Recombinase Polymerase Amplification Assay Research Articles

Phase Transition of Wax Enabling CRISPR Diagnostics for Automatic At-Home Testing of Multiple Sexually Transmitted Infection Pathogens.

Sexually transmitted infections (STIs) significantly impact women's reproductive health. Rapid, sensitive, and affordable detection of these pathogens is essential, especially for home-based self-testing, which is crucial for individuals who prioritize privacy or live in areas with limited access to healthcare services. Herein, an automated diagnostic system called Wax-CRISPR has been designed specifically for at-home testing of multiple STIs. This system employs a unique strategy by using the solid-to-liquid phase transition of wax to sequentially isolate and mix recombinase polymerase amplification (RPA) and CRISPR assays in a microfluidic chip. By incorporating a home-built controlling system, Wax-CRISPR achieves true one-pot multiplexed detection. The system can simultaneously detect six common critical gynecological pathogens (CT, MG, UU, NG, HPV 16, and HPV 18) within 30min, with a detection limit reaching 10-18 M. Clinical evaluation demonstrates that the system achieves a sensitivity of 96.8% and a specificity of 97.3% across 100 clinical samples. Importantly, eight randomly recruited untrained operators performe a double-blinded test and successfully identified the STI targets in 33 clinical samples. This wax-transition-based one-pot CRISPR assay offers advantages such as low-cost, high-stability, and user-friendliness, making it a useful platform for at-home or field-based testing of multiple pathogen infections.

A systematic approach for identifying unique genomic sequences for Fusarium oxysporum f. sp. lactucae race 1 and development of molecular diagnostic tools

Fusarium oxysporum f. sp. lactucae (FOLac) is a soil- and seedborne fungal pathogen that causes Fusarium wilt of lettuce, an important disease threatening global lettuce production. Based on pathogenicity on differential lettuce cultivars, four races (1-4) have been identified, with race 1 the only race detected in the United States, and the closely related, emerging race 4 known only in Europe. The development of race-specific diagnostic tools is hindered by insufficient genomic data to distinguish between the two races and FOLac from other F. oxysporum formae speciales and nonpathogenic isolates. Here, we describe a systematic approach for developing diagnostic markers for FOLac race 1 that utilized a comprehensive sequence database of F. oxysporum to identify 15 unique genomic sequences. Marker specificity was validated through an exhaustive screening process against genomic data from 797 F. oxysporum isolates representing 64 formae speciales and various plants and non-plant substrates. One of the unique sequences was used to develop a TaqMan quantitative polymerase chain reaction assay and a recombinase polymerase amplification assay, both exhibiting 100% sensitivity and specificity when tested against purified DNA from 171 F. oxysporum isolates and 69 lettuce samples. The relationship between qPCR Ct values and colony forming units (CFU)/g values was also determined. This study not only introduces a new marker for FOLac race 1 diagnostics and soil quantitation, but also underscores the value of an extensive genomic database and screening software pipeline for developing molecular diagnostics for F. oxysporum formae speciales and other fungal taxa.

Rapid Detection of Amoebic Liver Abscess Using Recombinase Polymerase Amplification Assay in Resource-Limited Settings.

Amoebic liver abscess (ALA) is a significant public health concern in tropical countries. Traditional diagnostic methods, such as microscopy, have low sensitivity, and nested polymerase chain reaction (nPCR) has lengthy turnaround times. In this study, we aimed to evaluate the effectiveness of the recombinase polymerase amplification (RPA) assay for detecting Entamoeba histolytica in amoebic liver pus samples. The assay was tested on 150 clinical pus samples collected from suspected ALA patients in the emergency department of a tertiary care center located in Chandigarh, northern India. For comparison with RPA, nPCR was also performed on these samples. Of 150 samples, 79 (53%) tested positive for E. histolytica using the RPA assay, and similar (79; 53%) number of samples tested positive with nested PCR. In addition, sequences obtained through Sanger sequencing showed high similarity with other sequences in the National Center for Biotechnology Information database and were submitted to the database. Our findings highlight the potential of RPA as a valuable tool for the accurate diagnosis of ALA.

