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Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by Clostridium tetani. C. tetani is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to identify the fragments of the neurotoxin gene of C. tetani. Primers and the exo probe targeting the conserved region were designed, and the resulting amplicons could be detected in less than 20 min, with a detection limit of 20 copies/reaction. The RPA assay displayed good selectivity, and there were no cross-reactions with other infectious bacteria common in penetrating wounds. Tests of target-spiked serum and pus extract revealed that RPA is robust to interfering factors and has great potential for further development for biological sample analysis. This method has been confirmed to be reliable for discriminating between toxic and nontoxic C. tetani strains. The RPA assay dramatically improves the diagnostic efficacy with simplified device architecture and is a promising alternative to real-time PCR for tetanus detection.

Bacterial wilt caused by Ralstonia solanacearum is a serious soilborne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 103 CFU/g artificially inoculated tobacco stems, and 104 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

Human norovirus (NOV) is a common and serious virus that accounts for sporadic cases and outbreaks of gastroenteritis. This study aimed to develop rapid, reliable and portable detection systems by coupling reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for NOV genotype GII.4. Here, three primers for RNA-dependent RNA polymerase gene of NOV were designed and screened. Then, RT-RPA products were detected using CRISPR-Cas12a system by combing with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 40 min, with the detection limit of up to 9.65 × 102copies/mL and no cross-reactivity with metapneumovirus, bocavirus, seoul virus, and respiratory syncytial virus. Furthermore, the detection coincidence rates of RT-RPA-Cas12a-based fluorescence and LF with qRT-PCR were 98.3%. Therefore, the present study suggests that both RT-RPA-Cas12a-based fluorescence and LF are promising sensitive, specific and alternative method for diagnosis of NOV genotype GII.4 without ancillary equipment.

Cucurbit leaf crumple virus (CuLCrV), a single-stranded DNA virus belonging to the species Cucurbit leaf crumple virus in the genus Begomovirus, causes cucurbit leaf crumple disease in many species of the Cucurbitaceae family. This report describes the development and standardization of field-based isothermal recombinase polymerase amplification (RPA) and exonuclease RPA (exo-RPA) assays for the detection and diagnosis of CuLCrV in the leaves of watermelon, squash and pumpkin, and the fruits of watermelon. The oligonucleotide primers were made to target a portion of the BL1 movement protein. The primer set used in the RPA was highly specific for CuLCrV and did not show a cross-reaction with other cucurbit viruses nor in tissues with co-infections of CuLCrV with cucurbit yellow stunting disorder virus (CYSDV) and/or squash vein yellowing virus (SqVYV). These assays were sensitive, producing a positive signal with 1 fg/uL of purified total DNA and at a dilution of 1 × 10–5 of crude extract from CuLCrV-infected watermelon plants. Incorporation of a 6-fluorescein amidite (6-FAM) probe into the exo-RPA assay enabled a total sample processing and detection time of 30 min and positive reactions were detectable in different tissues including watermelon leaves, fruits, and pips (seeds lacking an embryo). The exo-RPA was efficiently used in the detection of CuLCrV in 21 cucurbit plants growing in different regions of southeastern U.S. A sensitive, rapid and easy field-based method for the detection and diagnosis of CuLCrV in watermelon was developed.

Quantitative PCR (qPCR), loop-mediated amplification (LAMP), and lateral flow strip-based recombinase polymerase amplification (RPA-LFS) assays were assessed for early detection of Phytophthora infestans, the global causal agent of potato and tomato late blight, on passive wind-powered spore traps known as Spornados. Spore traps were deployed in potato and tomato fields during the 2018, 2019, and 2020 growing seasons in the provinces of Alberta, British Columbia, Manitoba, Prince Edward Island, and Ontario. All assays used DNA extracts from Spornado cassette membranes targeting the P. infestans nuclear ribosomal internal transcribed spacer. A total of 1,003 Spornado samples were qPCR tested, yielding 115 positive samples for P. infestans spores. In further assessment of these samples, LAMP detected P. infestans in 108 (93.9%) of 115 qPCR positive samples, and RPA-LFS detected it in 103 (89.6%). None of the assays showed cross-reaction with other Phytophthora species or pathogenic fungi known to infect potato and tomato. The qPCR detected ≤1 fg of P. infestans DNA, and LAMP and RPA-LFS amplified 10 fg in as little as 10 min. All assays detected P. infestans before the first report of late blight symptoms in commercial potato or tomato fields within each region or province. The combination of Spornado passive samplers with qPCR, LAMP, or RPA-LFS proved a valuable spore trapping system for early surveillance of late blight in potato and tomato. Both LAMP and RPA-LFS showed potential as alternative approaches to qPCR for in-field monitoring of P. infestans.

