Recombinase Polymerase Amplification Lateral Flow Research Articles

Visualized Detection of Tobacco Anthracnose by a Recombinase Polymerase Amplification-Lateral Flow Dipstick Assay.

Anthracnose caused by Colletotrichum spp. is a widespread fungal disease that is detrimental to tobacco growth and inflicts economic damage of up to 100 million in tobacco-growing regions in China. An early diagnostic tool is vital for the accurate determination and management of anthracnose in the field. This study investigated the diversity of Colletotrichum spp. on tobacco leaves with anthracnose and developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) diagnostic method for the rapid and equipment-independent detection of the main Colletotrichum spp. causing tobacco anthracnose. This assay targeted the chitin synthase gene (chs1) and could be performed in a few minutes (6 to 10 min). All isolates of C. karstii, C. fructicola, and C. gloeosporioides yielded positive results in the RPA-LFD assay, and no cross-reaction occurred with other fungal species from tobacco or other hosts. The detection threshold was 1 pg of genomic DNA under optimal reaction conditions. The entire RPA-LFD assay enabled the detection of pathogen visualization within 30 min without specialized equipment by combining a polyethylene glycol-KOH method for extracting gDNA rapidly from tobacco leaves infected with C. karstii, C. fructicola, and C. gloeosporioides. Based on these results, the RPA-LFD assay is easy to operate, rapid, and equipment-independent and is promising for development as a kit to diagnose tobacco anthracnose in resource-limited settings at point-of-care.

Rapid and visual detection of Pyricularia oryzae using coupled recombinase polymerase amplification-lateral flow dipstick assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive fungal diseases in rice, severely impacting rice production worldwide every year. Rapid, accurate and visual detection of P. oryzae is essential for more effective prevention and control. In this study, we developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay to detect P. oryzae. Species-specific RPA primer pairs and probe were designed based on target gene MGG_15975. The optimized reaction temperature and time were set at 37 °C and 25 min, respectively. Specificity analysis showed that the assay could specifically detect P. oryzae isolates from rice, whereas other fungal species or Pyricularia species from grasses were not detected. Additionally, this assay demonstrated highly sensitivity, capable of detecting as low as 10-2 ng/µL of P. oryzae genomic DNA, which was found to be 100 times more sensitive than conventional PCR. Furthermore, using this assay, P. oryzae was effectively detected in diseased leaves in rice fields, and could also be identified at an early stage of infection before obvious lesions appeared in artificially inoculated rice seedlings. Therefore, the RPA-LFD assay developed in our study for the detection of P. oryzae is rapid, highly sensitive and efficient, which has the potential application for early diagnosis of P. oryzae infection in rice fields.

The Recombinase Polymerase Amplification Test for Strongyloides stercoralis Is More Sensitive than Microscopy and Real-Time PCR in High-Risk Communities of Cusco, Peru.

Strongyloidiasis is a neglected, soil-transmitted helminth infection prevalent worldwide. The true burden of strongyloidiasis is unclear due to the lack of sensitive, field-friendly diagnostic tests. PCR tests to detect Strongyloides DNA in stool are sensitive and specific, but the need for expensive equipment limits their use in endemic regions. Isothermal PCR amplification tests are easier to deploy while maintaining sensitivity and specificity. We developed and evaluated a recombinase polymerase amplification lateral flow assay (RPA-LFA) to detect Strongyloides stercoralis in human stool samples. Three hundred stool samples were collected in three communities in the jungle of Cusco, Peru. Samples were tested for S. stercoralis larvae using microscopy (Baermann's, agar plate culture (APC), and rapid sedimentation), real-time PCR, and RPA-LF for Strongyloides DNA. The RPA-LFA showed an analytical limit of detection of 20 pg/µL. The prevalence of S. stercoralis was 27%, 38%, 46.3%, and 46% using microscopy, PCR, microscopy/PCR, and RPA-LFA, respectively. RPA-LFA had a sensitivity and specificity of 59.3% and 58.9%, 66.2% and 71.4%, and 77.2% and 73.1% when microscopy, microscopy/PCR, and real-time PCR were used as the gold standards, respectively. The Strongyloides RPA-LFA is a novel, fast, highly sensitive, and specific molecular method with the potential for deployment in endemic regions.

