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

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


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.


Neisseria gonorrhoeae; PCR

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