Development of real-time polymerase spiral reaction assay for rapid and visual detection of Treponema pallidum

Krishnamoorthy Priya; Aysika Das; Ramya Mohandass

doi:10.7324/JABB.2023.145559

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Abstract

Treponema pallidum, a bacterium, is the cause of the sexually transmitted disease syphilis. At present, the treatment of syphilis relies on serum-based detection methods and darkfield observation of T. pallidum. However, these methods of identification are less effective in the initial stage of infection. In this study, we have developed a real-time polymerase spiral reaction (PSR) detecting the polA gene of T. pallidum DNA. The PSR reaction conditions were optimized at 62°C for 45 min. The polA gene displayed a distinct melt peak with a Tm value of 80 ± 0.5°C in the real-time melt curve analysis of the PSR reaction. The detection limit of the developed method was found to be 100 × 10−12 μg/μL. The specificity was evaluated by utilizing several bacterial species, and this method was 100% specific only to T. pallidum. The developed method is rapid, efficient, and can be used as an additional method for diagnosing T. pallidum infection.

Keyword: Serological method Treponema pallidum Polymerase spiral reaction polA Real-time melt curve analysis

Citation:

Priya K, Das A, Mohandass R. Development of real-time polymerase spiral reaction assay for rapid and visual detection of Treponema pallidum. J App Biol Biotech. 2023;11(Suppl 1):34-39. http://doi.org/10.7324/JABB.2023.145559

