Recombinase-aided Amplification Research Articles

A rapid and visual detection assay for Senecavirus A based on recombinase-aided amplification and lateral flow dipstick

BackgroundSenecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease.MethodsWe designed multiple primer pairs targeting the most conserved region of the SVA 3D gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA.ResultsThe SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×101 copies/µL, 8.76×10-7 ng/µL, and 1×100.25 TCID50/mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays.ConclusionThe SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings.

Development of a Lateral Flow Strip-Based Recombinase-Aided Amplification for Active Streptococcus dysgalactiae emerging in channel catfish (Ictalurus punctatus)

Streptococcus dysgalactiae has become an important pathogen affecting mammals and fish (especially farmed fish). The development of a rapid, accurate, and sensitive diagnostic method will aid in the prevention of S. dysgalactiae infection. In this study, a method combining recombinase-aided amplification and a lateral flow dipstick (RAA-LFD) was developed to detect active S. dysgalactiae infection. The RAA-LFD detection was based on the ISP gene of S. dysgalactiae as the target, which could be accomplished with the lowest detection limit of 1.002×102 pg/μL reaction under optimal reaction conditions of 39 °C for 15 min. RAA-LFD and conventional PCR were used to detect 50 experimental samples invaded by S. dysgalactiae disease materials. The results showed that the positive concordance rate of the two methods was 97.6%, the negative concordance rate was 90.0%, and the total concordance rate was 98.0%. Compared with conventional PCR, the Area Under the ROC Curve (AUC) of RAA-LFD was 0.950, and the sensitivity and specificity were 100% and 90%, respectively. The RAA-LFD of S. dysgalactiae infection is convenient for rapid clinical detection.

Reverse-transcription recombinase-aided amplification and CRISPR/Cas12a-based universal detection for fast screening and accurate identification of six pospiviroids infecting Solanaceae crops.

Pospiviroids, members of the genus Pospiviroid, can cause severe diseases in tomato and other Solanaceae crops, causing considerable economic losses worldwide. Six pospiviroids including potato spindle tuber viroid (PSTVd), tomato chlorotic dwarf viroid (TCDVd), tomato planta macho viroid (TPMVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), and tomato apical stunt viroid (TASVd) are regulated in many countries and organizations. Rapid, accurate detection is thus crucial for controlling the spread of these pospiviroids. For simultaneous detection of these six pospiviroids, we developed a rapid, visual method that uses a reverse transcription recombinase-aided amplification (RT-RAA) assay coupled with a clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 12a (CRISPR/Cas12a) system. In particular, this technique could achieve both universal detection and specific identification of the six target pospiviroids within 40 min. The universal detection could diagnose the six target pospiviroids in a single reaction, and the specific identification could identify each target pospiviroid without cross-reactivity of other pospiviroids. The sensitivity limits for the target pospiviroids detection with the proposed detection method were higher than those of the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. We designed an RT-RAA-CRISPR/Cas12a-based universal detection method for both large-scale screening and accurate identification of the six target pospiviroids, which is appropriate for on-site detection. Our study results can aid in performing rapid, large-scale screening of multiple pests simultaneously. © 2024 Society of Chemical Industry.

RAA-CRISPR/Cas12a-Mediated Rapid, Sensitive, and Onsite Detection of Newcastle Disease in Pigeons.

Pigeon Newcastle disease, caused by pigeon paramyxovirus type 1 (PPMV-1), is a significant infectious disease in pigeons that can result in substantial mortality and poses a severe threat to the pigeon industry. The rapid and accurate onsite diagnosis of pigeon disease is crucial for timely diagnosis and the implementation of effective prevention and control measures. In this study, we established a rapid detection method for PPMV-1 based on recombinase-aided amplification (RAA) and CRISPR/Cas12a. The RAA primers target the conserved regions of the L gene for preamplification in clinical nucleic acid samples, followed by CRISPR/Cas12a detection of the target gene. Visualization could be achieved by combination with a lateral flow dipstick (LFD). This method demonstrated high specificity, showing no cross-reactivity with non-PPMV-1 samples. The sensitivity of the method assessed by fluorescence analysis reached 100 copies/µL, and when it was combined with an LFD, the sensitivity was 103 copies/µL. The constructed RAA-CRISPR/Cas12a-LFD visual detection method was applied to clinical sample testing and was found to enable the rapid and accurate detection of swab samples and tissue specimens. Its sensitivity was consistent with the current gold standard, quantitative real-time PCR results. The RAA-CRISPR/Cas12a-LFD detection method we developed provides a novel approach for the rapid, simple, precise, and specific onsite diagnosis of pigeon Newcastle disease.