Scale drop disease virus (SDDV) is a major pathogen of Asian sea bass that has emerged in many countries across the Asia Pacific since 1992 and carries the potential to cause drastic economic losses to the aquaculture sector. The lack of an approved vaccine for SDDV necessitates timely prevention as the first line of defence against the disease, but current diagnostic platforms still face challenges that render them incompatible with field applications, particularly in resource-limited settings. Here, we developed a novel detection platform for SDDV based on a CRISPR-Cas12a-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA-Cas12a). Using the viral adenosine triphosphatase (SDDV-ATPase) gene as a target, we achieved the detection limit of 40 copies per reaction and high specificity for SDDV. The coupling with fluorescence and lateral flow readouts enables naked-eye visualization and straightforward data interpretation requiring minimal scientific background. Compared with semi-nested PCR in field sample evaluation, our RPA-Cas12a assay is more sensitive and capable of detecting SDDV in asymptomatic fish. Importantly, the entire workflow can be carried out at a constant temperature of 37°C within an hour from start to finish, thus removing the need for an expensive thermal cycling apparatus and long turnaround times associated with PCR-based methods. Therefore, owing to its high accuracy, rapidity and user-friendliness, the developed RPA-Cas12a platform shows the potential for diagnosis of SDDV at point of need and could be a valuable tool to help protect fish farming communities from large-scale epidemics.

A multiplex assay based on recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a desirable tool for many areas. This multiplex assay could be efficiently realized using single-stranded (ss) DNAs located in separate zones on the test strip and bound complementary ssDNA tags of double-stranded (ds) DNA amplicons. Here, we investigate how to enrich multiplex assay capabilities using ssDNAs. Bifunctional oligonucleotide probes integrating (1) a forward primer for RPA, (2) a C9 spacer to stop polymerase, and (3) a ssDNA tag for binding at test strip are developed. The amplicons have a unique individual ssDNA tag at one end and a universal label of fluorescein introducing through a reverse primer at the other end. A conjugate of gold nanoparticles (GNP) with antibodies to fluorescein is used to detect all amplicons. The remainder of primers after RPA interacting with GNP conjugate was found to be a limiting factor for sensitive and specific multiplex assay. The addition of anti-RPA-primers before the use of test strips was proposed to simply and effectively eliminate remaining primers. This approach was successfully applied for the detection of three priority plant RNA viruses: potato virus Y (PVY), -S (PVS) and potato leafroll virus (PLRV). The total time of the assay is 30 min. The multiplex RPA-LFT detected at least 4 ng of PVY per g of plant leaves, 0.04 ng/g for PVS, and 0.04 ng/g for PLRV. The testing of healthy and infected potato samples showed concordance between the developed assay and reverse transcription-polymerase chain reaction. Thus, the capabilities of the proposed universal modules (ssDNA anchors, bifunctional probes, and blocking anti-primers) for multiplex detection of RNA analytes with high specificity and sensitivity were demonstrated.

Atlantic salmon (Salmo salar) is consumed worldwide for its flavor and high nutritional value. However, because S. salar is difficult and costly to breed and has low yield, it remains difficult to meet the global demand. Rainbow trout (Oncorhynchus mykiss) is closely related to S. salar, and their flesh is similar in appearance. Because the price difference between these two fish species is large, O. mykiss is sometimes sold as S. salar to maximize profits, which is a violation of consumer rights and could pose health risks. Freshwater farmed O. mykiss is at higher risk of infection by parasites that can easily infect humans, whereas those carried by marine-cultured S. salar cannot. Thus, there is an urgent need for rapid, simple, and sensitive detection methods to differentiate these two species. In this study, we developed a method that combines isothermal recombinase polymerase amplification and lateral flow strip (RPA-LFS) to identify S. salar. A standard sample of S. salar was used as a template, and the myoglobin gene was selected as a target gene to establish the RPA-LFS detection method. Frozen fish samples were purchased online and analyzed using the RPA-LFS system; the results were compared with those obtained using PCR amplification and cytochrome oxidase I (COI) gene sequencing. The RPA-LFS system successfully performed amplification at 37 °C, and the entire detection process required approximately 20 min. The minimum detection limit was 102 ng/mL or 103 copies/mL. The detection results for clinical samples were consistent with those obtained using DNA barcoding technology (32/36). Together, these findings indicate that the detection system developed in this study is applicable for rapid, high-precision, sensitive identification of S. salar.

Introduction: While PCR has been recognized as one of the appropriate ways to diagnose infectious diseases, Loop-mediated isothermal amplification (LAMP) which is a nucleic acid amplification method, can be considered as an alternative to PCR, and it is faster, cost-effective, and easier to perform than nested PCR. Patients and Methods: Keywords were searched in PubMed/MEDLINE, Scopus and Institute for Scientific Information Web of Science, as well as the search engine of Google Scholar. Keywords included PCR, LAMP, RAA, RPA, Virus and COVID-19. Results: LAMP technology has been extensively applied for the detection of human pathogenic bacteria, crop pests, pathogenic organisms and components in meat products. A new isotheral method, Recombinase polymerase amplification (RPA), can amplify the DNA as well as RPA. RPA involves benefits of isothermal PCR as well as simplicity and rapid amplification. Recombinase aided amplification (RAA) assay has been favorably used in the detection of bacterial and viral pathogens and solved the technical difficulties posed by DNA amplification methods because it does not need thermal denaturation of the template and involves a low and constant temperature. Conclusions: Reverse transcription polymerase chain reaction, digital PCR, LAMP, nicking endonuclease amplification reaction, recombinase polymerase amplification, and clustered regularly interspaced short palindromic repeats are different nucleic acid amplification tests of COVID-19. LAMP methods can be more specific than qPCR and immunoassays. The LAMP assay can be applied for rapid detection of SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza, because LAMP is a highly sensitive and specific DNA/RNA amplification technique.