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.

A Rapid, Equipment-Free Method for Detecting Avirulence Genes of Pyricularia oryzae Using a Lateral Flow Strip-Based RPA Assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control rice blast. Consequently, it is critical to monitor the distribution frequency of avirulence (Avr) genes in rice planting fields to facilitate the breeding of resistant rice varieties. In this study, we established a rapid recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection system for the identification of AvrPik, Avr-Piz-t, and Avr-Pi9. The optimized reaction temperature and duration were 37°C and 20 min, indicating that the reaction system could be initiated by body temperature without relying on any precision instruments. Specificity analysis showed that the primer and probe combinations targeting the three Avr genes exhibited a remarkable specificity at genus-level detection. Under the optimized condition, the lower detected thresholds of AvrPik, Avr-Piz-t, and Avr-Pi9 were 10 fg/μl, 100 fg/μl, and 10 pg/μl, respectively. Notably, the detection sensitivity of the three Avr genes was much higher than that of PCR. In addition, we also successfully detected the presence of AvrPik, Avr-Piz-t, and Avr-Pi9 in the leaf and panicle blast lesions with the RPA-LFD detection system. In particular, the genomic DNA was extracted using the simpler PEG-NaOH rapid extraction method. In summary, we developed an RPA detection system for AvrPik, Avr-Pi9, and Avr-Piz-t, combined with the PEG-NaOH rapid DNA extraction method. The innovative approach achieved rapid, real-time, and accurate detection of the three Avr genes in the field, which is helpful to understand the distribution frequency of the three Avr genes in the field and provide theoretical reference for the scientific layout of resistant rice varieties.

Recombinase polymerase amplification - lateral flow dipstick for rapid and visual detection of Blastocystis spp.

Blastocystis spp. is a ubiquitous protozoon in the intestinal tract of human and many animals. Microscopic examination is the main method of clinical diagnosis for Blastocystis spp., which is prone to false negative. A simple and rapid diagnosis of Blastocystis spp. infection is an important step to prevent and control blastocystosis. Here, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay was developed for rapid visual detection of Blastocystis spp. DNA amplification could be performed within 18min at 37°C. The minimum DNA detection limit was 1 pg/μL, and there was no cross-reactivity with 12 other non-target pathogens, which was consistent with the sensitivity of conventional PCR (cPCR). Furthermore, 56 fecal samples from the Third Affiliated Hospital of Xinxiang Medical University were tested using RPA and cPCR methods respectively, and the results were completely consistent. The results show that RPA-LFD method has high accuracy and visual results, which provides a new choice for the differential diagnosis and rapid field detection of Blastocystis spp.

Development of a duplex recombinase polymerase amplification-lateral flow strip assay with 1 min of DNA extraction for simultaneous identification of pork and chicken ingredients

Meat adulteration is a challenge faced by the global food industry. However, existing protein-based methods or DNA-based polymerase chain reactions are time-consuming and require specialist devices. Therefore, a rapid and on-site assay is urgently needed for identifying meat species. In this study, we developed a duplex recombinase polymerase amplification-lateral flow strip (dRPA-LFS) assay coupled with a rapid DNA extraction method (utilizing the UiO-66 coated stick as the DNA isolation device, and the isolation can be completed in 1 minute, including lysis, washing, and desorption in three steps) for the identification of pork and chicken ingredients. Targeted genes, the pork (Porcine) mitochondrial ND2 gene and chicken (Gallus gallus) cytochrome B gene, were designed with primers and probes. The whole dRPA-LFS detection procedure can be finished within 18.5 minutes (including 1 minute of rapid DNA extraction, 15 minutes of dRPA amplification, and 2.5 minutes of LFS semi-quantitative detection by the naked eye), and the result can also be quantified by ImageJ software within 15 minutes. No positive amplification was observed in beef, lamb, duck, rabbit, goose, ostrich, and horse meat DNA. This assay is sensitive and can detect as low as 0.1 % (wt %) of pork and chicken in simulated adulterated meat mixtures. This assay was successfully applied to the authentication identification of 30 commercial beef and lamb products.

