Rapid Identification of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum by RNA Amplification Lateral Flow Assay

Summary

Here, we present a protocol demonstrating RNA amplification lateral flow assay and evaluate the efficacy for rapidly detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasmaurealyticum.

Abstract

Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), and Ureaplasma urealyticum (UU) are common pathogens associated with sexually transmitted diseases (STDs). NG is the causative agent of gonorrhea, which can result in various disorders of the genitourinary system, including epididymitis, prostatitis, cervicitis, and infertility. CT is a microorganism responsible for non-gonococcal urethritis and pelvic inflammatory disease. UU is associated with non-gonococcal urethritis, cervicitis, prostatitis, and infertility. Current molecular technologies for the analysis of NG, CT, and UU are often complex and time-consuming. Rapid molecular testing for these pathogens may facilitate the early diagnosis of STDs. The RNA amplification lateral flow assay (RGT) is a method that does not require nucleic acid extraction. In this process, samples are lysed to release nucleic acid fragments, which are then amplified through reverse transcription and transcription. The amplified RNA product is recognized and captured by specific probes, forming an RNA-detection probe-gold probe complex that can be immobilized on a nitrocellulose membrane via lateral flow to produce visible bands. The entire procedure takes less than 1 h. The aim of this study was to evaluate the efficacy of RGT for the rapid detection of NG, CT, and UU. A total of 1,416 samples were included. The consistency of NG, CT, and UU when analyzed by RGT compared to the polymerase chain reaction (PCR) fluorescence probing method was 99.03% (307/310), 99.38% (159/160), and 99.02% (303/306), respectively. The consistency of NG, CT, and UU when analyzed by RGT compared to gene sequencing was 99.17% (238/240), 98.95% (188/190), and 98.30% (173/176), respectively. Compared with the pathogen isolation culture method, the detection rates for NG, CT, and UU assays were 100.00% (9/9, 17/17, 8/8). The high sensitivity and specificity, ease of use, and reduced time requirements make this assay ideal for the rapid and accurate detection of NG, CT, and UU.

Introduction

Sexually transmitted infections (STIs) represent significant global public health challenges, leading to serious genital and reproductive morbidity and mortality¹. Neisseria gonorrhoeae (NG) is the causative agent of gonorrhea, with approximately 87 million new cases reported annually worldwide². This pathogen primarily induces inflammation by invading the columnar epithelium of the urogenital tract³. Chlamydia trachomatis (CT), another microorganism, is responsible for non-gonococcal urethritis and pelvic infections, accounting for around 130 million new cases each year

Protocol

This study was conducted in accordance with the ethical guidelines and approval granted by the Institutional Review Board of the First People's Hospital of Foshan, Guangdong Province, China (Ethical Review Document No. LR (2025) 136). A total of 1,416 genital swab specimens (716 from female patients, 700 from male patients) were prospectively collected from individuals clinically suspected of STDs who had not undergone antibiotic therapy within the preceding 14 days (Supplementary Table 1). For each participant, paired swabs were obtained simultaneously and analyzed in parallel: one by the test method (RGT) and the other by reference method (PCR f....

Results

Consistency of the RGT and PCR fluorescence probing methods
A total of 310 samples were analyzed for NG, consisting of 161 females and 149 males. The positive coincidence rate was 98.78% (95%CI: 93.39%-99.97%), while the negative coincidence rate was 99.12% (95% CI: 96.87%-99.89%). The overall coincidence rate was 99.03% (95% CI: 97.20%-99.80%). The Kappa consistency value was 0.975 (95% CI: 0.947-1.003, Z = 17.171, P < 0.05) (Tab.......

Discussion

In this study, RGT was employed to detect STI pathogens, including NG, CT, and UU. When performed under stringent operational protocols, the RGT assay demonstrated strong concordance with established diagnostic methods such as PCR fluorescence probing, gene sequencing, and pathogen isolation culture techniques. The sensitivity and specificity of the RGT assay were comparable to or exceeded those of previously reported methodologies, as evidenced by comparative analyses with established benchmarks¹⁰