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

The northern root-knot nematode, Meloidogyne hapla, is a biotrophic parasite that infects many crops and causes severe economic losses worldwide. Rapid and accurate detection of M. hapla is crucial for disease forecasting and control. We developed a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay for rapid detection of M. hapla. The primers and probe were designed based on the effector gene 16D10 sequence and were highly specific to M. hapla. The RPA reaction was performed at a wide range of temperatures from 25 to 45°C within 5 to 25 min, and the amplicon was visualized directly on the LFD within 5 min. The detection limits of the RPA-LFD assay were 10−3 females and 10−2 second-stage juveniles/0.5 g of soil, which was 10 times more sensitive than the conventional PCR assay. In addition, the RPA-LFD assay can detect M. hapla from infested plant roots and soil samples, and the entire detection process can be completed within 1.5 h. These results indicate that the RPA-LFD assay is a simple, rapid, specific, sensitive, and visual method that can be used for rapid detection of M. hapla in the field and in resource-limited conditions.

Background Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor. Methods In this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases. Results The sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p Conclusions Considering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples. Graphical abstract

Heterosigma akashiwo is one of the main toxin-producing species that is globally known for frequently forming fish-killing harmful algal blooms. In this study, a novel technique referred to as recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) (RPA-LFD) was established for detection of H. akashiwo. RPA primers and an LFD probe were designed based on the sequence of the large ribosomal subunit (LSU rDNA) D1-D2 region of H. akashiwo. RAP/RPA-LFD was experimentally verified to be specific, displaying no cross-reaction with other control microalgae. It was demonstrated that the optimal amplification temperature and time for RPA were 41 °C and 40 min. The LFD assay was completed in only 5–10 min to analyze RPA products, and RPA results could be visualized directly. RPA-LFD was 100 times more sensitive than PCR, displaying a detection limit of 3.37 × 10−4 ng µL−1 for genomic DNA of H. akashiwo and 1.28 × 102 copies µL−1 for the recombinant plasmid containing the inserted LSU rDNA D1-D2 region of H. akashiwo. In this study, RPA-LFD’s detection limit was 1 cell mL−1, which is feasible to apply in the field test. Thus, the established RPA-LFD targeting the detection of H. akashiwo is characterized by simplicity, rapidity, sensitivity, specificity, and visualization.

The aim of the present study was to develop a sensitive and specific detection method for the rapid detection of apple scar skin viroid (ASSVd) in apple leaves. The resulting reverse transcription recombinase polymerase amplification (RT-RPA) assay can be completed in 10 min at 42oC, is 10 times more sensitive than conventional reverse transcription polymerase chain reaction, and can specifically amplify ASSVd without any cross-reactivity with other common apple viruses, including apple stem grooving virus, apple stem pitting virus, and apple chlorotic leaf spot virus. The reliability of the RT-RPA assay was assessed, and the findings suggested that it can be successfully utilized to detect ASSVd in field-collected samples. The RT-RPA assay developed in the present study provides a potentially valuable means for improving the detection of ASSVd in viroid-free certification programs, especially in resource-limited conditions.

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.

Acute hepatopancreatic necrosis disease (AHPND) is a significant deadly infectious disease in the shrimp farming industry, causing serious economic losses globally every year. Because of the rapid progress speed, lack of effective treatment and high mortality rate of AHPND, monitoring with frequent diagnostic tests is vital for a successful prevention. The conventional histopathological diagnosis fell far short of the requirement for efficient monitoring, and the polymerase chain reaction (PCR)-based molecular diagnostic methods that rely on sophisticated thermocycler and trained personnel are hardly applicable in the field. Combining the recombinase polymerase amplification (RPA) and the lateral flow strips (LFSs), a diagnostic method suitable for on-site everyday monitoring of AHPND has been established in this study. This RPA-LFS method targeted the binary toxic photorhabdus insect-related genes PirA and PirB on a virulence plasmid of the AHPND-causative Vibrio parahaemolyticus strains. The diagnostic test was completed within 30 min at 37°C and showed good specificity and good sensitivity of 20 fg DNA of the AHPND shrimp or one colony-forming unit of the causative bacterium per reaction, which was better than the administration-approved standard AP4 assay. Crude templates from sample boiling could be directly used. Tests of clinical samples showed 100% consistency of this method with the standard AP4 assay. This RPA-LFS method can be a good choice for on-site diagnosis of AHPND with quick response time, easy procedure and low demand for resources, and should have significant value for the control of spreading of this dangerous disease in farmed shrimp.

Background The diagnosis of cutaneous leishmaniasis (CL) is based on demonstration of the presence of the parasite in samples obtained from the lesions by direct examination (DE), culture or polymerase chain reaction (PCR)-based molecular tests. Recombinase polymerase amplification (RPA) represents an isothermal version of the conventional PCR (cPCR) technique, being ideal for detecting Leishmania DNA, especially in field conditions. Methods A prospective and cross-sectional study was conducted to evaluate the diagnostic performance of RPA in the health centres of rural endemic sites or the evaluation centre (EC) of 11 Colombian municipalities and in a reference centre (RC). Results Samples of 128 patients with suspected CL were included and processed for analysis by RPA vs DE in the EC and RPA vs DE and cPCR in the RC. The RPA performed at the EC was more sensitive (90.4% [95% confidence interval {CI} 81.9 to 95.7]) than the DE (42–67%) and the specificity was 72.7% (95% CI 57.2 to 85.0). Both the sensitivity and specificity increased to 100% when adjusting by the imperfect reference standard analysis method. In the RC, the sensitivity of RPA vs cPCR was 72% and the specificity was 69.8%, while the sensitivity of cPCR vs the DE test was 78.8% and the specificity was 81%. Conclusions The higher sensitivity and specificity shown by RPA in the EC, but also its ease and speed of use, justify performing RPA in the health centres of rural endemic sites. In addition, RPA eliminates the subjectivity inherent in the traditional DE.