Development of point-of-need colourimetric, isothermal diagnostic assays for specific detection of Bacillus subtilis using shikimate dehydrogenase gene.

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500pg/µL. DNA concentration as low as 10pg/µL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100pg/µL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

Rapid and Direct Detection of the Stubby Root Nematode, Paratrichodorus allius, from Soil DNA Extracts Using Recombinase Polymerase Amplification Assay.

The stubby root nematode, Paratrichodorus allius, is one of the most important plant-parasitic nematodes. Besides root feeding, P. allius also transmits the Tobacco rattle virus in potatoes, which causes corky ringspot disease. Rapid detection of P. allius is key for efficient management. This study was conducted to develop a real-time recombinase polymerase amplification (RPA) assay that is capable of detecting P. allius directly in DNA extracts from soil using a simple portable device in real time. A fluorophore-attached probe was designed to target the internal transcribed spacer (ITS)-rDNA of P. allius and was used along with primers designed previously. The real-time RPA assay had the ability to detect P. allius DNA extracted directly from infested soil with a sensitivity of one-sixteenth portion of a single nematode. This RPA assay was specific, as it did not produce positive signals from non-target nematodes tested. The real-time RPA was found to be rapid as it could even detect P. allius in as little as 7 min. Testing with 15 field soil samples validated the RPA assay developed in this study. This is the first report of P. allius detection directly from soil DNA using real-time RPA and is the fastest method for P. allius detection in soil to date.

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the New Root-lesion Nematode Pratylenchus dakotaensis on Soybean.

Root-lesion nematodes, Pratylenchus spp. are reported to cause serious yield losses in various crops including soybean. A new root-lesion nematode species was recently detected in a soybean field in North Dakota (ND) and named Pratylenchus dakotaensis. Nematode detection and differentiation from other species are critical in management strategies. Thus, a recombinase polymerase amplification (RPA) assay was developed for rapid detection of this nematode from field soils under isothermal conditions. New primers and probes were designed from ITS-rDNA region of the nematode genome and tested for both specificity and sensitivity. The RPA assay was able to detect DNA from a single adult nematode at 39.5°C in 20 minutes using both Basic and Exo kits. The specificity of the primers was initially confirmed through in silico analyses, followed by laboratory tests. The assay successfully amplified DNA from the target species, while no amplification occurred for other Pratylenchus spp. and non-Pratylenchus control species. Sensitivity testing with real-time RPA revealed its ability to detect DNA in dilutions equivalent to 1/32 of a single nematode from DNA extracted from inoculated sterile soil. To further validate the assay, it was tested with 19 field soil samples collected in ND. This assay amplified soil DNA extracts of all P. dakotaensis-infested field samples confirmed through conventional PCR. It did not amplify DNA from 13 other field soils infested with other Pratylenchus spp. This is the first report of RPA develoment for detecting a root-lesion nematode species. The RPA assay developed can help in the rapid detection of this nematode species for effective nematode management.

Graphene-Based Virus Enrichment Protocol Increases the Detection Sensitivity of Human Norovirus in Strawberry and Oyster Samples.

Human noroviruses (HuNoVs), the most prevalent viral contaminant in food, account for a substantial proportion of nonbacterial gastroenteritis cases. Extensive work has been focused on the diagnosis of HuNoVs in clinical samples, whereas the availability of sensitive detection methods for their detection in food is lacking. Here, we developed a virus enrichment approach utilizing graphene-based nanocomposites (CTAB-rGO-Fe3O4) that does not rely on large instruments and is suitable for on-site food pretreatment. The recovery efficiency of the developed virus enrichment procedure for serially diluted GII.4 norovirus ranged from 10.06 to 72.67% in strawberries and from 2.66 to 79.65% in oysters. Furthermore, we developed a real-time recombinase polymerase amplification (real-time RPA) assay, which can detect as low as 1.22 genome copies µL-1 of recombinant plasmid standard and has no cross-reactivity with genomes of astrovirus, rotavirus, adenovirus, and MS2 bacteriophage. Notably, the combined virus enrichment and real-time RPA detection assay enhanced the detection limits to 2.84 and 37.5 genome copies g-1 in strawberries and oysters, respectively, compared to those of qPCR. Our strategy, the graphene-based virus enrichment method combined with real-time RPA, presents a promising tool for sensitively detecting HuNoVs in food samples.

