Golden Gate-Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria.

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria.

Multiplex reverse transcription recombinase polymerase amplification combined with lateral flow biosensor for simultaneous detection of three viral pathogens in cattle.

Bovine viral diarrhea virus (BVDV), bovine epidemic fever virus (BEFV), and bovine respiratory syncytial virus (BRSV) cause respiratory symptoms in cattle. The absence of rapid, precise, and easily accessible diagnostic methods poses difficulties for herders and veterinary epidemiologists during outbreaks of major infectious animal diseases. Considering the mixed infection of viruses, a multiple-detection method, reverse transcription recombinase polymerase amplification (mRT-RPA) combined with a lateral flow biosensor (LFB), was established to simultaneously detect the three pathogens. This technique is based on the specific binding of three differently labeled RT-RPA products (DNA sequences) to antibodies on the three test lines of the LFB, achieving multiplex detection through the presence or absence of coloration on the LFB test lines. The fluorescence values of the LFB test lines are recorded by a test strip reader. The mRT-RPA-LFB assay completes detection at a constant temperature of 41 °C within 33 min. The limits of detection (LODs) for BVDV, BEFV and BRSV were 2.62 × 101, 2.42 × 101 and 2.56 × 101 copies/µL, respectively. No cross-reactivity was observed with the other six bovine viruses. The developed method showed satisfactory intra- and inter-assay precision, and the average coefficients of variation were ranged from 2.92 % to 3.99 %. The diagnostic sensitivity and specificity were 98.11 % and 100 %, respectively, which were highly consistent with the RT-qPCR assay, and the kappa value was 0.988 (95 % confidence interval, CI). In general, the mRT-RPA-LFB assay has the potential to become a powerful tool for rapid screening of cattle diseases because of its advantages such as fast detection speed, convenient operation, strong specificity, and high sensitivity.

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

BACKGROUND: Helicobacter 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. METHODS: In 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. RESULTS: A 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. CONCLUSION: The 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.

A gold nanoparticle-enhanced dCas9-mediated fluorescence resonance energy transfer for nucleic acid detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas proteins coupled with pre-amplification have shown great potential in molecular diagnoses. However, the current CRISPR-based methods require additional reporters and time-consuming process. Herein, a gold nanoparticle (AuNP)-enhanced CRISPR/dCas9-mediated fluorescence resonance energy transfer (FRET) termed Au-CFRET platform was proposed for rapid, sensitive, and specific detection of nucleic acid for the first time. In the Au-CFRET sensing platform, AuNP was functionalized with dCas9 and used as nanoprobe. Target DNA was amplified with FAM-labeled primers and then precisely bound with AuNP-dCas9. The formed complex rendered the distance between AuNP acceptor and FAM donor to be short enough for the occurrence of FRET, thus resulting in fluorescence quenching. Moreover, AuNPs were demonstrated to enhance binding efficiency of dCas9 to target DNA in Au-CFRET system. The key factors regarding the FRET efficiency were analyzed and characterized in detail, including the length of donor/acceptor and the size of AuNPs. Under the optimal conditions, Au-CFRET could determinate CaMV35S promoter of genetically modified rice as low as 21 copies µL-1. Moreover, Au-CFRET sensing system coupled with one-step extraction and recombinase polymerase amplification can identify the genuine plant seeds within 30 min from sampling to results at room/body temperature without expensive equipment or technical expertise, and requires no additional exogenous reporters. Therefore, the proposed sensing platform significantly simplified the system and shortened the assay time for nucleic acid diagnoses.

An Optimized CRISPR/Cas12a Assay to Facilitate the BRAF V600E Mutation Detection.

