Tamil Nadu, 603203, India. ramyam@srmist.edu.in.

PMID: 35648234
DOI: 10.1007/s00203-022-02973-z

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.

Keywords: LAMP; Multiplex HRM-LAMP; Serological methods; Treponema pallidum; polA; tprL.

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Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Sexually Transmitted Infections: A Review.
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Prospective evaluation of non-invasive saliva specimens for the diagnosis of syphilis and molecular surveillance of Treponema pallidum.
Imai K, Sato A, Tanaka M, Ohama Y, Nakayama S-i, Omachi R, Takeuchi K, Tarumoto N, Tokano M, Mesaki S, Maeda T, Akeda Y. Imai K, et al. J Clin Microbiol. 2024 Dec 11;62(12):e0080924. doi: 10.1128/jcm.00809-24. Epub 2024 Nov 6. J Clin Microbiol. 2024. PMID: 39503502 Free PMC article.
Multiplex Loop-Mediated Isothermal Amplification Coupled Lateral Flow Assay for Point-of-Care Detection of Syphilis.
Priya K, Saranya E, Kapoor A, Ramya M. Priya K, et al. Indian J Microbiol. 2024 Sep;64(3):1246-1256. doi: 10.1007/s12088-024-01308-4. Epub 2024 May 22. Indian J Microbiol. 2024. PMID: 39282190

References

1. 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
1. 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 - DOI - PubMed
1. 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 - DOI - PubMed - PMC
1. 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 - DOI
1. 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 - DOI - PubMed

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[1] 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

[2] 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

[3] 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 - DOI - PubMed

[4] 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 - DOI - PubMed

[5] 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 - DOI - PubMed - PMC

[6] 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 - DOI - PubMed - PMC

[7] 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 - DOI

[8] 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 - DOI

[9] 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 - DOI - PubMed

[10] 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 - DOI - PubMed

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Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum

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Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum

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