Fabrication and functional application of zein-based core-shell structures: A review.

Core-shell structures exhibit a number of distinct absorptive properties that make them attractive tools for use in a range of industrial contexts including pharmaceuticals, biotechnology, cosmetics, and food/agriculture. Several recent studies have focused on the development and fabrication of zein-based core-shell structures for a range of functional material deliveries. However, no recent review article has evaluated the fabrication of such core-shell structures for food-based applications. In this paper, we therefore survey current approaches to fabricating different zein-based platforms including particles, fibers, films, and hydrogels that have appeared in a variety of functionally relevant applications. In addition, we highlight certain challenges and future research directions in this field, thereby providing a novel perspective on zein-based core-shell structures.

A point-of-care testing platform for on-site identification of genetically modified crops.

Genetically modified (GM) food is still highly controversial nowadays. Due to the disparate policies and attitudes worldwide, demands for a rapid, cost-effective and user-friendly GM crop identification method are increasingly significant for import administration, market supervision, etc. However, as the most-recognized methods, nucleic acid-based identification approaches require bulky instruments, long turn-around times and trained personnel, which are only suitable in laboratories. To fulfil the urgent needs of on-site testing, we develop a point-of-care testing platform that is able to identify 12 types of GM crops in less than 40 minutes without using laboratory settings. Our system integrates sample pre-treatment modules in a microfluidic chip, performs DNA amplification via a battery-powered portable kit, and presents results via eye-recognized colorimetric change. A paraffin-based reflow method and a slip plate-based fluid switch are developed to encapsulate and release amplification primers in individual microwells on demand, thus enabling identification of varied targets simultaneously. Our system offers an efficient, affordable and convenient tool for GM crop identification, thus it will not only benefit customs and market administration bureaus, but also satisfy demands of numerous consumers.

Liquid-liquid biopolymers aqueous solution segregative phase separation in food: From fundamentals to applications-A review.

As a result of the spontaneous movement of molecules, liquid-liquid biopolymer segregative phase separation takes place in an aqueous solution. The efficacy of this type of separation can be optimized under conditions where variables such as pH, temperature, and molecular concentrations have minimal impact on its dynamics. Recently, interest in the applications of biopolymers and their segregative phase separation-associated molecular stratification has increased, particularly in the food industry, where these methods permit the purification of specific particles and the embedding of microcapsules. The present review offers a comprehensive examination of the theoretical mechanisms that regulate the liquid-liquid biopolymers aqueous solution segregative phase separation, the factors that may exert an impact on this procedure, and the importance of this particular separation method in the context of food science. These discussion points also address existing difficulties and future possibilities related to the use of segregative phase separation in food applications. This highlights the potential for the design of novel functional foods and the enhancement of food properties.

CRISPR/Cas12a integrated electrochemiluminescence biosensor for pufferfish authenticity detection based on NiCo2O4 NCs@Au as a coreaction accelerator.

Meat adulteration has brought economic losses, health risks, and religious concerns, making it a pressing global issue. Herein, combining the high amplification efficiency of polymerase chain reaction (PCR) and the accurate recognition of CRISPR/Cas12, a sensitive and reliable electrochemiluminescence (ECL) biosensor was developed for the detection of pufferfish authenticity using NiCo2O4 NCs@Au-ABEI as nanoemitters. In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a is activated upon specific recognition by crRNA, and then it cleaves dopamine-modified single stranded DNA (ssDNA-DA), triggering the ECL signal from the "off" to "on" state. However, without target DNA, the trans-cleavage activity of CRISPR/Cas12a is silenced. By rationally designing corresponding primers and crRNA, the biosensor was applied to specific identification of four species of pufferfish. Furthermore, as low as 0.1 % (w/w) adulterate pufferfish in mixture samples could be detected. Overall, this work provides a simple, low-cost and sensitive approach to trace pufferfish adulteration.

Formation, influencing factors, and applications of internal channels in starch: A review.

Starch, a natural polymer, has a complex internal structure. Some starches, such as corn and wheat starches, have well-developed surface pores and internal channels. These channel structures are considered crucial in connecting surface stomata and internal cavities and have adequate space for loading guest molecules. After processing or modification, the starch-containing channel structures can be used for food and drug encapsulation and delivery. This article reviews the formation and determination of starch internal channels, and the influence of different factors (such as starch species and processing conditions) on the channel structure. It also discusses relevant starch preparation methods (physical, chemical, enzymatic, and synergistic), and the encapsulation effect of starch containing internal channels on different substances. In addition, the role of internal channels in regulating the starch digestion rate and other aspects is also discussed here. This review highlights the significant multifunctional applications of starch with a channel structure.