Establishment and application of a rapid diagnostic method for BVDV and IBRV using recombinase polymerase amplification-lateral flow device.

Bovine Viral Diarrhea Virus (BVDV) and Infectious Bovine Rhinotracheitis Virus (IBRV) are the two most prevalent infectious diseases in cattle. They both can cause persistent infection and immunosuppression, resulting in significant economic losses in the livestock industry. Therefore, rapid detection of early BVDV and IBRV infections is crucial. In this study, a method for the rapid detection of BVDV and IBRV was established by using recombinase polymerase amplification (RPA) combined with lateral flow device (LFD). By optimizing the temperature and time conditions of the RPA reaction, the sensitivity, specificity, and clinical performance were evaluated. The results indicated that the RPA reaction could be completed at 40°C within 25 min. The LOD for BVDV and IBRV by RPA-LFD were 5.1 × 101 copies/μL and 6.65 × 101 copies/μL, respectively, with no cross-reactivity observed with other viruses such as CSFV, BRSV, BPIV3, BRV, and BCoV. Testing of 32 clinical samples showed consistent results between RPA-LFD and qPCR. The RPA-LFD method established in this study can be used for the rapid clinical detection of BVDV and IBRV, which providing a rapid and convenient molecular biology approach for on-site rapid detection and epidemiological investigations. Simultaneously, it offers technical support for the prevention and control of these viruses.

Rapid Visual Detection of Peronophythora litchii on Lychees Using Recombinase Polymerase Amplification Combined with Lateral Flow Assay Based on the Unique Target Gene Pl_101565.

Downy blight, caused by Peronophythora litchii, is a destructive disease that impacts lychee fruit throughout the pre-harvest, post-harvest, and transportation phases. Therefore, the prompt and precise identification of P. litchii is crucial for the effective management of the disease. A novel gene encoding a Rh-type ammonium transporter, Pl_101565, was identified in P. litchii through bioinformatic analysis in this study. Based on this gene, a coupled recombinase polymerase amplification-lateral flow (RPA-LF) assay for the rapid visual detection of P. litchii was developed. The assay has been shown to detect P. litchii accurately, without cross-reactivity to related pathogenic oomycetes or fungi. Moreover, it can be performed effectively within 15 to 25 min at temperatures ranging from 28 to 46 °C. Under optimized conditions, the RPA-LF assay could detect as low as 1 pg of P. litchii genomic DNA in a 25 μL reaction system. Furthermore, the RPA-LF assay successfully detected P. litchii in infected lychee samples within a 30 min timeframe. These attributes establish the RPA-LF assay as a rapid, sensitive, and specific method for diagnosing P. litchii early; it is particularly suitable for applications in resource-limited settings.

An integrated self-heating recombinase polymerase amplification lateral flow strip biosensor for quantification of enterohemorrhagic Escherichia coli O157:H7

The early and precise detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7, a major foodborne pathogen, is crucial to prevent outbreaks and ensure food safety. However, traditional diagnostic methods are often time-consuming, labor-intensive, and require sophisticated laboratory facilities, making them less feasible for point-of-care testing. Here, we present a novel, self-heating recombinase polymerase amplification (RPA)-based integrated lateral flow strip biosensor for the efficient detection of EHEC O157:H7. The self-heating mechanism based on calcium oxide provides a stable and optimal external temperature for RPA, avoiding the use of external power sources or complex temperature control devices. In addition, the color intensity of the test line of the lateral flow strip can be uploaded to the ImageJ software by taking photos, and the quantitative detection of EHEC O157:H7 can be realized by analyzing the gray value. Our results demonstrate that this biosensor has a visual detection limit of 1 × 102 CFU/mL and an analytical limit of detection of 36.23 CFU/mL for EHEC O157:H7, offering rapid detection within 30 min. Furthermore, the biosensor displayed exceptional specificity and exhibited high accuracy. The RSD of three varying concentrations were respectively 5.39 %, 3.85 % and 4.82 %.

Development of Recombinase Polymerase Amplification-Lateral Flow Dipstick (RPA-LFD) as a Rapid On-Site Detection Technique for Fusarium oxysporum.

