Recombinase Polymerase Amplification Reaction Research Articles

Establishment and application of a rapid visual diagnostic method for Streptococcus agalactiae based on recombinase polymerase amplification and lateral flow strips

This study aims to establish a rapid diagnostic method for Streptococcus agalactiae (GBS) based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). The best primer pairs designed by SIP gene were screened according to the basic RPA reaction, then the probe was designed. The reaction condition was optimized based on the color development of the LFS detection line. To ascertain the reaction specificity, 10 common clinical pathogens and 10 clinical specimens of GBS were tested. Furthermore, the reaction sensitivity was assessed by utilizing a tenfold gradient dilution of GBS genomic DNA as templates. RPA–LFS method was compared to the qPCR assay and biochemical culture method for the Kappa consistency test. The RPA–LFS technique was able to complete the amplification process within 30 min and the results were observed on lateral flow strips. The method is highly sensitive, with a minimum detection limit of 1.31 ng for GBS. The RPA–LFS method showed consistent accuracy of results compared to qPCR and the culture–biochemical method. The establishment of this method is conducive to the development of on-site immediate detection, which can provide information for the timely development of a reasonable antimicrobial treatment plan, and has a greater potential for clinical application.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay

BackgroundWhile Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection.MethodsMultiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo.ResultsUsing 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min.ConclusionCombined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/μL, and both assays were completed in 30min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Simple, sensitive, and visual detection of 12 respiratory pathogens with one-pot-RPA-CRISPR/Cas12a assay.

Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large-scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single-tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one-pot-RPA-CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including sixbacteria and sixviruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one-pot-RPA-CRISPR/Cas12a method is 2.5 × 100copies/μL plasmids, with no cross-reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one-pot-RPA-CRISPR/Cas12a method shows immense potential for accurate and large-scale detection of 12 common respiratory pathogens in point-of-care testing.

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.

Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta.

Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L-1 . Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA-LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10-1 ng μL-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry.

Efficient detection of Streptococcus pyogenes based on recombinase polymerase amplification and lateral flow strip.

This article aims to establish a rapid visual method for the detection of Streptococcus pyogenes (GAS) based on recombinase polymerase amplification (RPA) and lateral flow strip (LFS). Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out. The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6min at 33°C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908ng, and does not exhibit cross-reaction with other pathogenic bacteria. The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.

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.

Ni-Pt nanozyme-mediated relaxation and colorimetric sensor for dual-modality detection of norovirus

An NiPt nanozyme-mediated relaxation and colorimetric sensor is developed for dual-modality detection of norovirus (NoV). The relaxation modality is based on the “catalase-like” activity of the NiPt nanozyme, which adjusts the hydrogen peroxide (H2O2) mediated Fe (II)/Fe(III) conversion, thereby changing the relaxation signal. Poly-γ-glutamic acid (MW ≈ 1w) can enhance the relaxivity of Fe(III) (r1 = 7.11 mM−1 s−1; r2 = 8.94 mM−1 s−1). The colorimetric modality exploits the “peroxidase-like” activity of the NiPt nanozyme, which can catalyze the oxidation of colorless 3, 3′, 5, 5′-tetramethylbenzidine (TMB) to blue oxTMB in H2O2. Under optimal conditions, the relaxation modality exhibits a wide working range (1.0 × 101–1.0 × 104 fM) and a limit of detection (LOD) of 4.7 fM (equivalent to 2820 copies/μL). The spiked recoveries range from 99.593 to 106.442 %, and the relative standard deviation (RSD) is less than 5.124 %. The colorimetric modality exhibited the same working range with a lower LOD of 2.9 fM (equivalent to 1740 copies/μL) and an RSD of less than 2.611 %. Additionally, the recombinase polymerase amplification reaction enabled the detection of low NoV levels in food samples with a working range of 102–106 copies/mL and LOD of 102 copies/mL. The accuracy of the sensor in the analysis of spiked samples is consistent with the gold standard method (real-time quantitative reverse transcription-polymerase chain reaction), demonstrating the high accuracy and practical utility of the sensor.

