Recombinase Polymerase Amplification Reaction Research Articles

Highly Specific and Rapid Multiplex Identification of Candida Species Using Digital Microfluidics Integrated with a Semi-Nested Genoarray.

Candida species are the most common cause of fungal infections around the world, associated with superficial and even deep-seated infections. In clinical practice, there is great significance in identifying different Candida species because of their respective characteristics. However, current technologies have difficulty in onsite species identification due to long turnover time, high cost of reagents and instruments, or limited detection performance. We developed a semi-nested recombinase polymerase amplification (RPA) genoarray as well as an integrated system for highly specific identification of four Candida species with a simple design of primers, high detection sensitivity, fast turnover time, and good cost-effectiveness. The system constructed to perform the assay consists of a rapid sample processing module for nucleic acid release from fungal samples in 15 min and a digital microfluidic platform for precise and efficient detection reactions in 35 min. Therefore, our system could automatically identify specific Candida species, with a reagent consumption of only 2.5 μL of the RPA reaction mixture per target and no cross-reaction. Its detection sensitivity for four Candida species achieved 101-102 CFU/mL, which was 10-fold better than conventional RPA and even comparable to a common polymerase chain reaction. Evaluated by using cultured samples and 24 clinical samples, our system shows great applicability to onsite multiplex nucleic acid analysis.

Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales

The worldwide spread of carbapenemase-producing Enterobacterales (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (blaKPC and blaNDM) within 50 min, including 10 min for DNA extraction and 30–40 min for RCCS reaction (a 20 min RPA reaction with a 10–20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting blaKPC and blaNDM at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of Klebsiella pneumoniae and Escherichia coli; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments.

Rapid and multiple visual detection of Fasciola hepatica in feces via recombinase polymerase amplification integrated with CRISPR/Cas12a technology

Fasciola hepatica is a foodborne zoonotic parasite causing significant economic losses and impacting human and livestock health in resource-limited regions. We developed a rapid, reliable, and sensitive detection method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a, allowing visualization with the naked eye or a fluorescence reader. Multiple visual methods were used to analyze the assay results. Fluorescence signals were collected using a fluorescence reader or observed under UV or blue light. Lateral flow strips (LFS) were used for visual detection. Among seven primer pairs and three CRISPR RNA (crRNA) screened, F1/R1 and crRNA3 were optimal. The Cas12a reaction buffer was optimized with 50 mM Tris-HCl and 80 mM NaCl, with an RPA reaction time of 20 min. The assay showed high specificity and excellent sensitivity for F. hepatica, detecting 0.122 copies/μL with fluorescence and 8.6 copies/μL with LFS. Testing of 143 sheep and 43 human fecal samples showed 98.39 % consistency with qPCR and nested PCR, with prevalence rates of 52.45 % and 18.6 % in sheep and humans, respectively. Our assay offers substantial potential for point-of-care testing in resource-limited areas, addressing the need for rapid and accurate diagnosis of F. hepatica.

Development of an RPA-CRISPR/Cas12a Assay for Rapid and Sensitive Diagnosis of Plant Quarantine Fungus Setophoma terrestris.

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management.

Establishment of a Cas12a-Based Visual Detection Method Involving PMNT for the Colletotrichum gloeosporioides Species Complex.

Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of CGSC in strawberry seedlings. This method targets the β-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA (ssDNA). This method shows high sensitivity (ten copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37°C for 20 min), CRISPR/Cas12a detection (37°C for 10 min), and visual detection by the naked eye (1-2 min). Furthermore, the Cas12aVIP assay successfully detected CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.

Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within 30–40 min, comprising a 25–30 min RPA reaction and a 5–10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

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.

Rapid and Efficient Molecular Detection of Phytophthora nicotianae Based on RPA-CRISPR/Cas12a

Phytophthora nicotianae is a global and polyphagous pathogen with a wide host range. P. nicotianae can infect Areca catechu, Durio zibethinus L., Psidium guajava L., Hevea brasiliensis, and other tree species. The pathogen is capable of inducing butt rot and affecting aerial parts, including stems, leaves, and fruits. Compared to other Phytophthora species, P. nicotianae is more adaptable to abiotic stress. In this study, recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system was used for the detection of P. nicotianae, and achieved rapid and efficient detection of P. nicotianae. The assay was highly specific to P. nicotianae. All 4 tested isolates of P. nicotianae yielded positive results, whereas 30 isolates belonging to 17 other Phytophthora species, 8 fungal species, and 4 Bursaphelenchus xylophilus vermicules lacked detection. Under the conditions of 37 °C, after 20 min of RPA reaction and 25 min of Cas12a cleavage, a DNA concentration as low as 10 pg·μL−1 could be detected. In addition, it detected P. nicotianae from artificially inoculated leaves of Fatsia japonica. In this study, a novel method was established for the efficient and accurate detection of P. nicotianae based on the combination of RPA and the CRISPR/Cas12a system.

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.