BACKGROUND: Accurate detection of the BRAF V600E (1799T > A) mutation status can significantly contribute to selecting an optimal therapeutic strategy for diverse cancer types. CRISPR-based diagnostic platforms exhibit simple programming, cost-effectiveness, high sensitivity, and high specificity in detecting target sequences. The goal of this study is to develop a simple BRAF V600E mutation detection method. METHODS: We combined the CRISPR/Cas12a system with recombinase polymerase amplification (RPA). Subsequently, several parameters related to CRISPR/Cas12a reaction efficiency were evaluated. Then, we conducted a comparative analysis of three distinct approaches toward identifying BRAF V600E mutations in the clinical samples. RESULTS: Our data suggest that CRISPR/Cas detection is considerably responsive to variations in buffer conditions. Magnesium acetate (MgOAc) demonstrated superior performance compared to all other examined additive salts. It was observed using 150 nM guide RNA (gRNA) in an optimized reaction buffer containing 14 mM MgOAc, coupled with a reduction in the volumes of PCR and RPA products to 1 µL and 3 µL, respectively, resulted in an enhanced sensitivity. Detection time was decreased to 75 min with a 2% limit of detection (LOD), as evidenced by the results obtained from the blue light illuminator. The CRISPR/Cas12a assay confirmed the real-time PCR results in 31 of 32 clinical samples to identify the BRAF V600E mutation status, while Sanger sequencing detected BRAF V600E mutations with lower sensitivity. CONCLUSION: We propose a potential diagnostic approach that is facile, fast, and affordable with high fidelity. This method can detect BRAF V600E mutation with a 2% LOD without the need for a thermocycler.

The future in diagnostic tools for TB outbreaks: A review of the approaches with focus on LAMP and RPA diagnostics tests.

Tuberculosis (TB) is still the most frequent cause of morbidity and mortality in the world caused by Mycobacterium tuberculosis (MTB). Due to slow diagnostic and treatment options, the disease is a major concern for public health and also increases the burden on the global economy. Rapid, sensitive, and cheaper TB diagnosis test is urgent to lower their rates by point of care testing (POCT). Therefore, molecular detection techniques like recombinase polymerase assay (RPA) and Loop-mediated isothermal amplification (LAMP) play a significant role in this regard as they work on the principle of isothermal nucleic acid amplification. RPA and LAMP bridge the research gap between the previous PCR-based detection tool and other reported isothermal tools for MTB. In this review, we endeavor to provide an overview of the assay that will be a novel approach toward a rapid amplification and visualization of DNA by the naked eye in natural light. RPA and LAMP can prove to be a highly specific pathogen detection technique in combination with lateral flow (LF) strips and SYBR Green I. Optimization of amplification conditions also made the assay ideally suited to the resource-limited field application at POCT. Additionally, RPA and LAMP have paved the way for meeting a key component of the POC diagnosis of TB like universal drug susceptibility testing. However, RPA is more suitable at the POC level than LPA as it requires a lower amplification temperature of around 37-42 °C and a simpler primer design.

Validation of Recombinase Polymerase Amplification with In-House Lateral Flow Assay for mcr-1 Gene Detection of Colistin Resistant Escherichia coli Isolates.

BACKGROUND/OBJECTIVES: The emergence of the mobilized colistin resistance 1 (mcr-1) gene, which causes colistin resistance, is a serious concern in animal husbandry, particularly in pigs. Although antibiotic regulations in many countries have prohibited the use of colistin in livestock, the persistence and dissemination of this plasmid-mediated gene require effective and rapid monitoring. Therefore, a rapid, sensitive, and specific method combining recombinase polymerase amplification (RPA) with an in-house lateral flow assay (LFA) for the mcr-1 gene detection was developed. METHODS: The colistin agar test and broth microdilution were employed to screen 152 E. coli isolates from pig fecal samples of five antibiotic-used farms. The established RPA-in-house LFA was validated with PCR for mcr-1 gene detection. RESULTS: The RPA-in-house LFA was completed within 35 min (20 min of amplification and 5-15 min on LFA detection) at 37 °C. The sensitivity, specificity, and accuracy were entirely 100% in concordance with PCR results. No cross-reactivity was detected with seven common pathogenic bacteria or other mcr gene variants. CONCLUSIONS: Therefore, the in-house RPA-LFA serves as a point-of-care testing tool that is rapid, simple, and portable, facilitating effective surveillance of colistin resistance in both veterinary and clinical settings, thereby enhancing health outcomes.

