Rapid and visual detection of Lawsonia intracellularis with an improved recombinase polymerase amplification assay combined with a lateral flow dipstick

Abstract

BackgroundLawsonia intracellularis (L. intracellularis) is the etiologic agent of porcine proliferative enteropathy (PPE), which is reported in many swine breeding countries all over the world, and has caused enormous economic losses in intensive pig production systems. Therefore, the aim of this study was to develop a simple and rapid method for on-site detection of Lawsonia intracellularis (L. intracellularis). As the isothermal recombinase polymerase amplification (RPA) can be performed at a constant temperature and its product is directly observed on a lateral-flow dipstick (LFD) with naked eyes without electrophoresis, the RPA-LFD assay should be useful for field diagnosis of L. intracellularis as well as its detection from clinical samples.ResultsThe established RPA-LFD assay could be finished in 30 min at a wide temperature range of 25 to 40 °C, and the amplicons could be visualized by naked eyes. The developed RPA-LFD assay was specific to dnaA gene of L. intracellularis, and did not detect nucleic acids extracted from other common gastrointestinal pathogens. The minimum detection of this RPA-LFD method was 400 L. intracellularis per reaction, which was as sensitive as conventional PCR. Further, the RPA-LFD assay was performed with 150 clinical fecal samples and the detection results were compared with conventional PCR. Results showed that the coincidence rate of RPA-LFD and conventional PCR was 100%.ConclusionsThe combined RPA with LFD assay provides a simple, rapid, specific and sensitive alternative for field diagnosis of L. intracellularis infection.

Rapid and simple detection of Glaesserella parasuis in synovial fluid by recombinase polymerase amplification and lateral flow strip

“…The detection rate of RPA-LF-GPS is 98.1% (51/52), and that of PCR is 90.4% (47/52). Statistic analysis showed that RPA-LF-GPS and PCR had equal sensitivity ( P > 0.05), which is consistent with previous reports about RPA [29]. This result showed that the RPA-LF-GPS performed well with clinic samples, which make it a potential to replace PCR.…”

Development of recombinase polymerase amplification assays for rapid and visual detection of canine distemper virus infecting giant panda

“…Magnetic bead-based extraction methods will be used in future research to prepare the RPA templates from clinical samples. Extraction using magnetic beads is easy and time-saving, and it does not require potentially dangerous procedures or specialized laboratory equipment [ 26 , 27 ]. The combination of the RT-RPA-VF assay with magnetic bead-based nucleic acid extraction techniques represents a very promising point-of-care detection method.…”

Review of methods for the detection of Lawsonia intracellularis infection in pigs

“…2 ). 50 , 62 , 64 , 81 , 97 , 99 , 111 Pigs experimentally infected with L. intracellularis are expected to show L. intracellularis shedding in feces by 7 dpi and may sustain shedding of the bacterium for up to 10 wk after infection ( Fig. 2B ).…”

“… 31 , 96 The gene target commonly used is the 16S ribosomal DNA gene (16S rDNA), 64 although aspA 99 and dnaA 111 have also been used as markers for L. intracellularis . 42 , 64 , 111 Various primer pairs have been used for detection of L. intracellularis ( Table 2 ). “In situ” amplification in tissue samples has also been reported and could potentially be a good candidate to substitute for tissue IHC.…”