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% (w/v)  sodium hypochlorite	Preparation in the laboratory.	Preparation in the laboratory.	Diluted bleach solution for decontamination and inactivation of pathogens on equipment or surfaces.
75% (v/v) ethanol	Ante Food Co., Ltd (Anhui,China)	http://www.antegroup.com/product_detail_cn/id/81.html	Aqueous ethanol solution used for surface disinfection
Adjustable-volume Pipette (0.5–10 μL)	DLAB Scientific Co., Ltd.	7010101004	Single-channel micropipette for accurate handling of microliter-scale volumes.
Adjustable-volume Pipette (100–1000 μL)	DLAB Scientific Co., Ltd.	7010101014	Single-channel pipette calibrated for large-volume liquid handling.
Adjustable-volume Pipette (10–100 μL)	DLAB Scientific Co., Ltd.	7010101008	Single-channel pipette optimized for intermediate volumes, commonly used in molecular biology protocols.
Adjustable-volume Pipette (20–200 μL)	DLAB Scientific Co., Ltd.	7010101009	Single-channel pipette for medium-volume transfers, suitable for precise aliquoting of reagents.
Biosafety cabinet (HF Safe-1200)	HealForce Bio-Meditech Holdings Limited (Hong Kong, China)	HF Safe-1200	A Class II biosafety cabinet ensuring personnel, sample, and environmental protection during handling of pathogenic microorganisms or cell cultures.
Centrifuge Tubes	Haozhi Medical Devices Co., Ltd. (Guangzhou, China)	Guangzhou Yuexiu Medical Device Filing No. 20210414	For nucleic acid extraction, cell pelleting, and other laboratory procedures.
Chamydia trachomatis (CT) Nucleic Acid Assay (RNA Amplification-Lateral Flow Assay)	Wuhan Zhongzhi Biotechnologies INC.(Wuhan, China)	A.10.309-STD-RG-002	For qualitative detection of Chamydia trachomatis nucleic acid RNA in genital and urinary tract swab samples.
CO2 Incubator	Thermo Fisher Scientific(Asheville, USA) LLC	SN:300504406	Used for cell culture, providing a stable environment with controlled temperature (37°C), humidity, and CO2 concentration (typically 5%) to ensure optimal cell growth.
Culture Plate	Corning Incorporated (Corning, USA)	50 mm ´ 9 mm	For adherent cell culture and expansion.
Detection Kit for Nucleic Acid to Chamydia trachomatis (CT) (PCR-Fluorescence Probing)	Fosun Diagnostics (Shanghai, China) Co., Ltd	China Medical Device Registration Certificate No. 20143402234	For qualitative detection of nucleic acid of Chamydia trachomatis in genital and urinary tract swab samples.
Detection Kit for Nucleic Acid to Neisseria Gonorrhoeae (NG)(PCR-Fluorescence Probing)	Fosun Diagnostics (Shanghai, China) Co., Ltd	China Medical Device Registration Certificate No. 20173401080	For qualitative detection of nucleic acid of Neisseria gonorrhoeae in genital and urinary tract swab samples.
Detection Kit for Nucleic Acid to Ureaplasma Urealyticum (UU) (PCR-Fluorescence Probing)	Fosun Diagnostics (Shanghai, China) Co., Ltd	China Medical Device Registration Certificate No. 20173400536	For qualitative detection of nucleic acid of Ureaplasma Urealyticum in genital and urinary tract swab samples.
DMEM medium	Thermo Fisher Scientific Inc.(Waltham, USA)	11965092	For Chlamydia trachomatis culture.
Filter Pipette Tips (10 μL)	Guangzhou Surbiopure Biotechnology Co., Ltd (Guangzhou, China)	TF-10-BXL	For precise pipetting in the experiments.
Filter Pipette Tips (200 μL)	Guangzhou Surbiopure Biotechnology Co., Ltd (Guangzhou, China)	TF-200-BXL	For precise pipetting in the experiments.
Genital swab	Haozhi Medical Devices Co., Ltd. (Guangzhou, China)	Hz112	Sterile cotton-tipped swabs for collecting clinical specimens from genital mucosal surfaces.
Gibco Fetal Bovine Serum (FBS)	Thermo Fisher Scientific Inc. (Waltham, USA)	10270106	For Chlamydia trachomatis culture.
Glass slides	Haozhi Medical Devices Co., Ltd. (Guangzhou, China)	Guangzhou Yuexiu Medical Device Filing No. 20210414	Standard glass slides with anti-fade treatment for preparing cell smears or tissue sections, compatible with Gram staining protocols.
Goldenstar RT6 cDNA Synthesis Kit Ver2	Beijing Tsingke Biotech Co., Ltd (Beijing, China)	TSK302S	For the synthesis of the first strand of cDNA in the first step of the two-step RT-PCR. It contains all the reagents required for the synthesis of cDNA from RNA.