Duplex recombinase aided amplification-lateral flow dipstick assay for rapid distinction of Mycobacterium tuberculosis and Mycobacterium avium complex.

Objectives: This study aims to develop a novel diagnostic approach using the recombinase aided amplification-lateral flow dipstick(RAA-LFD) assay for the distinction of Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC), enabling rapid and convenient as well as accurate identification of them in clinical samples. Methods: Our study established a duplex RAA-LFD assay capable of discriminating between MTB and MAC. Based on the principles of RAA primer and probe design, specific primers and probes were developed targeting the MTB IS6110 and the MAC DT1 separately. Optimization of reaction time points and temperatures was conducted, followed by an evaluation of specificity, sensitivity, and reproducibility. The established detection method was then applied to clinical samples and compared with smear microscopy, liquid culture, LAMP, and Xpert/MTB RIF in terms of diagnostic performance. Results: The complete workflow allows for the effective amplification of the MTB IS6110 and MAC DT1 target sequences at constant 37°C within 20min, and the amplification products can be visually observed on the LFD test strip. This method exhibits high specificity, showing no cross-reactivity with nucleic acids from M. kansassi, M. abscessus, M. gordonae, M. chelonae, M. fortuitum, M. scrofulaceum, M. malmoense, M. chimaera, M. szulgai and common respiratory pathogens. It also demonstrates high sensitivity, with a detection limit as low as 102 CFU/mL. Additionally, the method's Coefficient of Variation (CV) is less than 5%, ensuring excellent repeatability and reliability. Furthermore, clinical performance evaluations, using Xpert/MTB RIF as the gold standard, demonstrated that the duplex RAA-LFD assay achieves a sensitivity of 92.86% and a specificity of 93.75%. It is also noteworthy that the assay exhibits considerable diagnostic efficacy in smear-negative patients. Conclusions: Our study introduces a rapid, specific, and sensitive duplex RAA-LFD assay for the discriminatory diagnosis of MTB and MAC. This method represents a significant advancement in the field of infectious disease diagnostics, offering a valuable tool for rapid detection and management of MTB and MAC infections. The implementation of this approach in point-of-care settings could greatly enhance TB control and prevention efforts, especially in resource-limited environments.

RAD51 recombinase and its paralogs: Orchestrating homologous recombination and unforeseen functions in protozoan parasites.

The DNA of protozoan parasites is highly susceptible to damage, either induced by environmental agents or spontaneously generated during cellular metabolism through reactive oxygen species (ROS). Certain phases of the cell cycle, such as meiotic recombination, and external factors like ionizing radiation (IR), ultraviolet light (UV), or chemical genotoxic agents further increase this susceptibility. Among the various types of DNA damage, double-stranded breaks (DSBs) are the most critical, as they are challenging to repair and can result in genetic instability or cell death. DSBs caused by environmental stressors are primarily repaired via one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). In multicellular eukaryotes, NHEJ predominates, but in unicellular eukaryotes such as protozoan parasites, HR seems to be the principal mechanism for DSB repair. The HR pathway is orchestrated by proteins from the RAD52 epistasis group, including RAD51, RAD52, RAD54, RAD55, and the MRN complex. This review focuses on elucidating the diverse roles and significance of RAD51 recombinase and its paralogs in protozoan parasites, such as Acanthamoeba castellanii, Entamoeba histolytica (Amoebozoa), apicomplexan parasites (Chromalveolata), Naegleria fowleri, Giardia spp., Trichomonas vaginalis, and trypanosomatids (Excavata), where they primarily function in HR. Additionally, we analyze the diversity of proteins involved in HR, both upstream and downstream of RAD51, and discuss the implications of these processes in parasitic protozoa.

CRISPR/Cas13a-mediated visual detection: A rapid and robust method for early detection of Nosema bombycis in silkworms.