Fusarium oxysporum can cause many important plant diseases worldwide, such as crown rot, wilt, and root rot. During the development of strawberry crown rot, this pathogenic fungus spreads from the mother plant to the strawberry seedling through the stolon, with obvious characteristics of latent infection. Therefore, the rapid and timely detection of F. oxysporum can significantly help achieve effective disease management. Here, we present a protocol for the recombinase polymerase amplification- lateral flow dipstick (RPA-LFD) detection technique for the rapid detection of F. oxysporum on strawberry, which only takes half an hour. A significant advantage of our RPA-LFD technique is the elimination of the involvement of professional teams and laboratories, which qualifies it for field detection. We test this protocol directly on plant samples with suspected infection by F. oxysporum in the field and greenhouse. It is worth noting that this protocol can quickly, sensitively, and specifically detect F. oxysporum in soils and plants including strawberry. Key features • This protocol is used to detect whether plants such as strawberry are infected with F. oxysporum. • This protocol has potential for application in portable nucleic acid detection. • It can complete the detection of samples in the field within 30 min.

Recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) designed for rapid detection of canine distemper virus.

In the present study, recombinase polymerase amplification (RPA) was combined with the colloidal gold lateral flow dipstick (LFD) method to establish a new, stable, and efficient assay for the detection of canine distemper virus (CDV). We designed a set of specific primers labeled with biotin and a specific probe labeled with dSpacer and C3 spacer, according to the conserved region in the N-terminal gene sequence of CDV. The reaction conditions and systems were then optimized, and the sensitivity and specificity were analyzed for potential clinical application. The results showed that the RPA-LFD assay for CDV detection was successfully established. We also found that the temperature in a closed fist (35°C) is optimal for the RPA reaction. The optimal ratio of primer to probe was 2:1. The minimum detection limit of the RPA-LFD assay was 1 × 101 the median tissue culture infective dose (TCID50)/mL. Using this assay with samples from experimentally infected dogs, CDV was detected in nasal secretions, eye secretions, and blood on the fourth day post infection. In summary, this novel RPA-LFD assay for CDV detection is simple to use, and preliminary findings indicate its high specificity and sensitivity.

Recombinase polymerase amplification-lateral flow assay (RPA-LFA) as a rapid and sensitive test for Escherichia coli O157:H7 detection in food and beverage: A comparative study

Recombinase polymerase amplification-lateral flow assay (RPA-LFA) as a rapid and sensitive test for Escherichia coli O157:H7 detection in food and beverage: A comparative study

Rapid and specific detection of Enterococcus faecium with an isothermal amplification and lateral flow strip combined method.

Enterococcus faecium is responsible for a highly contagious, drug-resistant nosocomial infection that often causes serious illness. In this study, a rapid and sensitive RPA-LFS (recombinase polymerase amplification-lateral flow strip) method for the detection of E. faecium was established based on specific primers and probes designed using the ddl gene. To verify the specificity and sensitivity of the method, 26 specific strains and 100-106CFU/μL E. faecium were selected for detection. The results show that the proposed method can specifically detect E. faecium, and the minimum detection limit is 100CFU/μL. To compare the clinical application of the method with qPCR, 181 clinical samples were collected for testing. RPA-LFS and qPCR had the same practical applicability, and 61 parts of E. faecium were detected in 183 clinical samples. The methods developed in this study not only have the advantages of rapid sensitivity and specificity but also meet the needs of remote areas with scarce medical resources.

Establishment of the Recombinase Polymerase Amplification-Lateral Flow Dipstick Detection Technique for Fusarium oxysporum.