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

1. Grange PA, Gressier L, Dion PL, Farhi D, Benhaddou N, Gerhardt P, et al. Evaluation of a PCR test for detection of Treponema pallidum in swabs and blood. J Clin Microbiol 2012;50:546-52. https://doi.org/10.1128/JCM.00702-11
2. Knauf S, Lüert S, Šmajs D, Strouhal M, Chuma IS, Frischmann S, et al. Gene target selection for loop-mediated isothermal amplification for rapid discrimination of Treponema pallidum subspecies. PLoS Negl Trop Dis 2018;12:e0006396. https://doi.org/10.1371/journal.pntd.0006396
3. Heymans R, van der Helm JJ, de Vries HJ, Fennema HS, Coutinho RA, Bruisten SM. Clinical value of Treponema pallidum real-time PCR for diagnosis of syphilis. J Clin Microbiol 2010;48:497-502. https://doi.org/10.1128/JCM.00720-09
4. Naidu NK, Bharucha ZS, Sonawane V, Ahmed I. Comparative study of Treponemal and non-Treponemal test for screening of blood donated at a blood center. Asian J Transfus Sci 2012;6:32-5. https://doi.org/10.4103/0973-6247.95048
5. Xiao Y, Xie Y, Xu M, Liu S, Jiang C, Zhao F, et al. 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 2017;97:1673-8. https://doi.org/10.4269/ajtmh.17-0051
6. Gayet-Ageron A, Sednaoui P, Lautenschlager S, Ferry T, Toutous- Trellu L, Cavassini M, et al. Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis. Emerg Infect Dis 2015;21:127-9. https://doi.org/10.3201/eid2101.140790
7. Wang C, Cheng Y, Liu B, Wang Y, Gong W, Qian Y, et al. Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay. Emerg Microbes Infect 2018;7:83. https://doi.org/10.1038/s41426-018-0085-2
8. Dubourg G, Edouard S, Prudent E, Fournier PE, Raoult D. Incidental syphilis diagnosed by real-time PCR screening of urine samples. J Clin Microbiol 2015;53:3707-8. https://doi.org/10.1128/JCM.01026-15
9. Gimenes F, Medina FS, Abreu AL, Irie MM, Esquiçati IB, Malagutti N, et al. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR. PLoS One 2014;9:e98862. https://doi.org/10.1371/journal.pone.0098862
10. Chen CY, Chi KH, Pillay A, Nachamkin E, Su JR, Ballard RC. Detection of the A2058G and A2059G 23S rRNA gene point mutations associated with azithromycin resistance in Treponema pallidum by use of a TaqMan real-time multiplex PCR assay. J Clin Microbiol 2013;51:908-13. https://doi.org/10.1128/JCM.02770-12
11. Priya K, Rathinasabapathi P, Arunraj R, Sugapriya D, Ramya M. Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum. Arch Microbiol 2022;204:355. https://doi.org/10.1007/s00203-022-02973-z
12. Kaewphinit T, Arunrut N, Kiatpathomchai W, Santiwatanakul S, Jaratsing P, Chansiri K. Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int 2013;2013:926230. https://doi.org/10.1155/2013/926230
13. Liu W, Dong D, Yang Z, Zou D, Chen Z, Yuan J, et al. Polymerase spiral reaction (PSR): A novel isothermal nucleic acid amplification method. Sci Rep 2015;5:12723. https://doi.org/10.1038/srep12723
14. Dong D, Zou D, Liu H, Yang Z, Huang S, Liu N, et al. Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China. Front Microbiol 2015;6:1100. https://doi.org/10.3389/fmicb.2015.01100
15. Becherer L, Bakheit M, Frischmann S, Stinco S, Borst N, Zengerle R, et al. Simplified real-time multiplex detection of loop-mediated isothermal amplification using novel mediator displacement probes with universal reporters. Anal Chem 2018;90:4741-8. https://doi.org/10.1021/acs.analchem.7b05371
16. He S, Jang H, Zhao C, Xu K, Wang J, Pang B, et al. Rapid visualized isothermal nucleic acid testing of Vibrio parahaemolyticus by polymerase spiral reaction. Anal Bioanal Chem 2020;412:93-101. https://doi.org/10.1007/s00216-019-02209-y
17. Momin KM, Milton AA, Ghatak S, Thomas SC, Priya GB, Das S, et al. Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products. Mol Cell Probes 2020;50:101510. https://doi.org/10.1016/j.mcp.2020.101510
18. Liu W, Zou D, He X, Ao D, Su Y, Yang Z, et al. Development and application of a rapid Mycobacterium tuberculosis detection technique using polymerase spiral reaction. Sci Rep 2018;8:3003. https://doi.org/10.1038/s41598-018-21376-z
19. Tomar PS, Kumar JS, Patel S, Sharma S. Polymerase spiral reaction assay for rapid and real time detection of West nile virus from clinical samples. Front Cell Infect Microbiol 2020;10:426. https://doi.org/10.3389/fcimb.2020.00426
20. Jiang X, Dong D, Bian L, Zou D, He X, Ao D, et al. Rapid detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples. Front Microbiol 2016;7:916. https://doi.org/10.3389/fmicb.2016.00916
21. Robertson T, Bibby S, O'Rourke D, Belfiore T, Lambie H, Noormohammadi AH. Characterization of Chlamydiaceae species using PCR and high resolution melt curve analysis of the 16S rRNA gene. J Appl Microbiol 2009;107:2017-28. https://doi.org/10.1111/j.1365-2672.2009.04388.x
22. Reja V, Kwok A, Stone G, Yang L, Missel A, Menzel C, et al. ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis. Methods 2010;50:S10-4. https://doi.org/10.1016/j.ymeth.2010.02.006
23. Sutton MY, Liu H, Steiner B, Pillay A, Mickey T, Finelli L, et al. Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: Use of specimens from ulcers and blood. J Infect Dis 2001;183:1601-6. https://doi.org/10.1086/320698
24. Zetola NM, Klausner JD. Syphilis and HIV infection: An update. Clin Infect Dis 2007;44:1222-8. https://doi.org/10.1086/513427
25. Martin IE, Gu W, Yang Y, Tsang RS. Macrolide resistance and molecular types of Treponema pallidum causing primary syphilis in Shanghai, China. Clin Infect Dis 2009;49:515-21. https://doi.org/10.1086/600878
26. Rodes B, Liu H, Johnson S, George R, Steiner B. Molecular cloning of a gene (poIA) coding for an unusual DNA polymerase I from Treponema pallidum. J Med Microbiol 2000;49:657-67. https://doi.org/10.1099/0022-1317-49-7-657
27. Milton AA, Momin KM, Ghatak S, Priya GB, Angappan M, Das S, et al. Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Clostridium perfringens in meat. Heliyon 2021;7:e05941. https://doi.org/10.1016/j.heliyon.2021.e05941
28. Xu W, Gao J, Zheng H, Yuan C, Hou J, Zhang L, et al. Establishment and Application of polymerase spiral reaction amplification for Salmonella detection in food. J Microbiol Biotechnol 2019;29: 1543-52. https://doi.org/10.4014/jmb.1906.06027
29. Milton AAP, Momin KM, Ghatak S, Thomas SC, Priya GB, Angappan M, et al. Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Staphylococcus aureus in meat. LWT 2021;139. https://doi.org/10.1016/j.lwt.2020.110507
30. Das A, Kumar B, Chakravarti S, Prakash C, Singh RP, Gupta V, et al. Rapid visual isothermal nucleic acid-based detection assay of Brucella species by polymerase spiral reaction. J Appl Microbiol 2018;125:646-54. https://doi.org/10.1111/jam.13882

