Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2)

Abstract

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A

“…Currently, the commonly used molecular detection techniques for PEDV, TGEV and PoRVA include RT-PCR, nested RT-PCR, qRT-PCR, reverse transcription loop-mediated isothermal amplification, reverse transcription recombinase-aided amplification, and CRISPR-Cas nucleic acid detection (Marthaler et al, 2014;Areekit et al, 2022;Wu et al, 2022;Lazov et al, 2023;Xia et al, 2024). RT-PCR and nested RT-PCR are not capable of quantitative analysis, and their operation is cumbersome, time-consuming and less sensitive.…”

Triplex-Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Immunoassay for the Simultaneous Detection of Three Pathogens of Porcine Viral Diarrhea Syndrome in Swine

“…The triplex LAMP–LFD assay developed in this study could simultaneously detect three target viruses within 30 min, with detection limits of 2.40 × 10 1 copies/μL, 2.89 × 10 1 copies/μL, and 2.52 × 10 1 copies/μL, respectively. When PEDV and PCV2 were detected simultaneously by a two-phase LAMP–LFD method, amplification was completed in approximately 20 min, and the detection limits were 0.1 ng/µL and 0.246 ng/µL, respectively [ 56 ]. Through conversion, the detection limit of the triplex LAMP–LFD assay was lower.…”

“…(A) Single LAMP–LFD sensitivity testing, (B) duplex LAMP–LFD sensitivity testing; Table S1: Sequences of qPCR primers and probes for detecting PEDV, PoRV and PBoV; Table S2: Informations of 125 animal feces samples; Table S3: Comparison of molecular methods for the detection of porcine diarrhea virus. References [ 25 , 50 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ] are cited in the supplementary materials.…”

Application of loop-mediated isothermal amplification combined with lateral flow assay visualization of Plasmodium falciparum kelch 13 C580Y mutation for artemisinin resistance detection in clinical samples

“…Loop-mediated isothermal amplification (LAMP) technology has been utilized to detect the presence of various pathogens, such as malaria parasites, fungi, viruses, and bacteria ( Areekit et al., 2022 ; Gomez-Gutierrez and Goodwin, 2022 ; Lee et al., 2020 ; Mohon et al., 2016 ; Ortega et al., 2020 ; Srisawat and Panbangred, 2015 ; Tang et al., 2011 ; Xiao and Li, 2021 ; Yin et al., 2016 ). LAMP technology can be used for SNP detection using the mismatch approach.…”