A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection

Abstract

A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients’ serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection

“…The developed lateral flow RPA for the detection of Borrelia burgdorferi had analytical sensitivity of one copy of constructed plasmid and 10 fg of DNA [35]. The sensitive LFD-RPA developed for detection of ORF virus with detection limit of 80 copies per reaction [27].…”

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

“…The exponential amplification is carried out on the target fragment by initiating a chain exchange reaction to form DNA synthesis [14]. The amplified products can typically be detected after operating only 5-10 min optimally at 37-40 • C. Since the first report in 2006, RPA has been emerging in medical diagnosis [15][16][17], foodborne pathogens [18,19], and genetically modified crops [20,21], as well as research on viruses [22,23]. For the detection of Salmonella, the fimY gene has been reported as unique to the Salmonella species and hence is suitable to use as an appropriate target gene for detecting Salmonella [24].…”

Direct Diagnostic Tests for Lyme Disease

“…Many polymerase chain reaction (PCR) assays have been geared to analyze small volumes of blood, plasma, or other sample types. In Lyme disease, outside the EM in skin, the infectious agent resides in very low numbers in tissue and body fluids, and standard PCR methods, as applied to B. burgdorferi detection, have suffered from poor sensitivity, especially in blood and cerebrospinal fluid [21][22][23][24][25]. An illustrative strategy to increase PCR sensitivity without diminishing specificity is to start with a larger specimen volume and/ or use target enrichment methods [26].…”