Rapid and visual detection of leptospira interrogans using polymerase spiral reaction assay

Archana Vishwakarma; Punith Chowdary; Mohandass Ramya

doi:10.7324/JABB.2023.144426

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Abstract

Leptospirosis is a waterborne zoonotic disease caused by pathogenic species of the genus Leptospira that affects humans and animals. Current diagnostic methods, such as culture and serology-based techniques, have limitations, including low sensitivity and time-consuming nature. This study presents the first implementation of the polymerase spiral reaction (PSR) assay for the rapid and visual detection of Leptospira interrogans. The assay utilizes Lipl32, a highly conserved gene that encodes an outer membrane lipoprotein, as a marker gene to detect pathogenic Leptospira spp. For visual detection, the assay was evaluated using a colorimetric dye and nucleic acid stain. The assay was optimized for various reaction parameters. The optimal conditions were determined to be 60°C for 60 min with 6U of Bst Polymerase, 5 mM of Mg2+, 2.5 mM of Betaine, and 10 mM of dNTPs. An artificial contamination study in tap water established the assay’s detection limit at 16 GEq/mL. Notably, the PSR assay showed high specificity and sensitivity compared to loop-mediated isothermal amplification (LAMP), requiring only a single primer pair in contrast to the six primers required for LAMP.

Keyword: Leptospira Zoonosis Waterborne Polymerase spiral reaction Lipl32

Citation:

Vishwakarma A, Chowdary P, Ramya M. Rapid and visual detection of leptospira interrogans using polymerase spiral reaction assay. J App Biol Biotech. 2023;11(Suppl 1):46-51. http://doi.org/10.7324/JABB.2023.144426

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

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2. Le Turnier P, Bonifay T, Mosnier E, Schaub R, Jolivet A, Demar M, et al. Usefulness of C-reactive protein in differentiating acute leptospirosis and dengue fever in French Guiana. Open Forum Infect Dis 2019;6:ofz323. https://doi.org/10.1093/ofid/ofz323
3. Nhan TX, Bonnieux E, Rovery C, De Pina JJ, Musso D. Fatal leptospirosis and chikungunya co-infection: Do not forget leptospirosis during chikungunya outbreaks. IDCases 2016;5:12-4. https://doi.org/10.1016/j.idcr.2016.06.003
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5. Goris MG, Hartskeerl RA. Leptospirosis serodiagnosis by the microscopic agglutination test. Curr Protoc Microbiol 2014;32:Unit 12E.5. https://doi.org/10.1002/9780471729259.mc12e05s32
6. Budihal SV, Perwez K. Leptospirosis diagnosis: Competancy of various laboratory tests. J Clin Diagn Res 2014;8:199-202. https://doi.org/10.7860/JCDR/2014/6593.3950
7. Pinto GV, Senthilkumar K, Rai P, Kabekkodu SP, Karunasagar I, Kumar BK. Current methods for the diagnosis of leptospirosis: Issues and challenges. J Microbiol Methods 2022;195:106438. https://doi.org/10.1016/j.mimet.2022.106438
8. Philip N, Affendy NB, Masri SN, Yuhana MY, Than LT, Sekawi Z, et al. Combined PCR and MAT improves the early diagnosis of the biphasic illness leptospirosis. PLoS One 2020;15:e0239069. https://doi.org/10.1371/journal.pone.0239069
9. Ahmed A, van der Linden H, Hartskeerl RA. Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira. Int J Environ Res Public Health 2014;11:4953-64. https://doi.org/10.3390/ijerph110504953
10. Li C, Shi Y, Yang G, Xia XS, Mao X, Fang Y, et al. Establishment of loop-mediated isothermal amplification for rapid detection of Pseudomonas aeruginosa. Exp Ther Med 2019;17:131-6. https://doi.org/10.3892/etm.2018.6910
11. 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
12. Hsu YH, Chou SJ, Chang CC, Pan MJ, Yang WC, Lin CF, et al. Development and validation of a new loop-mediated isothermal amplification for detection of pathogenic Leptospira species in clinical materials. J Microbiol Methods 2017;141:55-9. https://doi.org/10.1016/j.mimet.2017.07.010
13. Motohashi K. Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents. PLoS One 2019;14:e0222209. https://doi.org/10.1371/journal.pone.0222209
14. Kwong KM, Tam CC, Chan R, Lee SW, Ip P, Kwok J. Comparison of single nucleotide polymorphism genotyping of CYP2C19 by loop-mediated isothermal amplification and real-time PCR melting curve analysis. Clin Chim Acta 2018;478:45-50. https://doi.org/10.1016/j.cca.2017.12.013
15. Tubalinal GA, Balbin MM, Villanueva MA, Domingo CY, Mingala CN. Evaluation of LAMP for detection and/or screening of Leptospira spp. Infection among domestic animals in the Philippines. J Adv Vet Anim Res 2018;5:459-65. https://doi.org/10.5455/javar.2018.e299
16. Monica NI, Rathinasabapathi P, Ramya M. Development of real-time loop-mediated isothermal amplification (RealAmp) method for sensitive and rapid detection of pathogenic and nonpathogenic Leptospira. Lett Appl Microbiol 2019;68:196-203. https://doi.org/10.1111/lam.13108
17. Rashwan N, Diawara A, Scott ME, Prichard RK. Isothermal diagnostic assays for the detection of soil-transmitted helminths based on the SmartAmp2 method. Parasit Vectors 2017;10:496. https://doi.org/10.1186/s13071-017-2420-1

