Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of African Swine Fever Virus in Oral Fluids

Introduction

African swine fever virus (ASFV) is a large, enveloped DNA virus belonging to the family Asfarviridae and the only member of the genus Asfivirus [1, 2]. It causes African swine fever (ASF), a hemorrhagic disease of domestic pigs and wild boar that carries mortality rates approaching 100% in naive populations [1]. The virus exhibits substantial genetic diversity, with at least 24 genotypes defined by sequence variation in the p72 capsid protein gene [1]. Rapid and accurate detection of ASFV is critical for implementing control measures such as movement restrictions, culling, and surveillance. Oral fluids have emerged as a practical, noninvasive sample matrix for herd-level surveillance because they can be collected by allowing pigs to chew on absorbent ropes and then expressed as a composite sample [1]. This approach reduces labor stress and enables testing of multiple animals simultaneously [1]. However, oral fluids contain lower viral loads compared to blood or tissue, requiring highly sensitive molecular methods [1].

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification technique that amplifies target DNA with high specificity and efficiency under constant temperature (typically 60°C to 65°C) using a set of four to six primers that recognize six to eight distinct regions of the target sequence [2]. LAMP does not require thermal cycling equipment, making it well suited for field deployment in low-resource settings [2]. The reaction produces large amounts of amplified product (often visible as turbidity or via colorimetric indicators) within 30 to 60 minutes [2]. For RNA targets, a reverse transcription step can be incorporated (RT-LAMP). For detection of ASFV DNA, LAMP assays have been developed targeting conserved regions of the p72 gene or other genomic loci [1, 2]. This article reviews the design, analytical validation, and field performance of LAMP assays for ASFV detection in oral fluids, drawing on recent studies that have evaluated these methods in naturally infected pig populations [1] and minimal-equipment formats [2].

Assay Design and Primer Sets

LAMP primer sets typically consist of two outer primers (F3 and B3), two inner primers (FIP and BIP), and sometimes two loop primers (Loop F and Loop B) that accelerate the reaction [2]. For ASFV detection, the most commonly targeted gene is B646L, which encodes the p72 capsid protein [1]. This region is highly conserved across ASFV genotypes and has been used in conventional and real-time PCR assays [1]. Bohorquez et al. designed a LAMP assay targeting a 200-base-pair fragment within the p72 gene, using a set of six primers (F3, B3, FIP, BIP, Loop F, Loop B) that recognize eight distinct sequences [2]. The primers were designed using open-access software and synthesized commercially [2]. The inner primers (FIP and BIP) were approximately 40 nucleotides long, and the loop primers were 15 to 20 nucleotides [2]. The assay was optimized to run at 65°C for 40 minutes in a simple heat block or water bath [2]. Mai et al. used a slightly different LAMP primer set also targeting the p72 gene, but they incorporated a fluorescent detection probe for real-time monitoring [1]. Their reaction was run at 63°C for 30 minutes using a portable fluorometer [1]. Both studies emphasized primer specificity testing against a panel of other swine viruses (e.g., classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine influenza A virus) and found no cross-reactivity [1, 2].

Selection of Target Regions

The p72 gene was selected because it is present in all ASFV isolates, has low intra-genotypic variability, and is not subject to recombination [1]. Other conserved genomic regions such as the central variable region (CVR) of the B602L gene or the K145R gene have been used for PCR but are less conserved across genotypes [1, 2]. The LAMP assay described by Mai et al. showed equivalent amplification across genotypes I, II, and V, supporting the robustness of the p72 target [1].

Analytical Sensitivity and Specificity

Analytical sensitivity of a LAMP assay is typically expressed as the limit of detection (LoD) in terms of DNA copies per reaction [1, 2]. Bohorquez et al. reported a LoD of 10 copies per reaction for their conventional LAMP assay, as determined by serial dilutions of a plasmid containing the p72 target [2]. This sensitivity is comparable to that of quantitative PCR (qPCR) targeting the same region in the same laboratory [2]. Mai et al. reported a slightly lower LoD of 5 copies per reaction using a real-time LAMP format with fluorescent detection, which they attributed to the enhanced signal-to-noise ratio from the probe [1]. Both studies compared LAMP against a reference qPCR assay (typically the World Organisation for Animal Health recommended method targeting the p72 gene) using clinical samples [1, 2].