Carbapenemase-producing organisms (CPOs) pose a serious clinical threat and rapid detection tools are essential to aid in patient management. We developed rapid and simple molecular tests to detect blaNDM-type and blaVIM-type carbapenemase genes using recombinase polymerase amplification (RPA) combined with a lateral flow detection. The tests could provide results in approximately 15 min when using DNA extracts, with limits of detection of 9.2 copies/μl for the blaNDM-type assay and 7.5 copies/μl for blaVIM-type assay, and successfully detected all isolates harbouring the carbapenemase encoding genes in a panel of 57 isolates. These RPA tests may be suitable for use in low-resource settings to tailor rapid implementation of infection control precautions and antibiotic stewardship.

Recombinase Polymerase Amplification (RPA) assay was developed for specific detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV [potato]), causing potato apical leaf curl disease. The RPA assay was optimized with respect to its isothermal temperature, post-amplification treatment and its components of the reaction mix. The assay showed a clear and sharp expected amplicon at 40 °C for 30 min in the thermal cycle followed by post-amplification denaturation, i.e., heat treatment at 65 °C for 10 min. The target specificity of the developed assay was determined by sequencing the amplified product. Pairwise sequence alignment showed 73 numbers of significant hits with ToLCNDV [potato] isolates within the taxonomy of begomovirus with query coverage of 100% with 99% identity indicating that the assay is specific to ToLCNDV [potato]. The comparative sensitivity of the assay was ten times more sensitive than PCR. It was successfully applied for the detection of ToLCNDV [potato] from potato leaf samples, tissue culture raised plants and also from tuber and sprouts. The assay was proved to be rapid, sensitive, highly specific and suitable in diagnostic laboratories for virus indexing and selection of virus-free planting material. To the best of our knowledge, this is the first report of the development and application of RPA assay for the detection of ToLCNDV [potato].

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.

Salmonella spp. is one of the most serious foodborne pathogens causing millions of infection cases annually, especially in resource-limited areas. The standard culture method (2-3 days) and current nucleic acid amplification-based testing are not suitable for on-site testing in rural areas with heavy Salmonella spp. burden. Here, we developed a colorimetric recombinase polymerase amplification (RPA) method for fast and sensitive Salmonella spp. testing in 1 h. Specifically, the invA gene from the genomic DNA of Salmonella spp. was amplified isothermally to produce double-stranded DNA (dsDNA) amplicons, which were directly quantified by a photosensitization colorimetric assay. The proposed method offered the lowest detectable concentration of 5 × 103 colony-forming units/mL (cfu/mL), which is much lower than that of ELISA (105-107 cfu/mL). The detectable limit could be further pushed down to 3 cfu/mL upon coupling with bacteria pre-enrichment for 6 h. Analysis of synthetic milk samples confirmed the high precision (90%) and specificity (95%) of the method for Salmonella spp. testing. Moreover, use of a DNA releaser could further simplify the whole testing operation. Because RPA features low-temperature amplification (25-42 °C) without the need for specific instruments and the dsDNA-based photosensitization colorimetric assay served as a simple and facile readout for RPA, our method thus allows fast and low-cost Salmonella spp. testing in food samples.

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are known to produce several bioactive compounds including the potent neurotoxic ciguatoxins (CTXs) which are able to accumulate in fish and through the food web. When humans ingest fish contaminated with CTXs, it can result in an intoxication named ciguatera. Although not all the currently recognized species are able to produce toxins, G. australes and G. excentricus have been highlighted to be the most abundant and toxic among the species present in the Atlantic. Even though the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, recently their presence was recorded in subtropical and temperate regions. In this work, the development of three molecular assays for the detection of the Gambierdiscus and Fukuyoa genera and for G. australes and G. excentricus species, based on the combination of recombinase polymerase amplification with detection via hybridization, is successfully described. Furthermore, a remarkable limit of detection of a single cell was achieved. Additionally, six different species have been used to check the ability of each primer set to give an amplified product, even in presence of potentially interfering non-target DNAs. Therefore, these developments provide a rapid and cost-effective strategy for detection of both genera and two of the most toxic species, which will undoubtedly contribute to reliable screening of samples and ciguatera risk assessment, guaranteeing seafood safety and protection of human health. info:eu-repo/semantics/acceptedVersion

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.

Plum pox virus (PPV) is one of the most destructive viral pathogens infecting stone fruit trees worldwide. As PPV causes a viral disease that requires plants to be quarantined, the development of a reliable method of PPV detection is essential for preventing the spread of the disease. In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the detection of PPV in peaches. The major advantage of this RT-RPA assay is that it can be performed at 42 °C and can be completed in 10 min and has specificity for viruses that infect peaches. In addition, this assay was successfully performed using field-collected samples. This RT-RPA assay is a promising method with high efficiency for rapid PPV detection as part of quarantine inspection and certification program.