Electrochemical/photoelectrochemical dual-mode aptasensor for sensitive aflatoxin B1 assay based on distance-modulation strategy using Au NPs/PCZIF-8-ZnO as sensing substrate.

Aflatoxin B1 (AFB1), a hepatotoxic and carcinogenic food contaminant, is commonly found in agricultural food. Herein, Au NPs anchored ZIF-8-derived porous carbon-ZnO (Au NPs/PCZIF-8-ZnO) was firstly synthesized to act as the sensing substrate. Then, a ratiometric electrochemical (EC) and "off-on" photoelectrochemical (PEC) dual-mode paper-based aptasensor was presented for AFB1 detection based on a distance-modulation sensing strategy. The independent signal transduction mechanisms and output mode not only broaden the dynamic detection range but also provide a self-verification to assay results, improving the sensitivity and reliability. The wide detection ranges of 0.1 pg/mL-100 ng/mL (EC mode) and 0.02 pg/mL-100 ng/mL (PEC mode) were obtained using dual-mode aptasensor, with detection limits of 36.7 and 9.3 fg/mL, respectively. The fabricated aptasensor exhibited excellent selectivity, reproducibility and stability. Furthermore, it exhibited good practicability for AFB1 assays in real samples, demonstrating great potential applications for food safety evaluation.

In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria.

In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level.

Development and validation of sensitive and rapid CRISPR/Cas12-based PCR method to detect hazelnut in unlabeled products.

Hazelnut, one of the most popular tree nuts, is widely found in processed food and even very small amounts can trigger severe allergic reactions in susceptible people. Herein, we developed a sensitive and rapid method based on CRISPR and qPCR capable of detecting low-abundance hazelnut in processed food. The assay, known as CRISPR-based nucleic acid test method (Crinac) can detect 1 % of hazelnut in a mixture and allows the species to be identified in a complex processed sample. The detection process can be completed within 60 min. Contributed to amplification via PCR and CRISPR/Cas12a, enables end-fluorescence measurement for the quantification of hazelnut, thus reducing assay time and eliminating the need for costly real-time fluorescence PCR instruments. The assay based on CRISPR/Cas12 and PCR has potential as a sensitive and reliable analytical tool for the detection of food authenticity.

CRISPR/Cas12a-Derived Photoelectrochemical Aptasensor Based on Au Nanoparticle-Attached CdS/UiO-66-NH2 Heterostructures for the Rapid and Sensitive Detection of Ochratoxin A.

The sensitive and accurate detection of ochratoxin A (OTA) is crucial for public health due to its high toxicity. Herein, using Au nanoparticle (NP)-attached CdS/UiO-66-NH2 heterostructures as photoactive materials, a photoelectrochemical (PEC) aptasensor was presented for the ultrasensitive assay of OTA based on a competitive displacement reaction triggering the trans-cleavage ability of CRISPR/Cas12a. In this sensing strategy, methylene blue-labeled single-stranded DNA (MB-ssDNA) was immobilized on the Au NPs/CdS/UiO-66-NH2 electrode to accelerate the separation of the photogenerated carrier, thus producing a significantly increased PEC response. In the presence of OTA, it specifically bound with the aptamer (Apt) and resulted in the release of the activation chain, triggering the trans-cleavage characteristics of CRISPR/Cas12a. MB-ssDNA was cut randomly on the electrode surface to convert the PEC signal from the "on" to the "off" state, thereby achieving a quantitative and accurate detection of OTA. The CRISPR/Cas12a-derived PEC aptasensor exhibited excellent sensitivity and specificity, with a linear range from 100 to 50 ng/mL and a detection limit of 38 fg/mL. Overall, the proposed aptasensor could provide a rapid, accurate, and sensitive method for the determination of OTA in actual samples.

Tetrahedral DNA Nanostructure-Engineered Paper-Based Electrochemical Aptasensor for Fumonisin B1 Detection Coupled with Au@Pt Nanocrystals as an Amplification Label.

