Sensitive and visual identification of Chlamydia trachomatis using multiple cross displacement amplification integrated with a gold nanoparticle-based lateral flow biosensor for point-of-care use

Abstract

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection (STI) and remains a major public health challenge, especially in less-developed regions. Establishing a rapid, inexpensive, and easy-to-interpret point-of-care (POC) testing system for C. trachomatis could be critical for its treatment and limiting further transmission. Here, we devised a novel approach termed a multiple cross displacement amplification integrated with gold nanoparticle-based lateral flow biosensor (MCDA-AuNPs-LFB) for the highly specific, sensitive, user-friendly, and rapid identification of C. trachomatis in clinical samples. A suite of MCDA primers based on the C. trachomatis ompA gene from 14 serological variants (serovar A-K, L1, L2, and L3) were successfully designed and used to establish the assay. Optimal assay conditions were identified at 67°C, and the detection procedure, including nucleic acid preparation (approximately 5 min), MCDA amplification (30 min), and AuNPs-LFB visual readout (within 2 min), was completed within 40 min. The all-in cost for each test was approximately $5.5 USD. The limit of detection (LoD) was 10 copies/reaction, and no cross-reaction was observed with non-C. trachomatis microbes. A total of 135 suspected C. trachomatis-infection genital secretion samples were collected and simultaneously detected using real-time quantitative PCR (qPCR) in our assay. Compared with the qPCR technology, the MCDA-AuNPs-LFB sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 96.20%, 94.92%, and 100%, respectively. Hence, our MCDA-AuNP-LFB assay exhibited considerable potential for POC testing and could be used to identify C. trachomatis in clinical settings, particularly in low-income regions.

Keywords: Chlamydia trachomatis; gold nanoparticle-based lateral flow biosensor; isothermal amplification; multiple cross displacement amplification; point-of-care testing.

Figure 1

C. trachomatis-MCDA-AuNPs-LFB assay workflow. The workflow includes genomic DNA preparation, MCDA amplification, and AuNP-LFB visual interpretation, all completed within 40 min.

Figure 2

Schematic diagram showing AuNPs-LFB principles for the visual identification of C trachomatis-MCDA amplification products. (A) C trachomatis-MCDA amplification products (0.5 μl) and running buffer (100 μl) were simultaneously added to the sample pad. (B) Due to capillary action, the running buffer, containing (C) trachomatis-MCDA products, moved forward onto the conjugate pad and nitrocellulose (NC) membrane. Streptavidin-AuNPs were hydrated, rapidly released, and combined with C trachomatis-MCDA products at the conjugate pad. (C) FAM/biotin-labeled C trachomatis-MCDA products were arrested by anti-FAM at the TL strip, and streptavidin-DPNs were arrested at the biotin-BSA CL strip. (D) Interpretation of the C trachomatis-AuNP-LFB assay. For a positive result, both the CL and TL appeared on the biosensor. For a negative result, only the CL was observed on the AuNP-LFB. TL: test line; CL: control line.

Figure 3

Confirmation and verification of (C) trachomatis-MCDA products. C trachomatis-MCDA products were measured simultaneously using malachite green (MG) (A) and AuNPs-LFB (B). Tube 1/Biosensor 1: positive result for 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

Optimizing the temperature for the C. trachomatis-MCDA assay. C. trachomatis-MCDA amplification of ompA was monitored using real-time turbidity. Corresponding amplicon concentration curves are marked in graphs. Turbidity > 0.1 indicated a positive value. (A–H) Eight kinetic graphs were generated at different temperatures (63°C–70°C at 1°C intervals) with C. trachomatis ompA-plasmids at 1 × 10³ copies. Graph E (67°C) showed the fastest and most robust amplification.

Figure 5

Sensitivity analysis of C trachomatis-MCDA-AuNPs-LFB using C trachomatis ompA-plasmid serial dilutions. Serial dilutions (1.0 × 10⁴, 1.0 × 10³, 1.0 × 10², 1.0 × 10¹, 1.0 × 10⁰, and 1.0 × 10⁻¹ copies) of C trachomatis ompA-plasmids were used as templates, and distilled water (DW) was used as the negative control. Results were simultaneously analyzed by malachite green (MG) (A) and AuNPs-LFB (B). The limit of detection (LoD) for C trachomatis-MCDA-AuNP-LFB was 10 copies/test. CL, control line; TL, test line.

Figure 6

Optimal amplification time for the C. trachomatis-MCDA-AuNPs-LFB assay. Four reaction times (A, 10 min; B, 20 min; C, 30 min; and D, 40 min) were evaluated at 67°C. Tubes/biosensors 1–7 represented C. trachomatis ompA template levels: 1.0 × 10⁴, 1.0 × 10³, 1.0 × 10², 1.0 × 10¹, 1.0 × 10⁰, 1.0 × 10⁻¹ copies, and negative control (distilled water, DW), respectively. Results were simultaneously analyzed using malachite green (MG) and AuNP-LFB. The optimal limit of detection (LoD) occurred when the amplification lasted for 30 min (C). CL: control line; TL: test line.

Figure 7

Analytical specificity of the C. trachomatis-MCDA-AuNPs-LFB assay using different strains. Assay specificity was evaluated using different nucleic acids as temperatures, and products were tested using AuNPs-LFB. Biosensors 1–14, C. trachomatis serovars A, B, C, D, E, F, G, H, I, J, K, L1, L2, and L3 ompA-plasmids; Biosensors 15–21, C. trachomatis (clinical samples); Biosensor 22, Ureaplasma urealyticum; Biosensor 23, Neisseria gonorrhoeae; Biosensor 24, Escherichia coli; Biosensor 25, Staphylococcus aureus; Biosensor 26, Human papilloma virus; Biosensor 27, Human rhinovirus; Biosensor 28, Coxsackie virus CAV16; Biosensor 29, Human enterovirus EV71; Biosensor 30, Mycoplasma pneumoniae; Biosensor 31, Listeria monocytogenes; Biosensor 32, Haemophilus influenza; Biosensor 33, Cryptococcus neoformans; Biosensor 34, Bordetella pertussis; Biosensor 35, Streptococcus pyogenes; Biosensor 36, Candida glabrata; Biosensor 37, Pseudomonas aeruginosa; Biosensor 38, Shigella flexneri; Biosensor 39, Klebsiella pneumoniae; Biosensor 40, negative control (distilled water, DW). CL: control line; TL: test line.