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1. Grange PA, Gressier L, Dion PL, Farhi D, Benhaddou N, Gerhardt P, et al. Evaluation of a PCR test for detection of Treponema pallidum in swabs and blood. J Clin Microbiol 2012;50:546-52. https://doi.org/10.1128/JCM.00702-11

2. Knauf S, Lüert S, Šmajs D, Strouhal M, Chuma IS, Frischmann S, et al. Gene target selection for loop-mediated isothermal amplification for rapid discrimination of Treponema pallidum subspecies. PLoS Negl Trop Dis 2018;12:e0006396. https://doi.org/10.1371/journal.pntd.0006396

3. Heymans R, van der Helm JJ, de Vries HJ, Fennema HS, Coutinho RA, Bruisten SM. Clinical value of Treponema pallidum real-time PCR for diagnosis of syphilis. J Clin Microbiol 2010;48:497-502. https://doi.org/10.1128/JCM.00720-09

4. Naidu NK, Bharucha ZS, Sonawane V, Ahmed I. Comparative study of Treponemal and non-Treponemal test for screening of blood donated at a blood center. Asian J Transfus Sci 2012;6:32-5. https://doi.org/10.4103/0973-6247.95048

5. Xiao Y, Xie Y, Xu M, Liu S, Jiang C, Zhao F, et al. 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 2017;97:1673-8. https://doi.org/10.4269/ajtmh.17-0051

6. Gayet-Ageron A, Sednaoui P, Lautenschlager S, Ferry T, Toutous- Trellu L, Cavassini M, et al. Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis. Emerg Infect Dis 2015;21:127-9. https://doi.org/10.3201/eid2101.140790

7. Wang C, Cheng Y, Liu B, Wang Y, Gong W, Qian Y, et al. Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay. Emerg Microbes Infect 2018;7:83. https://doi.org/10.1038/s41426-018-0085-2

8. Dubourg G, Edouard S, Prudent E, Fournier PE, Raoult D. Incidental syphilis diagnosed by real-time PCR screening of urine samples. J Clin Microbiol 2015;53:3707-8. https://doi.org/10.1128/JCM.01026-15

9. Gimenes F, Medina FS, Abreu AL, Irie MM, Esquiçati IB, Malagutti N, et al. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR. PLoS One 2014;9:e98862. https://doi.org/10.1371/journal.pone.0098862

10. Chen CY, Chi KH, Pillay A, Nachamkin E, Su JR, Ballard RC. Detection of the A2058G and A2059G 23S rRNA gene point mutations associated with azithromycin resistance in Treponema pallidum by use of a TaqMan real-time multiplex PCR assay. J Clin Microbiol 2013;51:908-13. https://doi.org/10.1128/JCM.02770-12

11. Priya K, Rathinasabapathi P, Arunraj R, Sugapriya D, Ramya M. Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum. Arch Microbiol 2022;204:355. https://doi.org/10.1007/s00203-022-02973-z

12. Kaewphinit T, Arunrut N, Kiatpathomchai W, Santiwatanakul S, Jaratsing P, Chansiri K. Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples. Biomed Res Int 2013;2013:926230. https://doi.org/10.1155/2013/926230

13. Liu W, Dong D, Yang Z, Zou D, Chen Z, Yuan J, et al. Polymerase spiral reaction (PSR): A novel isothermal nucleic acid amplification method. Sci Rep 2015;5:12723. https://doi.org/10.1038/srep12723

14. Dong D, Zou D, Liu H, Yang Z, Huang S, Liu N, et al. Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China. Front Microbiol 2015;6:1100. https://doi.org/10.3389/fmicb.2015.01100