Sickle cell disease (SCD) is a group of common, life-threatening disorders caused by a point mutation in the β globin gene. Early diagnosis through newborn and early childhood screening, parental education, and preventive treatments are known to reduce mortality. However, the cost and complexity of conventional diagnostic methods limit the feasibility of early diagnosis for SCD in resource-limited areas worldwide. Although several point-of-care tests are commercially available, most are antibody-based tests, which cannot be used in patients who have recently received a blood transfusion. Here, we describe the development of a rapid, low-cost nucleic acid test that uses real-time fluorescence to detect the point mutation encoding hemoglobin S (HbS) in one round of isothermal recombinase polymerase amplification (RPA). When tested with a set of clinical samples from SCD patients and healthy volunteers, our assay demonstrated 100% sensitivity for both the βA globin and βS globin alleles and 94.7 and 97.1% specificities for the βA globin allele and βS globin allele, respectively (n = 91). Finally, we demonstrate proof-of-concept sample-to-answer genotyping of genomic DNA from capillary blood using an alkaline lysis procedure and direct input of diluted lysate into RPA. The workflow is performed in

Canine monocytic ehrlichiosis (CME), caused by transmitted Ehrlichia canis infection, is a major disease in dogs with worldwide distribution. Herein, a nucleic acid assay was established for the identification of E. canis infection employing a fluorescently labeled conformationally constrained pyrrolidinyl PNA probe (Flu-acpcPNA) designed to sequence-specifically target the 16S rRNA gene. The sensing principle is based on the excellent quenching ability of graphene oxide (GO) of the free PNA probe, that was diminished upon binding to the DNA target. The addition of DNase I improved the performance of the detection system by eliminating the nonspecific quenching capability of long-chain dsDNA and thus enhancing the fluorescence signaling. The assay was coupled with a recombinase polymerase amplification (RPA) technique, which could be performed under isothermal conditions (37 °C) without DNA denaturation and purification steps. The established method is simple to set up and execute, proving a rapid, specific, and sensitive detection of 16S rRNA gene of E. canis with a limit of detection at least 11.1 pM. This technique shows good potential for the visual detection of double-stranded DNA targets without the need for PCR or complicated instruments, which shows great promise for practical usage in resource limited areas.

This work presents a new approach to the rapid characterization of antibiotic resistance in environmental settings. For monitoring purposes and risk assessment, it is important to link the detectio...

Zika virus (ZIKV) has emerged as a particularly notorious mosquito-borne flavivirus, which can lead to a devastating congenital syndrome in the fetuses of pregnant mothers (e.g., microcephaly, spasticity, craniofacial disproportion, miscarriage, and ocular abnormalities) and cause the autoimmune disorder Guillain-Barre' syndrome of adults. Due to its severity and rapid dispersal over several continents, ZIKV has been acknowledged to be a global health concern by the World Health Organization. Unfortunately, the ZIKV has recently resurged in India with the potential for devastating effects. Researchers from all around the world have worked tirelessly to develop effective detection strategies and vaccines for the prevention and control of ZIKV infection. In this review, we comprehensively summarize the most recent research into ZIKV, including the structural biology and evolution, historical overview, pathogenesis, symptoms, and transmission. We then focus on the detection strategies for ZIKV, including viral isolation, serological assays, molecular assays, sensing methods, reverse transcription loop mediated isothermal amplification, transcription-mediated amplification technology, reverse transcription strand invasion based amplification, bioplasmonic paper-based device, and reverse transcription isothermal recombinase polymerase amplification. To conclude, we examine the limitations of currently available strategies for the detection of ZIKV, and outline future opportunities and research challenges.

The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/μL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

Chlamydia psittaci is a zoonotic agent of systemic wasting disease in birds and atypical pneumonia in mammalians including humans, constituting a public health risk. A rapid diagnostic assay would be beneficial in screening C. psittaci in the field. In this study, we developed a probe-based recombinase polymerase amplification (RPA) assay for the rapid detection of C. psittaci. The specific primer pairs and probe targeting the conserved region of the outer membrane protein A gene were designed and applied to the real-time real-time RPA assay. The test can be performed at 39°C for 20 min using a portable device, with sensitivities approaching 100 copies of DNA molecules per reaction, with no cross-reaction with other pathogens. The clinical performance of the RPA assay was evaluated in an outbreak of C. psittaci and has high accuracy levels in field applications. The epidemic C. psittaci strains were classed into 2 genotypes: A and C. Collectively, this study offers a promising approach in screening for C. psittaci both in a laboratory setting and in field settings, and RPA can be used as an effective clinical test to monitor outbreaks in domestic fowl populations.

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

Onion yellow dwarf virus (OYDV) is an important virus affecting Allium species like onion and garlic worldwide. Enzyme-linked immunosorbent assays (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) assays are two most commonly used assays for the detection of OYDV but the sensitivity of ELISA is less than that of RT-PCR. PCR based assays are often considered for their sensitivity, specificity but time consuming and require specific technical expertise and costly equipments. To counter these difficulties, a simple and rapid RT-RPA assay for the detection of OYDV was developed. A common set of primer was designed to amplify the coat protein region of OYDV to perform RT-PCR and RT-RPA. Specificity as well as sensitivity test using tenfold serial dilution series of the purified RNA was performed for both the assays. Both symptomatic and asymptomatic samples of different varieties of onion were collected from Delhi NCR region and evaluated for OYDV infection using RT-PCR along with RT-RPA assay. The comparative results have shown that sensitivity of RT-RPA was similar to RT-PCR. However, RT-RPA is simple, and rapid assay for the large scale virus indexing of onion.

