Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples

Abstract

Chlamydia trachomatis is the most common sexually transmitted human pathogen. Infection results in minimal to no symptoms in approximately two-thirds of women and therefore often goes undiagnosed. C. trachomatis infections are a major public health concern because of the potential severe long-term consequences, including an increased risk of ectopic pregnancy, chronic pelvic pain, and infertility. To date, several point-of-care tests have been developed for C. trachomatis diagnostics. Although many of them are fast and specific, they lack the required sensitivity for large-scale application. We describe a rapid and sensitive form of detection directly from urine samples. The assay uses recombinase polymerase amplification and has a minimum detection limit of 5 to 12 pathogens per test. Furthermore, it enables detection within 20 minutes directly from urine samples without DNA purification before the amplification reaction. Initial analysis of the assay from clinical patient samples had a specificity of 100% (95% CI, 92%-100%) and a sensitivity of 83% (95% CI, 51%-97%). The whole procedure is fairly simple and does not require specific machinery, making it potentially applicable in point-of-care settings.

Distribution of sexually transmitted diseases in a group of symptomatic male patients using urine samples and PCR technique / Distribuția bolilor cu transmitere sexuală într-un grup de barbați simptomatici utilizand probe de urina si tehnica PCR

“…The most frequently isolated STD pathogen was C. trachomatis (frequency of 32.69%, see Table I). Other studies also reported C. trachomatis as the most prevalent bacterial STD (26,28). Next two in the prevalence scale, U. urealyticum and N. gonorrhoeae (frequency of 17.31% and 13.46%), were found in relatively equal proportions, a finding also consistent with prevalence studies conducted in other countries (27,29).…”

Quantification of HIV-1 DNA Using Real-Time Recombinase Polymerase Amplification

“…3 Recombinase polymerase amplification (RPA) offers significant advantages over other isothermal amplification techniques for point-of-care applications: it requires a lower amplification temperature, is tolerant to impure samples, amplifies targets to detectable levels within minutes, and uses lyophilized enzymes to enable storage and transport at room temperature. 4,5 For these reasons, a number of recent reports have proposed RPA-based strategies for the detection of pathogens. [6][7][8][9][10] Although some papers demonstrate a relationship between nucleic acid concentration and onset of amplification, 10,11 to the best of our knowledge, RPA has not yet been implemented to quantify sample concentration using a standard curve.…”

Development of a real-time recombinase polymerase amplification assay for rapid and sensitive detection of porcine circovirus 2

“…Furthermore, RPA has demonstrated a certain tolerance to common PCR inhibitors [6], minimizing sample preparation requirements. The present study showed that genomic DNA or RNA, obtained from the urine and stool samples by direct dilution and boiling, could be successfully used as the template of RPA [7,12]. Our laboratory is also focusing on the simple and rapid genomic extraction to make RPA a useful in-field and point-of-care diagnostic technique.…”