Research Note: Development of a reverse transcriptase recombinase-aided amplification method for detection of Parrot Borna Virus 4

The Parrot Borna virus 4 (PaBV-4) is the primary causative agent of parrot developmental disorder (PDD), leading to symptoms such as bloating, undigested feed in stool, decreased appetite, diarrhea, and weight loss. Its impact on the parrot industry has been significant, therefore, it is imperative to develop a rapid detection method for PaBV-4. The detection of PaBV-4 was achieved through the development of an RT-RAA assay, which involved the design of specific probes and primers targeting the N gene. This method allows for detection at 41°C within 30 min and has a minimum detection threshold of 8.56 × 101 copies/μL. The RT-RAA method demonstrated specific detection of PaBV-4 without any cross-reactivity observed with H5N6, H7N9, H9N2 avian influenza virus, newcastle disease virus (NDV), avian infectious bronchitis virus (IBV) and Parrot Borna virus 2 (PaBV-2). The coefficient of variation for the 3 repeatability experiments was below 10%. Tissue samples from 28 suspected cases of PaBV related deaths in parrots were analyzed using both RT-RAA and RT-qPCR methods. The sensitivity and specificity of both methods were 100%, demonstrating perfect agreement between them as indicated by a kappa value of 1. In conclusion, this study created a RT-RAA method for PaBV-4 detection successfully.

Microfluidic-free droplet digital RAA/RT-RAA assisted CRISPR/Cas12a system for the detection of DNA/RNA viruses

Digital PCR (dPCR) is the most sensitive method for virus detection by accurate quantifying the nucleic acids. However, the requirement of sophisticated and expensive instrumentations greatly limits its application in clinical diagnosis particularly in poorly resourced settings. Herein, we developed a simple digital assay based on polydisperse droplet digital recombinase-aided amplification/reverse transcription recombinase-aided amplification dependent CRISPR/Cas12a system (PddRaaCas/PddRRaaCas) for viral DNA/RNA detection with minimal instruments. This assay does not require any microfluidic chip for droplet generation and can be completed about 1 hour. We found that the PddRaaCas and PddRRaaCas enabled to detect 0.015 fM target DNA and 10 fM target RNA respectively. In addition, we applied this method to detect human papillomavirus type 18 (HPV18) and severe acute respiratory syndrome coronavirus (SARS-CoV-2) in clinical samples, which represented the detection of DNA virus and RNA virus respectively. The sensitivity and specificity were 100 % and 100 % for both DNA and RNA virus detection respectively. Taken together, these results highlight that PddRaaCas/PddRRaaCas is a sensitive, rapid, and easy-to-use strategy for the detection of DNA/RNA viruses, which has great potential application for the control of infectious diseases.

Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa.

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay

BackgroundHelicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence. MethodsIn this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy. ResultsA RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10-2 ng/μL, 5.8 × 10-2 ng/μL and 1.4 × 10-2 ng/μL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay’s high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00. ConclusionThe dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.

Dye‐based recombinase‐aided amplification assay with enhanced sensitivity and specificity

AbstractObjectiveFluorescent recombinase‐aided amplification (RAA) assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity, compared with real‐time PCR (qPCR) assays, but they require a complex probe design. To eliminate the addition of fluorescent probes for RAA, an EvaGreen dye‐based recombinase‐aided amplification (EvaGreen‐RAA) assay using self‐avoiding molecular recognition system (SAMRS) primers was developed.MethodsThe SAMRS primers effectively avoided the production of primer dimers, thus improving the detection sensitivity, while EvaGreen dye was used to quantitatively measure the amplified products in real time. Using Staphylococcus aureus (SA) and Listeria monocytogenes (LM) as examples, EvaGreen‐RAA with SAMRS primers was developed. As a reference and comparison, a traditional fluorescence probe RAA method and a RAA with SAMRS primers (SAMRS‐RAA) for detecting SA and LM were also investigated. Serial dilutions of recombinant plasmids were used to evaluate the sensitivity of the assays. Unenriched and enriched simulated milk samples were used to evaluate the limits of detection (LOD) of these methods. Using high‐resolution melting (HRM) was used to explore the sensitivity of the dual EvaGreen‐RAA assay.ResultsThe sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS‐RAA and EvaGreen‐RAA for detecting SA and LM plasmids was 1 copies/μL. The LOD values of the EvaGreen‐RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL, respectively, and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL. EvaGreen‐RAA had linear amplification in real time in the range of 1–105 copies/μL of the plasmids of SA and LM. The sensitivity of the dual EvaGreen‐RAA assay for SA and LM was estimated to be 102 CFU/mL.ConclusionA real‐time quantitative EvaGreen‐RAA method for detecting SA and LM was developed, which eliminates the need to design complex RAA probes. This dye‐based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens, which has many potential applications.