Fumonisin B1 (FB1), as one of the highest toxicity mycotoxins, poses a serious threat to animal and human health, even at low concentrations. It is significant and challenging to develop a sensitive and reliable analytical device. Herein, a paper-based electrochemical aptasensor was designed utilizing tetrahedral DNA nanostructures (TDNs) to controllably anchor an aptamer (Apt), improving the recognition efficiency of Apt to its target. First, gold nanoparticles (AuNPs)@MXenes were used as a sensing substrate with good conductivity and modified on the electrode for immobilization of complementary DNA-TDNs (cDNA-TDNs). In the absence of FB1, numerous Apt-Au@Pt nanocrystals (NCs) was hybridized with cDNA and assembled on the sensing interface, which accelerated the oxidation of TMB with H2O2 and produced a highly amplified differential pulse voltammetry (DPV) signal. When the target FB1 specifically bound to its Apt, the electrochemical signal was decreased by releasing the Apt-Au@Pt NCs from double-stranded DNA (dsDNA). On account of the strand displacement reaction by FB1 triggering, the aptasensor had a wider dynamic linear range (from 50 fg/mL to 100 ng/mL) with a lower limit of detection (21 fg/mL) under the optimized conditions. More impressively, the designed FB1 aptasensor exhibited satisfactory performance in corn and wheat samples. Therefore, the TDN-engineered sensing platform opens an effective approach for sensitive and accurate analysis of FB1, holding strong potential in food safety and public health.

Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus.

Capripox viruses (CaPVs), including sheep pox virus (SPV), goat pox virus (GPV), and lumpy skin disease virus (LSDV), are the cause of sheep pox (SPP), goat pox (GTP), and lumpy skin disease (LSD) in cattle. These diseases are of great economic significance to farmers, as they are endemic on farms and are a major constraint to international trade in livestock and their products. Capripoxvirus (CaPV) infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) method for identifying CaPV in goats, sheep, and cattle. Clinical samples were tested and verified. The developed assay was highly specific for target viruses, including GPVSPV and LSDV, which had no cross-reaction with other viruses causing similar clinical symptoms. An artificially synthesized positive control plasmid using the CaPV 32 gene inserted into the vector pMD19-T was used as a template, and the correlation coefficient of the linear regression curve (R2) was 0.9916, the estimated amplification efficiency (E) was 96.06%, and the sensitivity (limit of detection, LOD) was 3.80 copies per reaction. Using the clinical samples as a template, the limit of detection (LOD) was 4.91 × 10-5 ng per reaction (1.60 × 10-5-2.13 × 10-3 ng, 95% confidence interval (CI)), which means that this method was one of the most sensitive detection assays for CaPVs. A total of 85 clinical samples from CaPV-infected animals (goats, sheep, and cattle) and 50 clinical samples from healthy animals were used to test and compare the diagnostic results using the Synergy Brands (SYBR) Green-based PCR method recommended by the World Organization of Animal Health (WOAH). Both diagnostic sensitivity (DSe) (95.8-100%, 95% CI) and diagnostic specificity (DSp) (92.9-100%, 95% CI) results of the real-time quantitative PCR (qPCR) and SYBR Green PCR were 100%, and the kappa value (κ) was 1.0 (1-1, 95% CI). In summary, the assay established based on TaqMan probes was advantageous in high specificity, sensitivity, and general applicability and could be a competitive candidate tool for the diagnosis of CaPV in clinically suspected animals.

Investigation of antibiotic resistance genotypic and phenotypic characteristics of marine aquaculture fish carried in the Dalian area of China.

Due to the long-term and irrational use of antibiotics for the prevention and control of bacterial diseases in aquaculture, antibiotic resistance genes have become a new source of pollution in aquatic products. Factors such as the spread of drug-resistant strains and the horizontal transfer of drug-resistant genes have led to multi-drug resistance in fish-infecting bacteria, which seriously affects the quality and safety of aquatic products. In this study, 50 samples of horse mackerel and puffer fish sold in Dalian aquatic products market and seafood supermarket were collected, and the phenotypic characteristics of the bacteria carried by the fish for drugs such as sulfonamides, amide alcohols, quinolones, aminoglycosides and tetracyclines were tested and analyzed, and the resistance genes carried by fish samples were detected by SYBG qPCR. Our statistical analyses demonstrated that the drug resistance phenotypes and genotypes of bacteria carried by mariculture horse mackerel and puffer fish in the Dalian area of China were complex, and the multi-drug resistance rate reached 80%. Among the examined antibiotics, the resistance rates to cotrimoxazole, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, levofloxacin, kanamycin, and florfenicol exceeded 50%, whereas the resistance rates to gentamicin and tobramycin were 26 and 16%, respectively. The detection rate of the drug resistance genes tetA, sul1, sul2, qnrA, qnrS, and floR exceeded 70% and all samples carried more than three drug resistance genes. The correlation analysis of drug resistance genes and drug resistance phenotypes showed that the detection of the drug resistance genes sul1, sul2, floR, and qnrD was correlated with the detection of drug resistance phenotypes (p < 0.01). However, the correlation between the resistance genes cmlA, cfr, tetA, qnrA, qnrS, and aac(6')-Ib-cr and the corresponding resistance phenotype was not significant (p > 0.05). In general, our findings indicated that the multi-drug resistance of bacteria carried by marine horse mackerel and puffer fish in the Dalian area was serious. From the perspective of drug resistance rate and drug resistance gene detection rate, the aminoglycosides gentamicin and tobramycin are still considered effective in controlling bacterial infection in marine fish in the study area. Collectively, our findings provide a scientific basis for the management of drug use in mariculture, which can prevent the transmission of drug resistance through the food chain and minimize the associated human health risks.