The sericulture industry faces a significant threat from the Pebrine disease of silkworms, caused by Nosema bombycis. Nonetheless, the current microscopic diagnostic methods can be time-consuming, labor-intensive, and lacking sensitivity and accuracy. Therefore, it is crucial to develop a novel detection approach that is efficient, highly sensitive, and low-cost. In this regard, the CRISPR/Cas system has the potential to be a fast, accurate, and highly specific method of detection. Herein, using a microplate reader, a portable fluorescence detection device, and test strips as signal output tools respectively, we have efficiently developed three rapid and facile visual detection methods for N. bombycis using a CRISPR/Cas13a system with conjugation of Recombinase polymerase amplification (RPA). We evaluated the sensitivity of this combined technology by comparing it with the positive plasmid standard and the genome standard of N. bombycis. Remarkably, the sensitivity of the CRISPR/Cas13a system for N. bombycis positive plasmid standard based on the microplate reader, portable fluorescence detection device, and test strips was 1 copy/µL, 10 copies/µL, and 1 copy/µL, respectively, while for the N. bombycis genome standards, the detection sensitivity was 10 fg/µL, 10 fg/µL, and 1 fg/µL, respectively. In addition, extensive evaluations have demonstrated that the established technology can accurately detect N. bombycis without cross-reactivity with other pathogens, ensuring a specificity rate of 100%. In brief, this study will provide a practical, efficient, and affordable method for early and rapid detection of N. bombycis in various settings.

Flagellar switch inverted repeat impacts flagellar invertibility and varies Clostridioides difficile RT027/MLST1 virulence.

Clostridioides difficile RT027 strains cause infections that vary in severity from asymptomatic to lethal, but the molecular basis for this variability is poorly understood. Through comparative analyses of RT027 clinical isolates, we determined that isolates that exhibit greater variability in their flagellar gene expression exhibit greater virulence in vivo. C. difficile flagellar genes are phase-variably expressed due to the site-specific inversion of the flgB 5'UTR region, which reversibly generates ON vs. OFF orientations for the flagellar switch. We found that longer inverted repeat (IR) sequences in this switch region correlate with greater disease severity, with RT027 strains carrying 6A/6T IR sequences exhibiting greater phenotypic heterogeneity in flagellar gene expression (60%-75% ON) and causing more severe disease than those with shorter IRs (> 99% ON or OFF). Taken together, our results reveal that phenotypic heterogeneity in flagellar gene expression may contribute to the variable disease severity observed in C. difficile patients.

Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages.

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.

Rapid and Ultrasensitive Detection of H. aduncum via the RPA-CRISPR/Cas12a Platform.

Hysterothylacium aduncum is one of six pathogens responsible for human anisakiasis. Infection with H. aduncum can cause acute abdominal symptoms and allergic reactions and is prone to misdiagnosis in clinical practice. This study aims to enhance the efficiency and accuracy of detecting H. aduncum in food ingredients. We targeted the internal transcribed spacer 1 (ITS 1) regions of Anisakis to develop a visual screening method for detecting H. aduncum using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system. By comparing the ITS 1 region sequences of eight nematode species, we designed specific primers and CRISPR RNA (crRNA). The specificity of RPA primers was screened and evaluated, and the CRISPR system was optimized. We assessed its specificity and sensitivity and performed testing on commercial samples. The results indicated that the alternative primer ADU 1 was the most effective. The final optimized concentrations were 250 nM for Cas12a, 500 nM for crRNA, and 500 nM for ssDNA. The complete test procedure was achievable within 45 min at 37 °C, with a limit of detection (LOD) of 1.27 pg/µL. The amplified product could be directly observed using a fluorescence microscope or ultraviolet lamp. Detection results for 15 Anisakis samples were entirely consistent with those obtained via Sanger sequencing, demonstrating the higher efficacy of this method for detecting and identifying H. aduncum. This visual detection method, characterized by simple operation, visual results, high sensitivity, and specificity, meets the requirements for food safety testing and enhances monitoring efficiency.