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1. Harada Y, Hayashi M. Severe leptospirosis in a patient with positive serological test for spotted fever rickettsiosis. BMJ Case Rep 2019;12:bcr-2018-226514. https://doi.org/10.1136/bcr-2018-226514

2. Le Turnier P, Bonifay T, Mosnier E, Schaub R, Jolivet A, Demar M, et al. Usefulness of C-reactive protein in differentiating acute leptospirosis and dengue fever in French Guiana. Open Forum Infect Dis 2019;6:ofz323. https://doi.org/10.1093/ofid/ofz323

3. Nhan TX, Bonnieux E, Rovery C, De Pina JJ, Musso D. Fatal leptospirosis and chikungunya co-infection: Do not forget leptospirosis during chikungunya outbreaks. IDCases 2016;5:12-4. https://doi.org/10.1016/j.idcr.2016.06.003

4. Gurumurthy K, Narasimha HB, Wyawahare M, Biswal N. Leptospirosis: A diagnostic conundrum. Trop Doct 2018;48:306-9. https://doi.org/10.1177/0049475518788470

5. Goris MG, Hartskeerl RA. Leptospirosis serodiagnosis by the microscopic agglutination test. Curr Protoc Microbiol 2014;32:Unit 12E.5. https://doi.org/10.1002/9780471729259.mc12e05s32

6. Budihal SV, Perwez K. Leptospirosis diagnosis: Competancy of various laboratory tests. J Clin Diagn Res 2014;8:199-202. https://doi.org/10.7860/JCDR/2014/6593.3950

7. Pinto GV, Senthilkumar K, Rai P, Kabekkodu SP, Karunasagar I, Kumar BK. Current methods for the diagnosis of leptospirosis: Issues and challenges. J Microbiol Methods 2022;195:106438. https://doi.org/10.1016/j.mimet.2022.106438

8. Philip N, Affendy NB, Masri SN, Yuhana MY, Than LT, Sekawi Z, et al. Combined PCR and MAT improves the early diagnosis of the biphasic illness leptospirosis. PLoS One 2020;15:e0239069. https://doi.org/10.1371/journal.pone.0239069

9. Ahmed A, van der Linden H, Hartskeerl RA. Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira. Int J Environ Res Public Health 2014;11:4953-64. https://doi.org/10.3390/ijerph110504953

10. Li C, Shi Y, Yang G, Xia XS, Mao X, Fang Y, et al. Establishment of loop-mediated isothermal amplification for rapid detection of Pseudomonas aeruginosa. Exp Ther Med 2019;17:131-6. https://doi.org/10.3892/etm.2018.6910

11. 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

12. Hsu YH, Chou SJ, Chang CC, Pan MJ, Yang WC, Lin CF, et al. Development and validation of a new loop-mediated isothermal amplification for detection of pathogenic Leptospira species in clinical materials. J Microbiol Methods 2017;141:55-9. https://doi.org/10.1016/j.mimet.2017.07.010

13. Motohashi K. Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents. PLoS One 2019;14:e0222209. https://doi.org/10.1371/journal.pone.0222209

14. Kwong KM, Tam CC, Chan R, Lee SW, Ip P, Kwok J. Comparison of single nucleotide polymorphism genotyping of CYP2C19 by loop-mediated isothermal amplification and real-time PCR melting curve analysis. Clin Chim Acta 2018;478:45-50. https://doi.org/10.1016/j.cca.2017.12.013

15. Tubalinal GA, Balbin MM, Villanueva MA, Domingo CY, Mingala CN. Evaluation of LAMP for detection and/or screening of Leptospira spp. Infection among domestic animals in the Philippines. J Adv Vet Anim Res 2018;5:459-65. https://doi.org/10.5455/javar.2018.e299

16. Monica NI, Rathinasabapathi P, Ramya M. Development of real-time loop-mediated isothermal amplification (RealAmp) method for sensitive and rapid detection of pathogenic and nonpathogenic Leptospira. Lett Appl Microbiol 2019;68:196-203. https://doi.org/10.1111/lam.13108

17. Rashwan N, Diawara A, Scott ME, Prichard RK. Isothermal diagnostic assays for the detection of soil-transmitted helminths based on the SmartAmp2 method. Parasit Vectors 2017;10:496. https://doi.org/10.1186/s13071-017-2420-1