Establishment of a Rapid LAMP Assay for Aeromonas hydrophila and Comparison with the Application of qPCR.

The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/µL (0.315-1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/µL (2.084-8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778-1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites.

Precise Authenticity of Quinoa, Coix Seed, Wild Rice and Chickpea Components Using Optimized TaqMan Real-Time PCR.

Functional food such as, quinoa, coix seed, wild rice and chickpea have experienced rapidly increasing demand globally and exhibit high economic values. Nevertheless, a method for rapid yet accurate detection of these source components is absent, making it difficult to identify commercially available food with labels indicating the presence of relevant components. In this study, we constructed a real-time quantitative polymerase chain reaction (qPCR) method for rapid detection of quinoa, coix seed, wild rice and chickpea in food to identify the authenticity of such food. Specific primers and probes were designed with 2S albumin genes of quinoa, SAD genes of coix seed, ITS genes of wild rice and CIA-2 genes of chickpea as the target genes. The qPCR method could specifically identify the four wild rice strains, yielding, LODs of 0.96, 1.14, 1.04 and 0.97 pg/µL quinoa, coix seed, wild rice and chickpea source components, respectively. Particularly, the method allowed the identification of the target component with content below 0.01%. A total of 24 commercially available food samples of different types were detected by using the method and the results indicate that the developed method is applicable to the detection of different food matrices, as well as authenticity verification in deeply processed food.

Real-time qPCR for the detection of puffer fish components from Lagocephalus in food: L. inermis, L. lagocephalus, L. gloveri, L. lunaris, and L. spadiceus.

Puffer fish is a type of precious high-end aquatic product, is widely popular in Asia, especially in China and Japan, even though it naturally harbors a neurotoxin known as tetrodotoxin (TTX) that is poisonous to humans and causes food poisoning. With the increasing trade demand, which frequently exceeds existing supply capacities, fostering fraudulent practices, such as adulteration of processed products with non-certified farmed wild puffer fish species. To determine the authenticity of puffer fish processed food, we developed a real-time qPCR method to detect five common puffer fish species in aquatic products: Lagocephalus inermis, Lagocephalus lagocephalus, Lagocephalus gloveri, Lagocephalus lunaris, and Lagocephalus spadiceus. The specificity, cross-reactivity, detection limit, efficiency, and robustness of the primers and probes created for five species of puffer fish using TaqMan technology have been determined. No cross-reactivity was detected in the DNA of non-target sample materials, and no false-positive signal was detected; the aquatic products containing 0.1% of a small amount of wild puffer fish materials without certification can be reliably tracked; the statistical p-value for each method's Ct value was greater than 0.05. The developed qPCR method was sensitive, highly specific, robust, and reproducibility, which could be used to validate the authenticity of wild puffer fish in aquatic products sold for commercial purposes.

CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity.

Food authenticity is a critical issue associated with the economy, religion, and food safety. Herein, we report a label-free and colorimetric nucleic acid assay for detecting DNA barcodes, enabling the determination of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR associated protein) to specifically recognize the polymerase chain reaction (PCR) products for further trans-cleavaging the peroxidase-mimicking G-quadruplex DNAzyme. Based on this principle, the presence of the cytochrome oxidase subunit I gene could be directly observed with the naked eye via the color change of 3,3',5,5'-tetramethylbenzidine sulfate (TMB). The whole detection process, including PCR amplification and TMB colorimetric analysis, can be completed within 90 min. The proposed assay can detect pufferfish concentrations diluted to 0.1% (w/w) in a raw pufferfish mixture, making it one of the most sensitive methods for food authenticity. The robustness of the assay was verified by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay is advantageous in easy signal readout, high sensitivity, and general applicability and thus could be a competitive candidate for food authenticity.