Gen Mutasyonunun Belirlenmesinde Rekombinaz Polimeraz Çoğaltım Tekniği Optimizasyonu Çalışmaları ve Sonuçları

Single nucleotide polymorphisms (SNPs) in human genes are very significant genetic changes and PCR (polymerase chain reaction) or NGS (next-generation sequencing) are extensively employed in SNP analysis. Thanks to the studies on the progress of new technologies, interest in the isothermal nucleic acid amplification approach has increased. As one of these methods, recombinase polymerase amplification (RPA) represents an attractive option for point-of-care nucleic acid quantification. The target SNPs selected within the scope of the study are mutations identified in the PIK3CA gene region (E542K, E545K), and DNA samples which were evaluated about PIK3CA mutations were isolated from the cancer cells MCF7, BT474, and also SKBr3. The optimization studies for the RPA reaction conditions were carried out for parameters such as assay time, temperature, primer, and also magnesium acetate concentration. According to the results of the reaction optimization studies, in which the RPA products can be obtained in the most efficient way, the assay time was determined as 20 min; the temperature as 40°C; the primer concentration as 10 µM and the MgOAc concentration as 140 mM.

RLP system: A single-tube two-step approach with dual amplification cascades for rapid identification of EGFR T790M

BackgroundThe detection of cancer gene mutations in biofluids plays a pivotal role in revolutionizing disease diagnosis. The presence of a large background of wild-type sequences poses a challenge to liquid biopsy of tumor mutation genes. Suppressing the detection of wild-type sequences can reduce their interference, however, due to the minimal difference between mutant and wild-type sequences (such as single nucleotide variants differing by only one nucleotide), how to suppress the detection of wild-type sequences to the greatest extent without compromising the sensitivity of mutant sequence detection remains to be explored. SignificanceThe RLP system addresses the incompatibility between RPA and RT-PCR reactions through a physical separation strategy. Besides, due to the remarkable flexibility of locked nucleic acid probes, the RLP system emerges as a potent tool for detecting mutations across diverse genes. It excels in sensitivity and speed, tolerates plasma matrix, and is cost-effective. This bodes well for advancing the field of precision medicine. ResultsThe recombinase-assisted locked nucleic acid (LNA) probe-mediated dual amplification biosensing platform (namely RLP), which combines recombinase polymerase amplification (RPA) and LNA clamp PCR method in one tube, enabling highly sensitive and selective detection of EGFR T790M mutation under the help of well-designed LNA probes. This technique can quantify DNA targets with a limit of detection (LoD) at the single copy level and identify point mutation with mutant allelic fractions as low as 0.007 % in 45 min. Moreover, RLP has the potential for the direct detection of plasma samples without the need for nucleic acid extraction and the cost of a single test is less than 1USD. Furthermore, the RLP system is a cascading dual amplification reaction conducted in a single tube, which eliminates the risk of cross-contamination associated with opening multiple tubes and ensures the reliability of the results.

Rapid and sensitive detection of two fungal pathogens in soybeans using the recombinase polymerase amplification/CRISPR-Cas12a method for potential on-site disease diagnosis.

Diaporthe aspalathi and Diaporthe caulivora are two of the fungal pathogens causing soybean stem canker (SSC) in soybean, which is one of the most widespread diseases in soybean growing regions and can cause 100% loss of yield. Current methods for the detection of fungal pathogens, including morphological identification and molecular detection, are mostly limited by the need for professional laboratories and staff. To develop a detection method for potential on-site diagnosis for two of the fungal pathogens causing SSC, we designed a rapid assay combining recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based diagnostics to specifically detect D. aspalathi and D. caulivora. The translation elongation factor 1-alpha gene was employed as the target gene to evaluate the specificity and sensitivity of this assay. The RPA/CRISPR-Cas12a system has excellent specificity to distinguish D. aspalathi and D. caulivora from closely related species. The sensitivities of RPA/CRISPR-Cas12a-based fluorescence detection and lateral flow assay for D. aspalathi and D. caulivora are 14.5 copies and 24.6 copies, respectively. This assay can detect hyphae in inoculated soybean stems at 12 days after inoculation and has a recovery as high as 86% for hyphae-spiked soybean seed powder. The total time from DNA extraction to detection was not more than 60 min. The method developed for rapid detection of plant pathogens includes DNA extraction with magnetic beads or rapid DNA extraction, isothermal nucleic acid amplification at 39 °C, CRISPR-Cas12a cleavage reaction at 37 °C, and lateral flow assay or endpoint fluorescence visualization at room temperature. The RPA and CRISPR-Cas12a reagents can be preloaded in the microcentrifuge tube to simplify the procedures in the field. Both RPA and CRISPR-Cas12a reaction can be realized on a portable incubator, and the results are visualized using lateral flow strips or portable flashlight. This method requires minimal equipment and operator training, and has promising applications for rapid on-site disease screening, port inspection, or controlling fungal pathogen transmission in crop. © 2023 Society of Chemical Industry.