The clinical value of rapidly detecting urinary exosomal lncRNA RMRP in bladder cancer with an RT-RAA-CRISPR/Cas12a method

Background and aimsBladder cancer (BCa) is a highly aggressive malignancy of the urinary system. Timely detection is imperative for enhancing BCa patient prognosis. Materials and methodsThis study introduces a novel approach for detecting long non-coding RNA (lncRNA) Mitochondrial RNA Processing Endoribonuclease (RMRP) in urine exosomes from BCa patients using the reverse transcription recombinase-aided amplification (RT-RAA) and clustered regularly interspaced short palindromic repeats and associated Cas12a proteins (CRISPR/Cas12a) technique. Various statistical methods were used to evaluate its diagnostic value for BCa. ResultsThe specificity of urine exosomal RMRP detection for BCa diagnosis was enhanced by using RT-RAA combined with CRISPR/Cas12a. The testing process duration was reduced to 30 min, which supports rapid detection. Moreover, this approach allows the identification of target signals in real-time using blue light, facilitating immediate detection. In clinical sample analysis, this methodology exhibited a high level of diagnostic efficacy. This was evidenced by larger area under the curve values with receiver operating characteristic curve analysis compared with using traditional RT-qPCR methods, indicating superior diagnostic accuracy and sensitivity. Furthermore, the combined analysis of RMRP expression in urine exosomes detected by RT-RAA-CRISPR/Cas12a and NMP-22 expression may further enhance diagnostic accuracy. ConclusionsThe RT-RAA-CRISPR/Cas12a technology is a swift, sensitive, and uncomplicated method for nucleic acid detection. Because of its convenient and non-invasive sampling approach, user-friendly operation, and reproducibility, this technology is very promising for automated detection and holds favorable application possibilities within clinical environments.

A New Dual Fluorescence Method for Rapid Detection of Infectious Bronchitis Virus at Constant Temperature.

Infectious bronchitis virus (IBV) causes infectious bronchitis in chicken, an acute, highly contagious respiratory infection. Because of genetic mutations and recombination, IBV forms many subtypes, which makes it difficult to treat the disease and apply commercial vaccines. Therefore, to detect IBV in time and stop the virus from spreading, a novel and convenient IBV detection technology based on reverse transcription recombinase-aided amplification (RT-RAA) was established in this study. According to the S1 gene of IBV CH I-V and Mass genotypes and S1 gene of IBV CH VI genotype, a set of optimal primers were designed and selected to establish a real-time dual fluorescence RT-RAA method. The lowest detection line was 10 copies/μL of RNA molecules and the method exhibited no cross-reactivity with avian reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV), and H9N2 avian influenza virus (H9N2), demonstrating high specificity. When compared to qPCR detection results, our method achieved a sensitivity of 96.67%, a specificity of 90%, and a Kappa value of 0.87 for the IBV CH I-V and Mass genotypes, and achieved a sensitivity of 100%, a specificity of 97.73%, and a Kappa value of 0.91 for the IBV CH VI genotype.

Research Note: Real-time fluorescence-based recombinase-aided amplification for rapid detection of Mycoplasma synoviae

Mycoplasma synoviae (MS) is an essential pathogenic mycoplasma in poultry worldwide, posing a serious threat to the poultry industry's health. Timely detection is imperative for early diagnosis, prevention, and control of MS infection. Current laboratory methods for MS detection are generally complicated, time-consuming, and require sophisticated equipment. Therefore, a simple and rapid method is urgently needed. This study developed a novel real-time fluorescence-based recombinase-aided amplification (RF-RAA) technique for detecting MS nucleic acids, enabling target gene amplification within 20 min at 39°C. The RF-RAA outcomes are interpretable in two modalities: real-time fluorescence monitoring employing a temperature-controlled fluorescence detector or direct visual inspection facilitated by a portable blue light transilluminator. This method exhibits robust specificity, demonstrating no cross-reactivity with various common poultry pathogens, and achieves high sensitivity, detecting as low as 10 copies/μL for the standard plasmid. Seventy-one clinical samples of chicken throat swabs were detected by RF-RAA and real-time fluorescence quantitative polymerase chain reaction (qPCR) methods. The diagnostic coincidence rates of qPCR with RF-RAA (fluorescence monitoring) and RF-RAA (visual observation) were determined to be 100% and 97.2% (69/71), respectively. In conclusion, the RF-RAA method developed in this study provides a rapid and visually observable approach for MS detection, offering a novel technique to diagnosing MS infection, especially in resource-limited settings.

