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.
Highlights
Lawsonia 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
The recombinase polymerase amplification (RPA) basic reaction was performed by using unlabeled primers at 37 °C for 25 min to test the usability of the primers
The sequencing results of RPA products were in good agreement with the dnaA gene of L. intracellularis
Summary
Lawsonia 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. Current available diagnostic methods for L. intracellularis infection in live animals include serological tests for detecting antibody against L. intracellularis, such as enzyme-linked immunosorbent assay (ELISA) [12,13,14], immunoperoxidase monolayer assay (IPMA) [15], indirect immunofluorescence assay (IFA) [16], and methods for detecting L. intracellularis in fecal samples, such as polymerase chain reaction (PCR)-based tests [17,18,19,20,21,22,23,24] and IFA [25] Among these methods, bacteria maintenance in vitro has limited the use of serological tests, since the cultivation of the obligate intracellular L. intracellularis requires establishment of suitable cell lines and L. intracellularis cannot be cultured in conventional cell-free medium. There is an urgent need for a rapid and reliable pen side diagnostic assay for a better detection and control of this economically important disease of swine
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