Development of a SYBR Green real-time PCR-based assay system for detection of Neisseria gonorrhoeae

https://doi.org/10.19106/JMedSci005401202201

Andi Yasmon(1*), Rela Febriani(2), Louisa Ivana Utami(3), Fithriyah Fithriyah(4), Yeva Rosana(5), Fera Ibrahim(6), Pratiwi Sudarmono(7)

(1) Department of Microbiology, Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital
(2) Department of Microbiology, Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital
(3) Master Program in Biomedical Science, Faculty of Medicine Universitas Indonesia, Jakarta
(4) Department of Microbiology, Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital
(5) Department of Microbiology, Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital
(6) Department of Microbiology, Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital
(7) Department of Microbiology, Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital
(*) Corresponding Author

Abstract


Diagnosis of Neisseria gonorrhoeae infection is needed for patient therapy and for reducing this bacterial transmission in the population. The culture method is a gold standard method for N. gonorrhoeae detection, however it has low sensitivity. Among molecular methods with high sensitivity and specificity, SYBR Green real-time PCR is the potential method for N. gonorrhoeae detection. In this study, we developed an SYBR Green real-time PCR-based system assay for N. gonorrhoeae detection. Several PCR conditions were optimized and analyzed including primer annealing temperature, DNA template volume, the limit of detection (LoD), cross-reaction with others (bacteria, viruses, fungus, protozoa), and quality assurance. The results showed that the annealing temperature and DNA template volume were 60oC and 5 µL, respectively. The LoD was 29 DNA copies corresponding to 3 bacterial cells per reaction. No cross-reaction was detected for other bacteria, viruses, fungus and protozoa. The external quality assurances enrolled in 2019 and 2021 showed 100% concordance. The preliminary testing for clinical samples was also 100% concordance. In conclusion, the SYBR Green real-time PCR-based system assay developed in this study is promising for application in clinical laboratories.