A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations

Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA–LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10–100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Understanding the dissemination of viruses and viroids identified through virome analysis of major grapevine rootstocks and RPA-based detection of prevalent grapevine virus B

Grapevine stocks are generally vegetatively propagated for the commercial cultivation. The propagation is predominantly done on rootstocks with >90 % of plant stocks propagated on Dogridge rootstock alone in India. Three additional rootstocks i.e. Paulsen, SO4 and Ramsey are also used for grapevine propagation on a limited scale. However, no information is available on the health status of these rootstocks being used by nurserymen and growers. Therefore, these rootstocks were collected from nurseries of five different locations and subjected to high-throughput sequencing (HTS) to determine the spectrum of viruses and viroids. The de novo assembly of unaligned reads revealed the presence of genome sequences of nine viruses viz. grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine leafroll-associated virus 4 (GLRaV-4), grapevine virus A (GVA), grapevine virus B (GVB), grapevine virus D (GVD), grapevine virus F (GVF), grapevine virus L (GVL), grapevine fleck virus (GFkV) and five viroids viz. hopstunt viroid (HSVd), grapevine yellow speckle viroid 1 and 2 (GYSVd-1 and GYSVd-2), Australian grapevine viroid (AGVd) and grapevine latent viroid (GLVd). The identified viruses and viroids were further confirmed through RT-PCR and Sanger sequencing. Indexing for the presence of these graft-transmissible pathogens in 43 rootstocks and 23 scion samples collected through field surveys indicated that infected rootstocks are the probable source for dissemination of these viruses and viroids in grapevine scions. As GVB was the most prevalent virus in all the rootstocks and scions as observed in RT-PCR testing, a simplified crude sap-based recombinase polymerase amplification (RPA) assay was optimized for its rapid detection. The present study highlights that grapevine rootstocks are reservoir of viruses and viroids and source for their dissemination in grapevine vineyards. Findings of the study including the optimized simplified cost-effective isothermal nucleic acid-based detection technique for GVB will be highly useful for production of certified clean rootstocks of grapevine in India.

A novel recombinase polymerase amplification approach with propidium iodide reporter for rapid detection of porcine adulteration in food

The adulteration of food products with porcine components is among the most widespread concerns in the global food industry. Apart from health and religious reasons, being able to detect porcine adulteration is crucial to warrant fair trading and legal compliance. Current detection methods are often constrained by their low sensitivity and expensive equipment. In this work, a recombinase polymerase amplification (RPA) assay for the rapid and sensitive detection of porcine is constructed, with propidium iodide (PI) dye as the reporter molecule. Under the optimized conditions, the RPA-PI assay achieved a wide linear range of 0.005–100 ng/μL and a detection limit of 0.005 ng/μL for porcine DNA with no cross-reactivity. Furthermore, the application of the assay to actual food samples demonstrated a 100 % consistency with conventional PCR results. This developed assay not only provides a novel porcine adulteration detection platform with simpler and faster protocols, but it also holds the potential to be developed further for point-of-care authenticity testing.

Refining the Schistosoma haematobium recombinase polymerase amplification (Sh-RPA) assay: moving towards point-of-care use in endemic settings

BackgroundUrogenital schistosomiasis is caused by the parasitic trematode Schistosoma haematobium. Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose S. haematobium has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered.MethodsUsing synthetic DNA, S. haematobium genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0 M, 1 M, 2.5 M, 12.5 M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single S. haematobium eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition.ResultsThe analytical sensitivity of the Sh-RPA with all betaine concentrations was ≥ 10 copies of the synthetic Dra1 standard and 0.1 pg of S. haematobium gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5 M of betaine together with the maximal possible sample volume of 12.7 µl proved to be the optimum reaction conditions. DNA was successfully isolated from a single S. haematobium egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions.ConclusionsThe addition of 2.5 M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single S. haematobium eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a molecular diagnostic platform to be robustly tested prior to implementation.Graphical

