Nanoparticle-Based Lateral Flow Biosensor Integrated With Loop-Mediated Isothermal Amplification for Rapid and Visual Identification of Chlamydia trachomatis for Point-of-Care Use

Abstract

Chlamydial infection, caused by Chlamydia trachomatis, is the most common bacterial sexually transmitted infection and remains a major public health problem worldwide, particularly in underdeveloped regions. Developing a rapid and sensitive point-of-care (POC) testing for accurate screening of C. trachomatis infection is critical for earlier treatment to prevent transmission. In this study, a novel diagnostic assay, loop-mediated isothermal amplification integrated with gold nanoparticle-based lateral flow biosensor (LAMP-LFB), was devised and applied for diagnosis of C. trachomatis in clinical samples. A set of LAMP primers based on the ompA gene from 14 C. trachomatis serological variants (serovar A-K, L1, L2, L3) was successfully designed and used for the development of C. trachomatis-LAMP-LFB assay. The optimal reaction system can be performed at a constant temperature of 67°C for 35 min. The total assay process, including genomic DNA extraction (~15 min), LAMP reaction (35 min), and LFB readout (~2 min), could be finished within 60 min. The C. trachomatis-LAMP-LFB could detect down to 50 copies/ml, and the specificity was 100%, no cross-reactions with other pathogens were observed. Hence, our C. trachomatis-LAMP-LFB was a rapid, reliable, sensitive, cost-effective, and easy-to-operate assay, which could offer an attractive POC testing tool for chlamydial infection screening, especially in resource starvation settings.

Keywords: Chlamydia trachomatis; gold nanoparticle-based lateral flow biosensor; limit of detection; loop-mediated isothermal amplification; point-of-care testing.

Figure 1

C. trachomatis-LAMP-LFB workflow. C. trachomatis-LAMP-LFB assay contains three steps: genomic DNA preparation (step 1), LAMP reaction (step 2), and LFB visually readout (step 3). The whole diagnosis procedure can be completed within 60 min.

Figure 2

Schematic diagram of the principle of LFB for visually readout C. trachomatis-LAMP products. (A) C. trachomatis-LAMP products and (1.0 μl) and running buffer (100 μl) were simultaneously added to the sample pad. (B) The running buffer and C. trachomatis-LAMP products move forward to conjugate pad and reaction region due to capillary action. (C) For positive results, the FAM/biotin-labeled ompA-LAMP products are arrested by anti-FAM at TL strip, and the streptavidin-DPNs are arrested through biotin-BSA at CL strip. For negative results, only the streptavidin-DPNs flow to reaction region and arrested by biotin-BSA at CL strip. (D) Interpretation of the C. trachomatis-LAMP-LFB assay results: negative—only the CL appears on the LFB; positive—CL and TL appear on biosensor.

Figure 3

Confirmation and verification of C. trachomatis-LAMP products. C. trachomatis-LAMP products were measured simultaneously through MG regents (A) and LFB (B). Tube 1/biosensor 1: positive result for the C. trachomatis ompA standard plasmids; tube 2/biosensor 2: negative result for Neisseria gonorrhoeae; tube 3/biosensor 3: negative result for Ureaplasma urealyticum; tube 4/biosensor 4: blank control (distilled water, DW). TL, test line; CL, control line.

Figure 4

Optimization of the temperature for C. trachomatis-LAMP reactions. C. trachomatis-LAMP reaction process was monitored using real-time turbidity (LA-500). The threshold value was 0.1, and the turbidity >0.1 was regarded as positive. Eight kinetic graphs (A–H) were yielded at various amplification temperatures (63–70°C at 1°C intervals) with C. trachomatis ompA-plasmids at the level of 5 × 10³ copies/ml. The graphs at 67°C showed robust amplification.

Figure 5

Assay sensitivity using serially diluted C. trachomatis ompA-plasmid templates. (A) Visual MG regents used for detecting the results; (B) LFB used for detecting the results. Tubes A1–A7 (biosensors B1–B7) represent the C. trachomatis ompA-plasmid levels of 5.0 × 10⁴, 5.0 × 10³, 5.0 × 10², 5.0 × 10¹, 5.0 × 10⁰, 5.0 × 10⁻¹, and 5.0 × 10⁻² copies/ml and blank control (distilled water). The template levels from 5.0 × 10⁴ to 5.0 × 10¹ copies/ml showed positive results. CL, control line; TL, test line.

Figure 6

Optimization of reaction time for C. trachomatis-LAMP-LFB assay. Reaction times (A, 15 min; B, 25 min; C, 35 min; D, 45 min) were tested at optimal reaction temperature of 67°C. Tube/biosensors B1–B7 represent plasmid levels of 5.0 × 10⁴, 5.0 × 10³, 5.0 × 10², 5.0 × 10¹, 5.0 × 10⁰, 5.0 × 10⁻¹, and 5.0 × 10⁻² copies/ml and blank control (distilled water). The LoD of plasmid template could be detected when the reaction lasted for 35 min (C). CL, control line; TL, test line.