Keywords


Neisseria gonorrhoeae; PCR

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References

Suay-García B, Pérez-Gracia MT. Neisseria gonorrhoeae infections. Pathogens 2020; 9(8):647.
https://doi.org/ 10.3390/pathogens9080647
2. Piszczek J, St Jean R, Khaliq Y. Gonorrhea: Treatment update for an increasingly resistant organism. Can Pharm J 2015; 148(2):82-9.
https://doi.org/10.1177/1715163515570111
3. Martín-Sánchez M, Fairley CK, Ong JJ, Maddaford K, Chen MY, Williamson DA, et al. Clinical presentation of asymptomatic and symptomatic women who tested positive for genital gonorrhoea at a sexual health service in Melbourne, Australia. Epidemiol Infect 2020; 148:e240.
https://doi.org/ 10.1017/S0950268820002265
4. Centers for Disease Control and Prevention (CDC). Gonorrhea - CDC Fact Sheet. 2017 June [cited 2021 Aug 22]. Available from https://www.cdc.gov/std/gonorrhea/Gonorrhea-FS.pdf
5. World Health Organization (WHO). Multi-drug resistant gonorrhoea. 2020 November 6 [cited 2021 Aug 28]. Available from https://www.who.int/news-room/fact-sheets/detail/multi-drug-resistant-gonorrhoea
6. Rowley J, Hoorn SV, Korenromp E, Low N, Unemo M, Abu-Raddad LJ, et al. Chlamydia, gonorrhoea, trichomoniasis and syphilis: Global prevalence and incidence estimates, 2016. Bull World Health Organ 2019; 97(8):548-62.
https://doi.org/10.2471/BLT.18.228486
7. European Centre for Disease Prevention and Control (ECDC). Gonorrhoea Annual Epidemiological Report for 2018. Stockholm, May 2020.
https://www.ecdc.europa.eu/sites/default/files/documents/gonorrhoea-annual-epidemiological-report-2018.pdf
8. Centers for Disease Control and Prevention (CDC). National Overview - Sexually Transmitted Disease Surveillance, 2019. 2021 April 13 [cited 2021 Aug 28]. Available from https://www.cdc.gov/std/statistics/2019/overview.htm
9. World Health Organization (WHO). Moving ahead on elimination of Sexually Transmitted Infections (STIs) in WHO South-East Asia Region - progress and challenges. 2019; 37:725-38.
https://apps.who.int/iris/handle/10665/330031
10. Sharma M, Rewari BB, Aditama TY, Turlapati P, Dallabetta G, Steen R. Control of sexually transmitted infections and global elimination targets, south-east asia region. Bull World Health Organ 2021; 99(4):304-11.
https://doi.org/10.2471/BLT.20.254003
11. Hananta IPY, van Dam AP, Bruisten SM, Van Der Loeff MFS, Soebono H, De Vries HJC. Gonorrhea in Indonesia: High prevalence of asymptomatic urogenital gonorrhea but no circulating extended spectrum cephalosporins-resistant Neisseria gonorrhoeae strains in Jakarta, Yogyakarta, and Denpasar, Indonesia. Sex Transm Dis 2016; 43(10):608-16.
https://doi.org/10.1097/OLQ.0000000000000510
12. Meyer T, Buder S. The laboratory diagnosis of Neisseria gonorrhoeae: current testing and future demands. Pathogens 2020; 9(2):91.
https://doi.org/10.3390/pathogens9020091
13. Geraats-Peters CWM, Brouwers M, Schneeberger PM, Van Der Zanden AGM, Bruisten SM, Weers-Pothoff G, et al. Specific and sensitive detection of Neisseria gonorrhoeae in clinical specimens by real-time PCR. J Clin Microbiol 2005; 43(11):5653-9.
https://doi.org/10.1128/JCM.43.11.5653-5659.2005
14. Ng LK, Martin IE. The laboratory diagnosis of Neisseria gonorrhoeae. Can J Infect Dis Med Microbiol 2005; 16(1):15-25.
https://doi.org/10.1155/2005/323082
15. Tajadini M, Panjehpour M, Javanmard SH. Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res 2014; 3(1):85.
https://doi.org/10.4103/2277-9175.127998
16. Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, et al. Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunol Med Microbiol 2003; 39(1):81-6.
https://doi.org/10.1016/S0928-8244(03)00224-4
17. Kumar B, Kumar P, Rajput R, Daga MK, Khanna M. OL-055 Evaluation of SYBR Green I and TaqMan real-time PCR chemistries for specific detection of influenza A viruses. Int J Infect Dis 2011; 15(7):S33.
https://doi.org/10.1016/S1201-9712(11)60117-3
18. Bhat KS, Gibbs CP, Barrera O, Morrison SG, Jähnig F, Stern A, et al. The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes. Mol Microbiol 1991; 5(8):1889-901.
https://doi.org/10.1111/j.1365-2958.1991.tb00813.x
19. Rychlik W, Spencer WJ, Rhoads RE. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 1990; 18(21):6409-12.
https://doi.org/10.1093/nar/18.21.6409
20. Pruvot M, Kamyingkird K, Desquesnes M, Sarataphan N, Jittapalapong S. The effect of the DNA preparation method on the sensitivity of PCR for the detection of Trypanosoma evansi in rodents and implications for epidemiological surveillance efforts. Vet Parasitol 2013; 191(3-4):203-8.
https://doi.org/10.1016/j.vetpar.2012.09.010
21. Chuang LY, Cheng YH, Yang CH. Specific primer design for the polymerase chain reaction. Biotechnol Lett 2013; 35(10):1541-9.
https://doi.org/10.1007/s10529-013-1249-8
22. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol 2012; 113(5):1014-26.
https://doi.org/10.1111/j.1365-2672.2012.05384.x
23. Sidstedt M, Rådström P, Hedman J. PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions. Anal Bioanal Chem 2020; 412(9):2009-23.
https://doi.org/10.1007/s00216-020-02490-2
24. Verma R, Sood S, Bala M, Mahajan N, Kapil A, Sharma VK, et al. Evaluation of an opa gene-based nucleic acid amplification test for detection of Neisseria gonorrhoeae in urogenital samples in North India. Epidemiol Infect 2012; 140(11):2110-6.
https://doi.org/10.1017/S0950268811002883
25. Bruisten SM, Noordhoek GT, Van Den Brule AJC, Duim B, Boel CHE, El-Faouzi K, et al. Multicenter validation of the cppB gene as a PCR target for detection of Neisseria gonorrhoeae. J Clin Microbiol 2004; 42(9):4332-4.
https://doi.org/10.1128/JCM.42.9.4332-4334.2004
26. World Health Organization (WHO). Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus. 2013.
27. Chui L, Chiu T, Kakulphimp J, Tyrrell GJ. A comparison of three real-time PCR assays for the confirmation of Neisseria gonorrhoeae following detection of N. gonorrhoeae using Roche COBAS AMPLICOR. Clin Microbiol Infect 2008; 14(5):473-9.
https://doi.org/10.1111/j.1469-0691.2008.01950.x
28. Chen L, Shin DJ, Zheng S, Melendez JH, Gaydos CA, Wang T. Direct-qPCR assay for coupled identification and antimicrobial susceptibility testing of Neisseria gonorrhoeae. ACS Infect Dis 2018; 4(9):1377-84.
https://doi.org/10.1021/acsinfecdis.8b00104



DOI: https://doi.org/10.19106/JMedSci005401202201

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