Faecal microbial diversity in a cattle herd infected by Mycobacterium avium subsp. paratuberculosis: a possible effect of production status

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease, or paratuberculosis (PTB) in ruminants, besides having zoonotic potential. It possibly changes the gut microbiome, but no conclusive data are available yet. This study aimed at investigating the influence of MAP on the faecal microbiome of cattle naturally infected with PTB. In a follow up period of 10 months, PTB status was investigated in a herd of dairy cattle with history of clinical cases. Each animal was tested for MAP infection using serum and milk ELISA for MAP anti-bodies and IS900 real-time PCR and recombinase polymerase amplification assays for MAP DNA in the faeces and milk monthly for 4 successive months, then a last one after 6 months. The faecal samples were subjected to 16S rDNA metagenomic analysis using Oxford Nanopore Sequencing Technology. The microbial content was compared between animal groups based on MAP positivity rate and production status. All animals were MAP positive by one or more tests, but two animals were consistently negative for MAP DNA in the faeces. In all animals, the phyla firmicutes and bacteroidetes were highly enriched with a small contribution of proteobacteria, and increased abundance of the families Oscillospiraceae, Planococcaceae, and Streptococcacaceae was noted. Animals with high MAP positivity rate showed comparable faecal microbial content, although MAP faecal positivity had no significant effect (p > 0.05) on the microbiome. Generally, richness and evenness indices decreased with increasing positivity rate. A significantly different microbial content was found between dry cows and heifers (p < 0.05). Particularly, Oscillospiraceae and Rikenellaceae were enriched in heifers, while Planococcaceae and Streptococcaceae were overrepresented in dry cows. Furthermore, abundance of 72 genera was significantly different between these two groups (p < 0.05). Changes in faecal microbiome composition were notably associated with increasing MAP shedding in the faeces. The present findings suggest a combined influence of the production status and MAP on the cattle faecal microbiome. This possibly correlates with the fate of the infection, the concern in disease control, again remains for further investigations.

A novel rapid detection method for chicken adulteration based on recombinant polymerase amplification and multicomponent nuclease (MNAzyme)

The identification of meat adulteration is of great significance to food safety. This work reports a novel rapid detection platform for chicken adulteration based on recombinase polymerase amplification (RPA) and multicomponent nuclease (MNAzyme). The conserved sequence target of the Cytb gene was used to design RPA primers and MNAzyme. First, the RPA amplification products were introduced into the detection system, which included MNAzyme and DNA-RNA composite probes. When the chicken-derived RPA amplification product was present in the sample, it generated an “input” signal that triggered MNAzyme's catalytic core to cleave the DNA-RNA complex probe, leading to a fluorescence signal. The entire procedure, from nucleic acid extraction to detection, took just 60 min, and the results could be easily interpreted using a portable fluorescent thermostatic amplifier. The RPA-MNAzyme assay detected 8 copies/µL of chicken DNA in 60 min, which is consistent with the results of the RPA assay, demonstrating that the method is highly accurate and specific (does not cross-react with nucleic acids from other animal sources). And it could detect as low as 0.5% chicken in beef and lamb. By harnessing MNAzyme to specifically identify target genes in RPA amplification products, the platform eliminates the need for RPA primer screening and RPA probe design, reducing experimental costs.

Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops

IntroductionGenetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. ObjectiveIn this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. MethodsThe RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. ResultsDetection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. ConclusionThe development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

A portable fluorescence-based recombinase polymerase amplification assay for the detection of mal secco disease by Plenodomus tracheiphilus

In this study, a new diagnostic assay to detect Plenodomus tracheiphilus, the causative agent of mal secco of citrus, was developed based on the recombinase polymerase amplification (RPA) technology. Mal secco is a well-known and damaging vascular disease, affecting primarily lemon (Citrus limon) and, to a lesser extent, other citrus species, including those in the genera Citrus, Fortunella, Poncirus and Severina. The disease poses a considerable threat to lemon production in most of the citrus-producing countries of the Mediterranean region and in the Black Sea area. RPA primers and probes were designed to amplify a 142 bp amplicon from the ITS regions of P. tracheiphilus. The inclusivity and specificity of the RPA assay were tested on gDNA isolated from a panel including 29 strains of various origin of P. tracheiphilus and 18 non-target fungal and oomycete plant pathogens typically isolated from citrus trees. The assay was specific to P. tracheiphilus and had a detection threshold of 1.0 pg of gDNA. Preliminary tests carried out on plant crude extract highlighted RPA's potential for the rapid, user-friendly, and cost-effective field diagnosis of mal secco.

Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification

Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively.DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.

Rapid and specific detection of Pentastiridius leporinus by recombinase polymerase amplification assay.

Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of an emerging and fast spreading sugar beet disease, the syndrome 'basses richesses' (SBR), in different European countries. The disease is caused by the γ-3-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the phytoplasma 'Candidatus Phytoplasma solani' which are exclusively transmitted by planthoppers and can lead to a significant loss of sugar content and yield. Monitoring of this insect vector is important for disease management. However, the morphological identification is time consuming and challenging as two additional cixiid species Reptalus quinquecostatus and Hyalesthes obsoletus with a very close morphology have been reported in sugar beet fields. Further, identification of females and nymphs of P. leporinus at species level based on taxonomic key is not possible. In this study, an isothermal nucleic acid amplification based on recombinase polymerase amplification (RPA) was developed to specifically detect P. leporinus. In addition, real-time RPA was developed to detect both adults (male and female) and nymph stages using pure or crude nucleic acid extracts. The sensitivity of the real-time RPA for detection of P. leporinus was comparable to real-time PCR, but a shorter time (< 7 min) was required. This is a first report for real-time RPA application for P. leporinus detection using crude nucleic acid templates which can be applied for fast and specific detection of this vector in the field.

Simultaneous detection of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) using recombinase polymerase amplification

Bovine viral diarrhea virus (BVDV) is considered to be the most common agent of severe diarrhea in cattle worldwide, causing fever, diarrhea, ulcers, and abortion. Bovine herpesvirus 1 (BoHV-1) is also a major bovine respiratory disease agent that spreads worldwide and causes extensive damage to the livestock industry. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method with the advantages of high efficiency, rapidity and sensitivity, which has been widely used in the diagnosis of infectious diseases. A dual RPA assay was developed for the simultaneous detection of BVDV and BoHV-1. The assay was completed at a constant temperature of 37 °C for 30 min. It was highly sensitive and had no cross-reactivity with other common bovine viruses. The detection rate of BVDV RPA in clinical samples (36.67%) was higher than that of PCR (33.33%), the detection rate of BoHV-1 RPA and PCR were equal. Therefore, the established dual RPA assay for BVDV and BoHV-1 could be a potential candidate for use as an immediate diagnostic.

Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero

Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.

Establishment of rapid extraction and sensitive detection system of trace corn syrup DNA in honey

Honey adulteration with exogenous syrup has become a common phenomenon, and current detection techniques that require large instruments are cumbersome and time-consuming. In this study, a simple and efficient method was developed by integrating the rapid extraction of nucleic acids (REMD) and recombinase polymerase amplification (RPA), known as REMD-RPA, for the rapid screening of syrup adulteration in honey. First, a rapid extraction method was developed to rapidly extract corn syrup DNA in five minutes to meet the requirements of PCR and RPA assays. Then, the RPA method for detecting endogenous maize genes (ZssIIb) was established, which could detect 12 copies/μL of the endogenous maize gene within 30 min without cross-reacting with other plant-derived genes. This indicated that the RPA technique exhibited high sensitivity and specificity. Finally, the REMD-RPA detection platform was used to detect different concentrations of corn syrup adulteration, and 1 % adulteration could be detected within 30 min. The 22 commercially available samples were tested to validate the efficacy of this method, and the established RPA was able to detect seven adulterated samples in less than 30 min. Overall, the developed method is rapid, sensitive, and specific, providing technical support for the rapid field detection of honey adulteration and can serve as a reference for developing other field test methods.