African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-020-00323-1) contains supplementary material, which is available to authorized users.

Antibiotic resistant bacteria complicate infection treatment worldwide. Rapid and inexpensive detection of the current occurrence of antibiotic resistant bacteria in surface and irrigation water as well as treated wastewater is essential to minimize exposure and further spread. To reduce cost and analysis time compared to current qPCR (quantitative polymerase chain reaction), isothermal nucleic acid amplification tests are promising bioanalytical methods which can be integrated in simplified molecular biological detection systems. This study establishes heterogeneous asymmetric recombinase polymerase amplification (haRPA) for the detection of antibiotic resistance genes in water. After DNA extraction of bacteria cultivated from water, the target DNA for blaCTX-M cluster 1 was amplified at 39 °C for 40 min on a microfluidic DNA chip. The amplified DNA on each spot was quantified by a flow-based chemiluminescence reaction. Even though slightly less sensitive than conventional qPCR, the haRPA method was successful in identifying the blaCTX-M cluster 1 in bacterial isolates with a limit of detection of 0.013 ng μL-1. In a proof-of-principle study, 37 bacterial isolates from environmental water samples were classified according to blaCTX-M cluster 1 occurrence and gave 100% agreement in cross-reference with PCR. Importantly, haRPA allows for a quick in-field monitoring at low incubation temperatures and by an easy visual readout. This study paves the path to establish haRPA as a quick on-site monitoring option for antibiotic resistance gene occurrence without the need for a thermal cycling device or long data processing.

The analysis of mold in indoor air is a prominent topic but it is hardly dealt with. The most affected fields of this issue are residential- and occupational safety since mold can have a number of impacts on human health. To date the most used methods for quantification of microorganism contamination in indoor air are culture- or microscopy-based and are not capable of translating the on-site situation to analytical data reliably. Here we present a chemiluminescence-based method to detect mycotoxin producers through isothermal amplification of mycotoxin biosynthesis genes using glass and polycarbonate carriers. In this proof-of-principle study, zearalenone producers were aimed to be detected by heterogeneous asymmetric recombinase polymerase amplification (haRPA). For this, an appropriate lysis method for fungal spores was developed allowing rapid access to DNA. A system calibration with spores of Fusarium culmorum as zearalenone-producing organism resulted in an LOD of 2.7 × 105 spores per ml. The system was shown to be specific for zearalenone producers. This work presents the first application of a heterogeneous isothermal amplification for rapid detection and quantification of mycotoxin producers. In the future, a multiplex detection can be possible by haRPA.

Rice bacterial leaf blight (BLB) by Xanthomonas oryzae pv. oryzae (Xoo) is considered to be one of the major rice diseases steadily occurring around the rice-producing countries. In this study, we developed a recombinase polymerase amplification (RPA) assay for the rapid, convenient and specific diagnosis of Xoo by targeting Xoo-specific transposase A gene. As the target gene can be amplified in 10 min without DNA extraction process and special equipment for temperature control, RPA for BLB can be useful and practical component for on-site diagnosis.

Caused by Salmonella pullorum, pullorosis is a bacterial disease threatening the poultry industry and has been listed as the bacterial disease to be eliminated by the government. However, antibiotic treatment of pullorosis has become increasingly difficult, resulting in severe influences on the sustainable development of poultry. Abuse of antibiotics may cause global drug-resistant problems. Hence, early diagnosis of young chickens and accurate treatment of sick chickens are urgently needed. Traditional serotyping for Salmonella detection is costly and labor-intensive, whereas other commonly used plate agglutination test methods often cause physical injury of chickens. Therefore, a rapid and nondamaging detection method is of great significance for early diagnosis, which is the key step in accurate medication and elimination of pullorosis. In this study, we propose a novel lateral flow nucleic acid assay (LFNAA) system combining recombinase polymerase amplification (RPA) for the detection of S. pullorum. In this method, the DNA of S. pullorum strains was quickly amplified by RPA under 37°C, and then, the RPA products were added onto the LFNAA sample pad until the final results could be observed by naked eyes within 3 min. The proposed assay is fast and delivers visible results to naked eyes in field test. The limit of detection for genomic DNA was 5 × 10−3 ng/μL, indicating high sensitivity. In addition, the proposed LFNAA system is cost-effective, efficient, and nondamaging to chicks in the field test. This system provides technical support for early diagnosis of S. pullorum in the poultry and paves a way for future precision medicine to avoid the global drug-resistance issues.