Gram Stain Kit	Baso Diagnostics,Inc. (Zhuhai, China)	BA4012	A staining kit containing crystal violet, iodine, decolorizer, and safranin for differentiating Neisseria Gonorrhoeae.
IBM SPSS Statistics (V25.0)	International Business Machines Corporation (New York, USA)	Version 25.0	For data analysis.
Infrared sterilizer	Huaxixinrui (Qingdao) Analytical Instruments Co., Ltd.	MJ-200W	For instant decontamination of inoculation loops or forceps, minimizing cross-contamination in biosafety cabinets.
Inoculation loop	BKMAM Biotechnology Co., Ltd.(Hunan, China)	110306002	For microbial sample transfer, streaking, or inoculation.
Inverted Microscope (IX-51)	Olympus Corporation (Tokyo, Japan)	Asset Code of Labortory:  71807101	For observing adherent cells or live-cell dynamics in culture dishes.
MALDI Biotyper automated microbial mass spectrometry system	Bruker Daltonics GmbH & Co. KG (Bremen, Germany)	National Medical Device Import Registration No. 20182222017	A MALDI-TOF mass spectrometry-based system for rapid microbial identification via protein profiling, enabling species identification of bacteria and fungi in clinical microbiology laboratories.
McCoy cell lines	Preserved in the laboratory.	Preserved in the laboratory.	For Chlamydia trachomatis culture.
Microplate Thermostatic Incubator Leopard70-4 (4 Plate Capacity)	Leaptec Scientific Instruments (Beijing) Co., Ltd.	Leopard70-4	A microplate incubator accommodating up to 4 standard microplates, with a temperature range from ambient to 70°C,  used for the incubation steps.
Microscope (OLYMPUS CX43)	Olympus Corporation (Tokyo, Japan)	https://lifescience.evidentscientific.com.cn/zh/microscopes/upright/cx43-33/	For clinical sample observation (e.g., cell morphology, stained specimens).
Mini Metal Bath MiniG-R100	Leaptec Scientific Instruments (Beijing) Co., Ltd.	MiniG-R100	For rapid temperature regulation during the experiment, such as heating, incubating, or terminating reactions in small-volume samples.
Neisseria gonorrhoeae (NG) Nucleic Acid Assay (RNA Amplification-Lateral Flow Assay)	Wuhan Zhongzhi Biotechnologies INC.(Wuhan, China)	A.10.309-STD-RG-001	For qualitative detection of Neisseria gonorrhoeae nucleic acid RNA in genital and urinary tract swab samples.
Nucleic Acid Extraction Reagent	Wuhan Zhongzhi Biotechnologies INC.(Wuhan, China)	A.10.105-SC-001	For the extraction, enrichment and purification of nucleic acids.
Specimen Transport Medium	QIAGEN N.V. (Venlo, Netherlands)	5128-1220	This medium is specifically formulated for the collection, preservation, and transport of clinical specimens, ensuring the viability of Chlamydia trachomatis and Neisseria gonorrhoeae for subsequent diagnostic testing.
Thermostatic Centrifuge	Beckman Coulter, Inc. (Brea, USA)	SN: MRZ21L037	For cell, microbial, or nucleic acid pelleting.
TM medium	Shanghai Yihua Medical Science & Technology Co., Ltd (Shanghai, China)	Shanghai Pu Medical Device Filing No. 20140081	TM medium can be used for the isolation, cultivation and identification of Neisseria gonorrhoeae from clinical samples to assist in the diagnosis of gonorrhea.
Trypsin-EDTA (0.25%)	Thermo Fisher Scientific Inc.(Waltham, USA)	25200072	For gentle dissociation of adherent cells during subculturing or experimental procedures.
Ureaplasma urealyticum (UU) Nucleic Acid Assay (RNA Amplification-Lateral Flow Assay)	Wuhan Zhongzhi Biotechnologies INC.(Wuhan, China)	A.10.309-STD-RG-003	For qualitative detection of Ureaplasma Urealyticum nucleic acid RNA in genital and urinary tract swab samples.
Ureaplasma urealyticum and Mycoplasma hominis Selective Isolation Culture, Identification and Drug Sensitivity Kit	Hebei Zhongaisheng Biotechnology Co., Ltd.(Hebei, China)	Hebei Medical Device Registration No. 20152400168	For the selective isolation, culture, identification, and drug sensitivity determination of Ureaplasma urealyticum and Mycoplasma hominis.
Vortex mixer	Xinkang Medical Instrument Co. Ltd (Jiangsu, China)	XK80-A	Laboratory device for homogenizing liquid samples by rapid agitation.