Visual LAMP method for the detection of Vibrio vulnificus in aquatic products and environmental water.

BACKGROUND: A visual, rapid, simple method was developed based on a loop-mediated isothermal amplification (LAMP) assay to detect Vibrio vulnificus in aquatic products and aquaculture waters. RESULTS: Genomic DNA was extracted from Vibrio vulnificus using the boiling method, and optimized primers were used to detect the gyrB gene using a visual LAMP method. The sensitivity of the assay was 10 fg/µL, and the obtained results were stable and reliable. Out of 655 aquatic product samples and 558 aquaculture water samples, the positive rates of Vibrio vulnificus detection were 9.01% and 8.60%, respectively, which are markedly higher than those of the traditional culture identification methods. CONCLUSION: The relatively simple technical requirements, low equipment cost, and rapid detection make the visual LAMP method for the detection of Vibrio vulnificus a convenient choice for field detection in the aquaculture industry.

High-Efficiency Reducing Strain for Producing Selenium Nanoparticles Isolated from Marine Sediment.

Selenium nanoparticles (SeNPs) are all important for research because they exhibit a higher degree of absorption and lower toxicity than that of their organic and inorganic forms. At present, there are few reports on marine strains that can reduce Se(IV) to generate Se(0). In this study, a strain that reduces sodium selenite to SeNPs with high efficiency was screened from 40 marine strains. The SeNPs-S produced by the whole cells and SeNPs-E produced by the extracellular extract were characterized by FTIR, UV, Raman, XRD and SEM. Based on the results, the two kinds of SeNPs exhibited obvious differences in morphology, and their surfaces were capped with different biomacromolecules. Due to the difference in shape and surface coating, opposite results were obtained for the antibacterial activity of SeNPs-S and SeNPs-E against Gram-positive and Gram-negative bacteria. Both SeNPs-S and SeNPs-E exhibited no obvious cytotoxicity at concentrations up to 100 µg/mL, but SeNPs-E retained lower cytotoxicity when its concentration increased to 200 µg/mL. This is the first report on the detailed difference between the SeNPs produced by whole cells and cell extracts.

Validation of qPCR from a crude extract for the rapid detection of white spot syndrome virus.

White spot disease (WSD) has posed a serious threat to the China and the global shrimp aquaculture. In order to diagnose white spot syndrome virus (WSSV) early and prevent the spread and outbreak of WSD, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay from the crude extract of samples heated lysis was established, which was further compared with the universal qPCR assay to verify the shrimp samples. The limit of detection (LOD) of pre-amplification qPCR assay and universal qPCR assay was 2.80 copies and 20.57 copies per reaction at 95% CI, respectively. It had good WSSV specificity and did not show cross-detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreas necrosis disease (AHPND), necrotizing hepatopancreatitis bacteria (NHPB), and decapod iridescent virus 1 (DIV1). A total of 36 shrimp samples were detected as WSSV DNA positive by pre-amplification qPCR with crude extract from samples heated lysis and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5 ~ 99.9%, 95% CI) and 100% (81.5 ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms. Supplementary Information: The online version contains supplementary material available at 10.1007/s10499-022-00920-9.

Clinical Validation of DNA Extraction-Free qPCR, Visual LAMP, and Fluorescent LAMP Assays for the Rapid Detection of African Swine Fever Virus.

The global pig industry and food safety are seriously threatened by outbreaks of African swine fever (ASF). To permit early diagnosis of African swine fever virus (ASFV), prevent its spread, and limit its outbreaks, a highly sensitive diagnostic method that can be performed at pig farms is required. Herein, we established DNA extraction-free real-time PCR (qPCR), visual loop-mediated isothermal amplification (LAMP), and fluorescent LAMP assays, which were compared with the results of World Organization for Animal Health (OIE) qPCR to assess ASFV-infected clinical samples. Based on plasmid DNA, the limit of detection for the three assays and OIE qPCR were 5.8 copies/µL. All four assays had good ASFV specificity and showed no cross-reactivity with other tested viruses. These assays were used to diagnose 100 clinical samples. The assays showed good diagnostic consistency, with kappa values of 1.0, 0.84, and 0.88, respectively. Compared with OIE qPCR, the diagnostic specificity/sensitivity of DNA extraction-free qPCR, visual LAMP, and fluorescent LAMP assays were 100%/100%, 100%/87.1%, and 100%/90.32%, respectively. The assays eliminated the need for DNA extraction and are more suitable for ASF diagnosis by inexperienced farmers in low-resource environments, making them a good choice for on-site monitoring of pig farms.