Angiostrongylus cantonensis is a parasitic nematode known to infect humans through the ingestion of third stage larvae which can cause inflammation and damage to the central nervous system. Currently, polymerase chain reaction (PCR) is one of the most reliable diagnostic methods for detecting A. cantonensis in humans as well as in gastropod hosts, but requires expensive and specialized equipment. Here, we compare the sensitivity and accuracy of a recombinase polymerase amplification Exo (RPA-EXO) assay, and a recombinase polymerase amplification lateral flow assay (RPA-LFA) with a traditional quantitative PCR (qPCR) assay currently available. The three assays were used to test 35 slugs from Hawai‘i for the presence of A. cantonensis DNA. Consistent results among the three tests were shown in 23/35 samples (65.7%), while 7/35 (20%) were discordant in low infection level samples (μL−1 to ~1 copy μL−1. All three assays consistently detected 50–100 copies μL−1 in triplicate and qPCR was able to detect ~13 copies μL−1 in triplicate. RPA-EXO was able to detect 25 copies μL−1 in triplicate and RPA-LFA was not able to amplify consistently below 50 copies μL−1. Thus, our RPA-EXO and RPA-LFA assays do not appear as sensitive as the current qPCR assay at low DNA concentrations; however, these tests have numerous advantages that may make them useful alternatives to qPCR.

Late blight, caused by the oomycete Phytophthora infestans, is a major constraint on the production of potatoes and tomatoes as well as a constant threat to global food security. An early diagnostic tool is important for the effective management of late blight in the field. Here, in combination with a simplified DNA extraction method, we developed a lateral flow strip-based recombinase polymerase amplification (LF-RPA) assay for the rapid, equipment-free detection of P. infestans. This assay targets the Ras-related protein (Ypt1) gene and can be performed over a wide range of temperatures (25 to 45°C). All 12 P. infestans isolates yielded positive detection results using the LF-RPA assay, and no cross-reaction occurred with related oomycetes or fungal species. With this assay, the detection limit was 500 fg of genomic DNA in optimized conditions. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire LF-RPA assay enabled the detection of P. infestans within 30 min with no specialized equipment. When applied to field samples, it successfully detected P. infestans in naturally diseased potato plants from eight different fields in China. Therefore, the LF-RPA assay is simple, rapid, and cost-effective and has potential for further development as a kit for diagnosing late blight in resource-limited settings or even on-site.

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3′ polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management.

The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

Human norovirus is the leading cause of viral gastroenteritis worldwide. Rapid detection facilitates management of disease outbreaks, but field diagnosis is difficult to achieve due to the lack of reliable and portable methods. Recombinase polymerase amplification (RPA) is a robust isothermal amplification method that is capable of rapidly amplifying and detecting nucleic acids using simple equipment. In this study, RPA combined with lateral flow (LF) strips specific for human genogroup II (GII) noroviruses was established and evaluated. The assay specifically detects purified GII noroviruses as well as RNA in boiled human stool samples, with a sensitivity of 50 norovirus genome copies per reaction. The whole detection procedure of the one-step RT-RPA-LF is completed within 20 min, which is eight times faster than that of the standard real-time RT-PCR. The RT-RPA-LF method described here is suitable for rapid field diagnosis of all GII noroviruses in human stool samples.

Phytophthora fragariae causes drastic damage in strawberry crops. P. fragariae infects strawberry roots and causes red stele root rot. Although P. fragariae is a quarantine organism, its spread in Finland continues as more and more fields contract the disease. The spread can be halted through developing rapid and reliable detection assays. We have developed a rapid recombinase polymerase amplification (RPA) assay for P. fragariae targeting the Phytophthora mitochondrial DNA intergenic atp9-nad9 marker. The assay is DNA-extraction free and capable of detecting as low as 10 fg of P. fragariae genomic DNA. We found the assay reliable for diagnosing field plants when samples are adequately collected. We also applied the RPA assay to the detection of the pathogen in the soil through coupling the assay with baiting with the host plant. The results suggest that if only a small number of samples are analysed, the baiting results will not be reliable.

Dickeya spp. cause blackleg and soft rot diseases of potato and several other plant species worldwide, resulting in high economic losses. Rapid detection and identification of the pathogen is essential for facilitating efficient disease management. Our aim in this research was to develop a rapid and field-deployable recombinase polymerase amplification (RPA) assay coupled with a lateral flow device (LFD) that will accurately detect Dickeya spp. in infected plant tissues without the need for DNA isolation. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific robust primers and probes for an RPA assay. Assay specificity was validated with 34 representative strains from all Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed when 1 µl of host sap, macerated in Tris-EDTA buffer, was added to each reaction in the sensitivity tests. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.

Background Milk vetch dwarf virus (MDV) is an important ssDNA virus which causes yellowing, stunting and leaf rolling symptoms on legumes. In China, the virus causes great economic losses and has recently been found to infect tobacco. The expansion of its host range and its ability to spread rapidly has given rise to the urgent need for a sensitive, specific and rapid diagnostic assay that can assist in effective disease control. Methods Assays based on the polymerase chain reaction combined with lateral flow strip detection (PCR-LFS) and recombinase polymerase amplification combined with LFS (RPA-LFS) were developed targeting the coat protein (CP) gene of MDV. Results The PCR and RPA assays could detect respectively 103 copies or 101 copies of MDV by agarose gel electrophoresis. The PCR-LFS and RPA-LFS assays developed could both detect as few as 101 copies per reaction at 37 °C. Both methods could detect MDV in crude leaf extracts. Conclusions The RPA-LFS assay developed is a rapid, sensitive and specific method for detecting MDV, which is convenient and has great potential for use in the field.