A point-of-care single nucleotide variation assay based on strand-displacement-triggered recombinase polymerase amplification

The single nucleotide variation (SNV) assay holds significant value in the diagnosis of genetic diseases. However, the wide application of SNV analysis in clinics meets many difficulties because of the long test time and the limited experimental conditions. In this study, we develop a lateral flow-based strand-displacement-triggered recombinase polymerase amplification (SD-triggered RPA) assay for detecting β-thalassemia-related SNV (codon 17 (A>T) mutation), which is sensitive, fast, simple and easy to interpret in clinics. By innovatively integrating toehold-mediated strand displacement into recombinase polymerase amplification (RPA), selective strand displacement starts with the specific discrimination of SNV and triggers subsequent RPA reaction, releasing the displaced strands to contribute more D-loop structures for the strand-displacement probes to hybridize with the template strands in next amplification cycle. The limitations of current RPA SNV assays regarding specificity and sensitivity have been overcome in this method, as low as 4 pg/μL codon 17 (A>T) mutant genomic DNA (nearly 6 copies) can be accurately determined and robust behavior in clinical specimen analysis was also demonstrated. Moreover, the clinical samples can be analyzed within 35 min from sample extraction to obtaining results. Thus, the SD-triggered RPA strategy provides a pragmatic visualization approach for the rapid, sensitive, and easy detection of codon 17 (A>T) mutation, which is a promising method for point-of-care (POC) SNV assay and exhibits significant potential for future application in clinics.

Capillary-driven microfluidic device integrating recombinase polymerase amplification for human papillomavirus detection

Nucleic acid amplification tests (NAATs) have been widely used in clinical diagnosis. However, current NAATs lack of user-friendly operation, require expensive systems and rely on laboratory-based methods. Here, we present low-cost and easy-to-fabricate capillary-driven microfluidic device for detecting DNA targeting of Human papillomavirus (HPV) type 16. The combination of recombinase polymerase amplification (RPA), reagent mixing, and detection of amplified DNA can be completed in a single microfluidic platform. We made the microfluidic circuit by stacking layers of transparent film and double-sided adhesive film, creating capillary in the gap between two layers of transparent film that drives the flow. The different channel height in each part of microfluidic channel creates push valve that allowed sequential fluid flow to be stopped for RPA reaction for 30 min. Users open the valve by pressing it to transfer RPA product into the detection system and automatically mix with SYBR Green I for fluorometric readout. The microfluidic device could detect HPV 16 DNA within the range of 0.002–1.0 ng/μL, with a detection limit of 0.24 pg/μL. The device demonstrated great performance when applied to clinical sample. The innovative device design transforms NAATs development for real-world point-of-care use through streamlined single-device operation.

Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Stewart’s vascular wilt and leaf blight of sweet corn is caused by the Gram-negative enteric bacterium Pantoea stewartii subsp. stewartii. Stewart’s wilt results in substantial yield losses worldwide warranting rapid and accurate disease diagnosis. Recombinase polymerase amplification (RPA) is an isothermal technique that is tolerant to host plant-derived inhibitors and is, therefore, ideally suited for rapid in-field detection vis-à-vis traditional polymerase chain reaction-based molecular assays. An RPA assay coupled with a Lateral Flow Device (LFD) was developed for rapid, accurate, and sensitive real-time detection of P. stewartii subsp. stewartii directly from the infected host offering in-field pathogen detection, timely disease management, and satisfying quarantine and phytosanitary requirements. Twelve novel primer sets were designed against conserved genomic regions of P. stewartii subsp. Stewartii; however, only the primers for amplification of the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB were discernibly unique and adequate for unambiguous identification of P. stewartii subsp. stewartii. The P. stewartii subsp. stewartii-specific primers were further validated in a simplex RPA assay for specificity against twenty-six bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii subsp. stewartii with an empirical detection limit of 0.0005 pg μL−1 bacterial DNA and 1 × 102 CFU mL−1 (app. two bacterial cells used per RPA reaction) in minimally processed samples for accurate, low-cost, and point-of-need diagnosis of the quarantine pathogen P. stewartii subsp. stewartii.

Application of recombinant human pyruvate kinase in recombinase polymerase amplification

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the invitro synthesized urease subunit β (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20ng/μL and 10mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.

New and rapid visual detection assay for Trogoderma granarium Everts based on recombinase polymerase amplification and CRISPR/Cas12a.

Khapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a system. We designed and selected the first khapra beetle-specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA=100 nM/500 nM). With only a 37°C-heat-source and a blue light torch, RPA and CRISPR/CAS12a-based visualization assays can be completed within 40 min to differentiate among khapra beetle and other nine similar Dermestidae species. After DNA extraction using a kit (4-5 h) or a simple method (5 min), the specific amplicons were obtained after a 15 min-RPA-reaction at 37°C, followed by a 15 min-color-reaction under 37°C in dark using CRISPR/CAS12a system and a fluorescent probe (5'-FAM/3'-BHQ1 labeled). This method is ingenious to low levels of DNA (10-1 ng/μL) and meets the sensitivity requirements for detecting a single khapra beetle's egg (about 0.7 mm). Our specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37°C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management and provides value as a reference for monitoring and identification of other pests. This article is protected by copyright. All rights reserved.

A lateral flow immunochromatographic strip based on RPA and quantum dot nanobeads for rapid identification of pathogenic Yersinia enterocolitica

To detect pathogenic Yersinia enterocolitica in meat, a lateral flow immunochromatographic strip targeting on virulence gene ail was developed, which was based on recombinase polymerase amplification (RPA) and quantum dot nanobeads (QDNBs). Under optimal conditions, the minimum detection limit of the strip was 9.6 × 103 CFU/mL, with a standard curve equation of y = 0.144x - 0.5098 (R2 = 0.9863), and the detection times including the RPA reaction at hand temperature (35–36.5 °C), could be controlled in 10 min. When testing spiked sample, the recoveries (%) of Yersinia enterocolitica at 9.6 × 104 CFU/mL were 44.27 ± 2.08 and 45.31 ± 3.12 in beef and pork samples and was 107.55 ± 1.30 in chicken samples. When testing 15 actual samples, only one pork sample was tested positive, consistent with polymerase chain reaction (PCR) results. The strips are body temperature-triggered and rapidly visualized, suitable for rapid field detection of pathogenic Yersinia enterocolitica.

A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics.

Persistent infection with human papillomavirus (HPV) is the leading cause of cervical cancer, and early diagnosis is crucial for clinical management. However, the easy and rapid on-site diagnostic for HPV genotyping remains challenging. Here, we develop a Cas12a-based fluorescent microfluidic detection system for diagnosing six HPV subtypes (HPV6, HPV11, HPV16, HPV18, HPV31, andHPV33). A panel of crRNAs and recombinase polymerase amplification (RPA) primers targeting the HPV L1 gene was screened for sensitive and specific detection. Furthermore, a one-pot RPA reaction was developed to amplify the six HPV subtypes without cross-reactivity. For on-site detection, we integrated the RPA-Cas12a detection into a microfluidic device, enabling the detection of processed clinical samples within 35 minutes. The assay was validated using 112 clinical swab samples and obtained consistent results with the qPCR assay, with a concordance rate of 99.1%. Overall, our diagnostic method offers a rapid, sensitive, and easy-to-use on-site assay for detecting HPV genotypes and holds promise for improving cervical cancer screening and prevention. KEY POINTS: • The Cas12a-based fluorescent microfluidic detection system for the diagnosis of six HPV subtypes. • A one-pot RPA reaction for amplifying the six HPV subtypes without cross-reactivity. • The RPA-Cas12a-microfluidic system provides results within 35 minutes for on-site detection.

Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system.

Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen involved in a wide range of infections and antimicrobial resistance. Rapid and reliable diagnostic methods are of vital important for early identification, treatment, and stop of P. aeruginosa infections. In this study, we developed a simple, rapid, sensitive, and specific detection platform for P. aeruginosa infection diagnosis. The method integrated recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 12a (Cas12a) biosensing system and was termed P. aeruginosa-CRISPR-RPA assay. The P. aeruginosa-CRISPR-RPA assay was subject to optimization of reaction conditions and evaluation of sensitivity, specificity, and clinical feasibility with the serial dilutions of P. aeruginosa genomic DNA, the non-P. aeruginosa strains, and the clinical samples. As a result, the P. aeruginosa-CRISPR-RPA assay was able to complete P. aeruginosa detection within half an hour, including RPA reaction at 42°C for 20min and CRISPR-Cas12a detection at 37°C for 10min. The diagnostic method exhibited high sensitivity (60 fg per reaction, ~8 copies) and specificity (100%). The results of the clinical samples by P. aeruginosa-CRISPR-RPA assay were consistent to that of the initial result by microfluidic chip method. These data demonstrated that the newly developed P. aeruginosa-CRISPR-RPA assay was reliable for P. aeruginosa detection. In summary, the P. aeruginosa-CRISPR-RPA assay is a promising tool to early and rapid diagnose P. aeruginosa infection and stop its wide spread especially in the hospital settings.