15. Becherer L, Bakheit M, Frischmann S, Stinco S, Borst N, Zengerle R, et al. Simplified real-time multiplex detection of loop-mediated isothermal amplification using novel mediator displacement probes with universal reporters. Anal Chem 2018;90:4741-8. https://doi.org/10.1021/acs.analchem.7b05371

16. He S, Jang H, Zhao C, Xu K, Wang J, Pang B, et al. Rapid visualized isothermal nucleic acid testing of Vibrio parahaemolyticus by polymerase spiral reaction. Anal Bioanal Chem 2020;412:93-101. https://doi.org/10.1007/s00216-019-02209-y

17. Momin KM, Milton AA, Ghatak S, Thomas SC, Priya GB, Das S, et al. Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products. Mol Cell Probes 2020;50:101510. https://doi.org/10.1016/j.mcp.2020.101510

18. Liu W, Zou D, He X, Ao D, Su Y, Yang Z, et al. Development and application of a rapid Mycobacterium tuberculosis detection technique using polymerase spiral reaction. Sci Rep 2018;8:3003. https://doi.org/10.1038/s41598-018-21376-z

19. Tomar PS, Kumar JS, Patel S, Sharma S. Polymerase spiral reaction assay for rapid and real time detection of West nile virus from clinical samples. Front Cell Infect Microbiol 2020;10:426. https://doi.org/10.3389/fcimb.2020.00426

20. Jiang X, Dong D, Bian L, Zou D, He X, Ao D, et al. Rapid detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples. Front Microbiol 2016;7:916. https://doi.org/10.3389/fmicb.2016.00916

21. Robertson T, Bibby S, O'Rourke D, Belfiore T, Lambie H, Noormohammadi AH. Characterization of Chlamydiaceae species using PCR and high resolution melt curve analysis of the 16S rRNA gene. J Appl Microbiol 2009;107:2017-28. https://doi.org/10.1111/j.1365-2672.2009.04388.x

22. Reja V, Kwok A, Stone G, Yang L, Missel A, Menzel C, et al. ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis. Methods 2010;50:S10-4. https://doi.org/10.1016/j.ymeth.2010.02.006

23. Sutton MY, Liu H, Steiner B, Pillay A, Mickey T, Finelli L, et al. Molecular subtyping of Treponema pallidum in an Arizona County with increasing syphilis morbidity: Use of specimens from ulcers and blood. J Infect Dis 2001;183:1601-6. https://doi.org/10.1086/320698

24. Zetola NM, Klausner JD. Syphilis and HIV infection: An update. Clin Infect Dis 2007;44:1222-8. https://doi.org/10.1086/513427

25. Martin IE, Gu W, Yang Y, Tsang RS. Macrolide resistance and molecular types of Treponema pallidum causing primary syphilis in Shanghai, China. Clin Infect Dis 2009;49:515-21. https://doi.org/10.1086/600878

26. Rodes B, Liu H, Johnson S, George R, Steiner B. Molecular cloning of a gene (poIA) coding for an unusual DNA polymerase I from Treponema pallidum. J Med Microbiol 2000;49:657-67. https://doi.org/10.1099/0022-1317-49-7-657

27. Milton AA, Momin KM, Ghatak S, Priya GB, Angappan M, Das S, et al. Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Clostridium perfringens in meat. Heliyon 2021;7:e05941. https://doi.org/10.1016/j.heliyon.2021.e05941

28. Xu W, Gao J, Zheng H, Yuan C, Hou J, Zhang L, et al. Establishment and Application of polymerase spiral reaction amplification for Salmonella detection in food. J Microbiol Biotechnol 2019;29: 1543-52. https://doi.org/10.4014/jmb.1906.06027

29. Milton AAP, Momin KM, Ghatak S, Thomas SC, Priya GB, Angappan M, et al. Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Staphylococcus aureus in meat. LWT 2021;139. https://doi.org/10.1016/j.lwt.2020.110507

30. Das A, Kumar B, Chakravarti S, Prakash C, Singh RP, Gupta V, et al. Rapid visual isothermal nucleic acid-based detection assay of Brucella species by polymerase spiral reaction. J Appl Microbiol 2018;125:646-54. https://doi.org/10.1111/jam.13882