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.

Biosensor‐based multiple cross displacement amplification platform for visual and rapid identification of hepatitis C virus

“…However, the two former approaches need expensive facilities and are not suitable for a POC diagnostic platform. The L‐HNB method was visual and equipment‐free, but need more amplification time for detection of the LoD (Figure 6B,C), and the visual result was ambiguous when the amplicons were low 23,25 . Our AuNPs‐LFB strip was convenient operation and inexpensive (approximately US$2.0 per test).…”

“…The HCV‐positive samples were also identified with Sanger sequencing (Dian Medical Laboratory Center Co. Ltd.). Online statistical software MedCalc (http://www.medcalc.org/calc/diagnostic_test.php) was used to analyze the conventional HCV‐RT‐qPCR and HCV‐RT‐LAMP‐AuNPs‐LFB methods 23 …”

“…equipment-free, but need more amplification time for detection of the LoD (Figure6B,C), and the visual result was ambiguous when the amplicons were low 23,25. Our AuNPs-LFB strip was convenient operation and inexpensive (approximately US$2.0 per test).…”

Rapid point of care testing for four bacterial sexually transmitted infections using the portable isothermal loop-mediated nucleic acid amplification eazyplex platform

“…Novel POC test systems using loop-mediated amplification have increasingly been introduced in the last years for different infectious agents, e.g. for malaria in pregnant women [ 8 ], SARS-CoV-2 [ 9 ], or C. trachomatis [ 10 ]. With a rising incidence of STI in selected populations the need for POC tests has increased for this indication with accuracy and reasonable pricing being the main factors required for implementation [ 11 ].…”

Rapid Point of Care Testing for Sexually Transmitted Infections Using a Portable Isothermal Loop Mediated Nucleic Acid Amplification Platform

“…Novel POC test systems using loop-mediated ampli cation have increasingly been introduced in the last years for different infectious agents, e.g. for malaria in pregnant women [5], SARS-CoV-2 [6], or C. trachomatis [7]. With a rising incidence of STI the need of POC tests has increased for this indication with accuracy and reasonable pricing being main factors required for implementation [8].…”