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris.

Candida auris (C. auris) was first discovered in Japan in 2009 and has since spread worldwide. It exhibits strong transmission ability, high multidrug resistance, blood infectivity, and mortality rates. Traditional diagnostic techniques for C. auris have shortcomings, leading to difficulty in its timely diagnosis and identification. Therefore, timely and accurate diagnostic assays for clinical samples are crucial. We developed a novel, rapid recombinase-aided amplification (RAA) assay targeting the 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA genes for C. auris identification. This assay can rapidly amplify DNA at 39 °C in 20 min. The analytical sensitivity and specificity were evaluated. From 241 clinical samples collected from pediatric inpatients, none were detected as C. auris-positive. We then prepared simulated clinical samples by adding 10-fold serial dilutions of C. auris into the samples to test the RAA assay's efficacy and compared it with that of real-time PCR. The assay demonstrated an analytical sensitivity of 10 copies/μL and an analytical specificity of 100%. The lower detection limit of the RAA assay for simulated clinical samples was 101 CFU/mL, which was better than that of real-time PCR (102-103 CFU/mL), demonstrating that the RAA assay may have a better detection efficacy for clinical samples. In summary, the RAA assay has high sensitivity, specificity, and detection efficacy. This assay is a potential new method for detecting C. auris, with simple reaction condition requirements, thus helping to manage C. auris epidemics.

Rapid detection of Ebolavirus using isothermal recombinase-aided amplification.

Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.

High-Performance Detection of Mycobacterium bovis in Milk Using Recombinase-Aided Amplification-Clustered Regularly Interspaced Short Palindromic Repeat-Cas13a-Lateral Flow Detection.

Mycobacterium bovis (M. bovis), the microorganism responsible for bovine tuberculosis (bTB), is transferred to people by the ingestion of unpasteurized milk and unprocessed fermented milk products obtained from animals with the infection. The identification of M. bovis in milk samples is of the utmost importance to successfully prevent zoonotic diseases and maintain food safety. This study presents a comprehensive description of a highly efficient molecular test utilizing recombinase-aided amplification (RPA)-clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) 13a-lateral flow detection (LFD) for M. bovis detection. In contrast to ELISA, RPA-CRISPR-Cas13a-LFD exhibited greater accuracy and sensitivity in the detection of M. bovis in milk, presenting a detection limit of 2 × 100 copies/μL within a 2 h time frame. The two tests exhibited a moderate level of agreement, as shown by a kappa value of 0.452 (95%CI: 0.287-0.617, p < 0.001). RPA-CRISPR-Cas13a-LFD holds significant potential as a robust platform for pathogen detection in complex samples, thereby enabling the more dependable regulation of food safety examination, epidemiology research, and medical diagnosis.

A multiplex method for the detection of food microorganisms based on suspension microarrays combined with recombinase-aided amplification

Foodborne diseases caused by foodborne pathogenic microorganisms have long been a severe public health problem. Timely and accurate detection of food-borne microorganisms can minimize their harm and enable the development of efficient control strategies. In this study, we establish a multiplex nucleic acid detection method based on recombinase-aided amplification reaction on a suspension microarray (referred to as SC-RAA) to identify and quantify microorganisms. The experimental results confirmed that this approach has an outstanding sensitivity, with a limit of detection (LOD) of 0.8 fM and a linear range of 0.8 fM–80 pM, spanning five orders of magnitude. Moreover, the prepared method also showed exquisite specificity, which the amplified signal can be detected only when the target nucleic acid is present. The recovery rate of the SC-RAA assay was 82.2%–113.4%, suggesting that the SC-RAA assay is performing well and accurately measuring the concentration of the substance in the sample. In addition, the consistency analysis showed that the results of the SC-RAA detection were also in good agreement with those of traditional qPCR detection (R2 > 0.95). These results highlight the potential of the SC-RAA assay to be applied as a multiplex and sensitive DNA detection platform for identifying and quantifying foodborne pathogenic microorganisms.

Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus

Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/μL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9

BackgroundAvian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA.ResultsThe results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%.ConclusionsRT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.