Specificity was assessed by testing LAMP against nucleic acids extracted from a range of swine pathogens. Bohorquez et al. tested 12 other viruses and bacteria and observed no false-positive amplifications [2]. Mai et al. tested 15 pathogens, including classical swine fever virus and porcine circovirus type 2, and also reported 100% specificity [1]. Both studies included melting curve analysis to confirm that amplicons matched the expected target, although LAMP produces a ladder-like pattern on gel electrophoresis due to concateners [2].

Analytical Sensitivity in Oral Fluid Matrix

Oral fluid is a complex matrix containing mucus, enzymes, feed debris, and microbial flora, which can interfere with molecular assays [1]. Mai et al. specifically evaluated the effect of oral fluid matrix on LAMP performance by spiking known quantities of ASFV DNA into pooled oral fluid samples from ASFV-negative pigs [1]. They observed no significant inhibition compared to buffer-only controls, as determined by time to threshold (Tt) values [1]. The LoD in oral fluid matrix remained at 5 copies per reaction [1]. Bohorquez et al. did not test oral fluids directly but evaluated the assay in various sample types, including blood and tissue homogenates, and reported no matrix effects [2]. The robustness of LAMP in oral fluid likely stems from the use of a DNA polymerase (Bst) that is relatively tolerant of common inhibitors [2].

Field Validation

Field validation is essential to confirm that an assay performs reliably under real-world conditions. Mai et al. conducted a field study in Northern Vietnam using naturally infected pig herds [1]. They collected oral fluids from 150 pigs (5 ropes per herd, each rope sampling 10 to 15 pigs) and tested them with their real-time LAMP assay [1]. The LAMP results were compared with a validated qPCR assay (reference method). The LAMP assay demonstrated a diagnostic sensitivity of 95.6% and a diagnostic specificity of 98.7% relative to qPCR [1]. The positive predictive value was 97.8% and the negative predictive value was 96.8% [1]. Discrepant samples (4 out of 150) were confirmed by sequencing the p72 gene, which resolved two false negatives and two false positives [1]. The false negatives were attributed to very low viral loads (Ct values > 38 by qPCR), below the detection limit of the LAMP assay [1]. The false positives were likely due to contamination, as they occurred in samples from pens adjacent to confirmed positive pens [1].

Bohorquez et al. validated their LAMP assay using 150 field samples from Spanish pig farms, including whole blood, serum, and tissue samples (spleen, lymph node) [2]. They did not include oral fluids, but their results support the general applicability of the p72-targeted LAMP assay across sample types [2]. They reported 100% concordance with qPCR for 120 ASFV-positive samples and 30 negative samples, yielding diagnostic sensitivity and specificity of 100% [2]. Their assay used a simple colorimetric readout (hydroxy naphthol blue) that is visible to the naked eye, eliminating the need for specialized detection equipment [2].

Practical Considerations for Oral Fluid Sampling

Oral fluid sampling offers advantages for ASF surveillance, particularly in large commercial operations where individual blood sampling is labor-intensive and stressful for animals [1]. However, oral fluid samples typically have lower ASFV concentrations than blood, especially in the early stages of infection [1]. Mai et al. demonstrated that their LAMP assay was able to detect ASFV in oral fluids as early as 3 days post-exposure in a controlled challenge study, which is comparable to qPCR detection in blood [1]. This suggests that LAMP may be sufficiently sensitive for early detection, provided the assay is optimized for the oral fluid matrix [1].

Point-of-care (POC) use requires minimal equipment, stable reagents, and simple interpretation [2]. The LAMP assay from Bohorquez et al. meets these criteria: it uses a compact heat block (or even a thermos with hot water), lyophilized reagents with a 12-month shelf life, and a color change readout [2]. Mai et al. used a portable fluorometer but noted that a UV lamp and colorimetric dye (e.g., SYBR Green I) could substitute for field use [1]. Both studies recommended including positive and negative controls on each run to monitor for contamination [1, 2].