Haemophilus parasuis is the etiological agent of Glässer's disease in swine, which associates with severe economic losses in the swine industry worldwide. A real‐time recombinase polymerase amplification assay (real‐time RPA) was developed for direct and rapid detection of H. parasuis basing on the translation‐initiation factor IF2 (infB) gene. The assay was performed successfully at 39°C for 20 min in Genie III, which is portable and chargeable by battery. The developed assay was highly specific for H. parasuis, and the limit of detection of the assay was 6.0 × 103 fg of H. parasuis genomic DNA, which was the same as that of a real‐time PCR developed previously. The assay was further evaluated on 68 pig tissue samples, and 18 (26.5%), 20 (29.4%), and 8 (11.8%) samples were positive for H. parasuis by the real‐time RPA, real‐time PCR and bacterial isolation, respectively. With the bacteria isolation as the reference method, the real‐time RPA showed a diagnostic specificity of 83.33% and a diagnostic sensitivity of 100%. The above data demonstrated the well‐potentiality and usefulness of the developed real‐time RPA assay in reliable diagnosis of swine Glässer's disease, especially in resource limited settings., Haemophilus parasuis is the etiological agent of Glässer’s disease in swine, which associates with severe economic losses in the swine industry worldwide. A real‐time recombinase polymerase amplification assay (real‐time RPA) was developed for direct and rapid detection of H. parasuis basing on the infB gene.

Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and mariculture. Rapid and accurate detection technologies are critical for effective control of its outbreak and spreading. Conventional technologies and polymerase chain reaction (PCR)-based approaches have limited usage because of the requirement of laboratory instruments and trained personnel. Using the isothermal recombinase polymerase amplification (RPA) technology, several detection assays have been developed with added convenience. Combining the lateral flow strip (LFS) test with RPA can further simplify the detection. In this study, an improved RPA assay using LFS for visual detection of V. parahaemolyticus was developed. Primers were designed targeting the virulence genes and screened for amplification efficiency, nonspecific amplification, and primer-dimer formation. Probes were designed for the best primer pairs, and the weakness of LFS tests, being easily affected by primer-dependent artifacts, was overcome by sequence modifications on primers and probe. The RPA-LFS assay took 25 min at 35 to 45 °C, and showed excellent specificity. It detected as low as one colony forming unit (CFU) of V. parahaemolyticus per reaction without DNA purification, or 10 CFU/10 g spiked food samples with 2 hr of enrichment. The detection limit was better than the currently available RPA-based detection methods. Application of the RPA-LFS assay for simulated samples or real clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. The RPA-LFS assay provided a rapid, accurate, and convenient V. parahaemolyticus detection method suitable for on-site detection in resource-limited conditions. PRACTICAL APPLICATION: This research developed a rapid and visual detection technology for Vibrio parahaemolyticus that is not dependent on complicated equipment. The detection process takes 25 min and the result is read with the naked eye. A detection kit can be developed based on this technology for on-site detection of V. parahaemolyticus in resource-limited regions for food safety management and mariculture.

With the large-scale planting of genetically modified (GM) crops, the development of a rapid and convenient method for on-site monitoring GM crops is needed. In this study, a duplex recombinase polymerase amplification (DRPA)-based, quick and simple detection system is presented for on-site detection of GM crops. In this system, a rapid DNA extraction method, a DRPA, and a lateral flow biosensor (LFB) are integrated to allow for rapid DNA extraction and amplification, and fast visualization of the detection results. Using the rapid DNA extraction method, high-quality DNA suitable for RPA and polymerase chain reaction (PCR) was quickly isolated from various crops within 5 min. Utilizing the optimal DRPA assay, the universal screening sequences (Cauliflower mosaic virus 35S promoter [35S] and Agrobacterium tumefaciens NOS terminator [NOS]) were rapidly and simultaneously amplified with high selectivity and sensitivity. The sensitivity threshold of the DRPA assay was ∼10 copies for GM soybean genomic DNA and 100 ng DNA of 0.1% GM soybean. In combination with the LFB in an enclosed cassette, the entire detection process was performed in approximately 20–30 min and eliminated the carryover contamination. No special or expensive equipment was needed for the detection process. The system was successfully applied and validated for on-site detection of GM rice, demonstrating its suitability for on-site testing of GM crops and high potential for application to other fields, especially in low-resource regions that require rapid detection of DNA targets.

Background Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. Since then, PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. Therefore, it is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV. Results In the present study, a highly sensitive and specific diagnostic method using recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed for rapid and visual detection of PDCoV. The system can be performed under a broad range of temperature conditions from 10 to 37 °C, and the detection of PDCoV can be completed in 10 min at 37 °C. The sensitivity of this assay was 10 times higher than that of conventional PCR with a lower detection limit of 1 × 102 copies/µl of PDCoV. Meanwhile, the LFD-RPA assay specifically amplified PDCoV, while there was no cross-amplification with other swine-associated viruses, including Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine kobuvirus (PKoV), Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2), Classical swine fever virus (CSFV) and Seneca valley virus (SVV). The repeatability of the test results indicated that this assay had good repeatability. In addition, 68 clinical samples (48 fecal swab specimens and 20 intestinal specimens) were further tested by LFD-RPA and RT-PCR assay. The positive rate of LFD-RPA clinical samples was 26.47% higher than that of conventional PCR (23.53%). Conclusions The LFD-RPA assay successfully detected PDCoV in less than 20 min in this study, providing a potentially valuable tool to improve molecular detection for PDCoV and to monitor the outbreak of PDCoV, especially in low-resource areas and laboratories.