Fusarium oxysporum causes crown rot, wilt, root rot, and many other major plant diseases worldwide. During the progression of strawberry crown rot disease, the pathogen is transmitted from the mother plant to the seedling through the stolon, with obvious characteristics of latent infection. Therefore, rapid and timely detection of F. oxysporum is important for efficient disease management. In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection technique was developed for the rapid detection of F. oxysporum on strawberry plants by targeting the CYP51C gene, which is unique to Fusarium spp. Because this RPA-LFD detection technique was highly specific to F. oxysporum, other Fusarium and non-Fusarium fungi were not detected. The optimal reaction temperature and time for this technique were 39°C and 8 min, respectively. The detection limit was 1 pg of F. oxysporum genomic DNA in a 50-μl reaction system. A total of 46 strawberry plants with or without crown rot symptoms collected from Jiande, Changxing, and Haining in Zhejiang Province were further assessed for F. oxysporum infection using both RPA-LFD and traditional tissue isolation techniques. The RPA-LFD test showed that 32 of the 46 strawberry plants tested were positive for F. oxysporum, while in the traditional isolation technique, F. oxysporum was isolated from 30 of the 46 strawberry plants. These results suggest that our established RPA-LFD method is rapid, sensitive, and highly specific in detecting F. oxysporum infection in strawberry plants.

Rapid onsite detection of piper yellow mottle virus infecting black pepper by recombinase polymerase amplification-lateral flow assay (RPA-LFA)

Piper yellow mottle virus (PYMoV) is a pararetrovirus associated with stunt disease in black pepper. As the primary spread of the virus occurs through vegetative propagation, effective diagnostics are required for the production of virus-free plants. Currently available assays are time-consuming, require expensive equipment, and are not suitable for on-site detection. In the present study, two rapid assays based on the recombinase polymerase amplification (RPA) coupled with lateral flow assay (LFA) using (i) 6-carboxyfluorescein (FAM) labeled nfo probe and biotin-labeled reverse primer and (ii) FAM labeled forward and biotin-labeled reverse primer was developed for the detection of PYMoV. The assays were performed using TwistAmp DNA amplification reagents and crude extract from the infected plant and mealybug as templates. Both assays were optimized for parameters like concentration of magnesium acetate, temperature, and time. The RPA product was then diluted and applied to the sample pad of a lateral flow device for visualizing the results. The formation of a colored line at the test line was considered positive for PYMoV. The entire process from sample preparation to visualization of results could be completed in about 30 min. The developed assays were specific and 10 times more sensitive than PCR. The assays were validated using field samples of black pepper and mealybug vectors.

A Preliminary Study to Compare Recombinase Polymerase Amplification-Lateral Flow and Quantitative PCR in the Detection of Cutaneous Leishmania in Communities from the Volta Region of Ghana.

Background: Leishmaniasis is a parasitic disease that mostly affects populations in tropical and subtropical countries. In Ghana, cutaneous leishmaniasis (CL) is the most common form of the disease affecting communities of the Volta Region. Conventional parasitological method (microscopy) is the commonly used test for CL diagnosis in many endemic countries, but has low sensitivity in chronic cases. Therefore, there is a clear need for a sensitive and easy-to-use point-of-care diagnostic method like an isothermal recombinase polymerase amplification-lateral flow (RPA-LF) test, suitable for use in austere and low-resource settings for the identification of CL cases. This study compared the efficacy of RPA-LF test with quantitative PCR (qPCR) in detecting Leishmania in suspected CL cases from the Volta Region. Methods: Twenty-five participants between 5 and 14 years were enrolled in the study from whom a total of 26 samples were obtained. Lesion samples were collected using FTA® filter papers applied to ulcerated lesions for molecular diagnosis. DNA isolated from filter papers was used for both the RPA-LF test and qPCR. Results: Twenty-two participants (88%) presented with one or two ulcerated active lesions per individual, while the rest of them had plaques or dried lesions. Among the 26 samples, 19/26 (73%) had concordant results when comparing the two diagnostic methods. Conclusion: Data from this study suggest that the RPA-LF test can be used in addition to a conventional parasitological diagnostic test (microscopy) to detect CL cases in communities of the Volta Region.

Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation.

The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10min and visualization of the amplicons through LF strips for 5min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40CFU per reaction. The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

Rapid sample preparation and low-resource molecular detection of hepatopancreatic parvoviruses (HPV) by recombinase polymerase amplification lateral flow detection assay in shrimps (Fenneropenaeus merguiensis).

Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments. We developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity. The rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized F. merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity (95% confidence interval, CI: 86-100%) and 100% specificity (95% CI: 66-100%) for detection of HPV. The rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from samples and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture.

Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip.

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.