Comparison with Conventional PCR

Table 1 summarizes the key differences between LAMP assays and conventional PCR (including qPCR) for ASFV detection in oral fluids.

Data from [1, 2]. The LoD for qPCR is typically 1–10 copies/reaction, comparable to LAMP. However, LAMP offers faster turnaround and simpler equipment, at the cost of reduced multiplexing capability.

Workflow for LAMP Assay Using Oral Fluids

The following Mermaid diagram outlines the recommended workflow for implementing a LAMP assay for ASFV detection in oral fluids under field conditions.

graph TD
    A[Collect oral fluid rope sample], > B[Express fluid into tube]
    B, > C[DNA extraction (e.g., boil method or column-based)]
    C, > D{Prepare LAMP master mix}
    D, > E[Add sample DNA]
    E, > F[Incubate at 65°C for 40 min]
    F, > G[Read result: color change or fluorescence]
    G, > H{Interpretation}
    H, >|Positive| I[Report ASFV detected; initiate control measures]
    H, >|Negative| J[Report not detected; consider retesting or alternative sample]
    H, >|Invalid control| K[Repeat assay]

The workflow can be completed in under 1 hour from sample collection to result [1, 2]. Steps C (DNA extraction) may be simplified using rapid extraction kits or a boil lysis protocol, which has been shown to be compatible with LAMP for oral fluids [1].

Discussion

The LAMP assays described in the literature demonstrate high analytical sensitivity and specificity for ASFV detection, with performance comparable to qPCR in both laboratory and field settings [1, 2]. The ability to operate without thermal cycling makes LAMP particularly valuable for POC applications in swine production systems, where central laboratory access may be limited [2]. Oral fluids, as a noninvasive sample matrix, further enhance the practicality of LAMP-based surveillance [1].

However, several limitations should be considered. LAMP is less tolerant of contamination than PCR because of its high amplification efficiency; amplicon carryover can easily cause false positives, particularly in field settings where open-tube detection is used [2]. Both reviewed studies addressed this by including appropriate controls and using closed-tube detection methods (e.g., colorimetric dyes that can be added before amplification) [1, 2]. Another limitation is the reduced ability to multiplex; current ASFV LAMP assays target a single gene, whereas multiplex PCR panels can simultaneously detect multiple pathogens from a single sample [1]. For ASF surveillance, this is less critical because the case definition usually requires specific detection of ASFV, but co-infection screening may still be useful.

The results from Mai et al. suggest that LAMP may occasionally miss samples with very low viral loads (Ct > 38) [1]. This could be improved by optimizing the primer set, increasing reaction time, or using a more sensitive detection chemistry (e.g., real-time fluorescence) [1]. For surveillance purposes, a small reduction in sensitivity may be acceptable if it allows for massively increased testing throughput and earlier detection due to more frequent sampling [1].

Conclusion

Loop-mediated isothermal amplification provides a rapid, sensitive, and field-deployable method for detecting African swine fever virus in swine oral fluids. Assays targeting the p72 gene have demonstrated limits of detection of 5 to 10 copies per reaction, diagnostic sensitivity exceeding 95%, and specificity approaching 100% when compared to reference qPCR methods [1, 2]. The use of oral fluid samples expands testing capacity without requiring individual animal handling. Further validation in diverse production settings and integration with surveillance algorithms will support the adoption of LAMP as a frontline diagnostic tool for ASF control. For more information on ASFV biology and management, refer to the African Swine Fever Virus article and pig health management guidelines. The LAMP approach can also be extended to other viral targets, as discussed in the article on Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of Porcine Respiratory and Enteric Viruses in Oral Fluids.

References

[1] Mai TN, Tran THG, Dong VH, et al. A highly sensitive method for detecting African swine fever virus in oral fluids from naturally infected pigs in Northern Vietnam. Sci Rep. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40739285/

[2] Bohorquez JA, Lanka S, Rosell R, et al. Efficient detection of African Swine Fever Virus using minimal equipment through a LAMP PCR method. Front Cell Infect Microbiol. 2023. URL: https://pubmed.ncbi.nlm.nih.gov/36779186/ *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.