Capillary flow velocity-based length identification of PCR and RPA products on paper microfluidic chips.

This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γLV) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R2 values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.

Establishment of recombinase polymerase amplification detection method for Dactylobotrys graminicola.

Hulless barley sheath rot is a spike disease caused by Dactylobotrys graminicola. In recent years, it has generally occurred in hulless barley growing areas in China, resulting in reduced hulless barley yields. In this study, primers and probes were designed based on conserved genome sequences, and a method was established using recombinant enzyme polymerase amplification-lateral flow burette (RPA-LFD) technology for the rapid diagnosis of sheath rot in hulless barley. The method can be successfully established in five minutes at a constant temperature of 39℃, and the results are consistent with those of normal PCR analysis. The method demonstrated high sensitivity, with a detection limit of 10 fg/µL. Furthermore, the rapid method was able to successfully detect D. graminicola in hulless barley during field incubation, which highlighted the significant advantage of the method in practical applications. In conclusion, the RPA method established in this study exhibited several advantageous characteristics, including high efficiency, simplicity, rapidity and practicality, which provide a theoretical basis for the early detection and prevention of D. graminicola.

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.

Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a.

Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 104 copies/µL and 103 copies/µL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/µL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2-3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance.

Establishment of a platform based on dual RPA combined with CRISPR/Cas12a for the detection of Klebsiella pneumoniae and its KPC resistance gene.

Carbapenem resistant Klebsiella pneumoniae (CRKP) can cause serious hospital- and community-acquired infections. Treatment for CRKP infection is limited, resulting in prolonged hospitalization and high consultation costs. The KPC genotype has the highest detection rate of CRKP, and its mortality rate is higher than the overall mortality rate of CRKP. However, traditional testing methods have disadvantages such as long time and reliance on complex and sophisticated instruments, which are not conducive to rapid screening for CRKP. Therefore, this study aimed to establish a detection platform for early screening of CRKP so that effective antimicrobial therapy could be administered promptly to prevent the widespread spread of CRKP. We integrated dual RPA with CRISPR/Cas12a to establish a dual platform for the detection of K. pneumoniae (Kp) rcsA-specific gene and KPC resistance gene. Four result reading methods were established, including fluorescence detection (FD), blue light irradiation detection (BLID), ultraviolet irradiation detection (UID), and lateral flow test strips (LFTS). For the rcsA gene, the LOD of FD was 1 × 10 pg/µL, and the other three methods could detect 1 × 101 pg/µL of bacterial DNA. As for the KPC gene, four resultant readout methods were able to detect 1 × 102 pg/µL of bacterial DNA. In 59 clinical strains tested, the dual RPA-CRISPR/Cas12a detection of the rcsA had 100% sensitivity, specificity, and accuracy compared to the culture method. Compared with the drug sensitivity test, the sensitivity of dual RPA-CRISPR/Cas12a detection for the KPC was 85.71%, the specificity was 100%, and the accuracy was 94.92%. In summary, our dual RPA-CRISPR/Cas12a platform proved to be rapid, precise, and convenient for the efficient detection of Kp with KPC in the laboratory or at the point of care.

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.

Rapid and equipment-free identification of papaya mealybug Paracoccus marginatus based on RPA-CRISPR/Cas12a.

BACKGROUND: Paracoccus marginatus has invaded many countries, spreading rapidly and causing significant economic losses to crops. Accurate detection during the monitoring process is critical to prevent its expansion into new areas, therefore it is necessary to develop efficient and reliable detection methods. Traditional detection methods are time-consuming and instrument-dependent owing to the morphological similarities and small sizes of P. marginatus and other mealybugs, therefore establishing an efficient, rapid, and sensitive method for field detection in resource-limited settings is critical. RESULTS: A sensitive and rapid detection system was developed to detect P. marginatus using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. The RPA-CRISPR/Cas12a assay distinguished P. marginatus from 10 other mealybugs. The entire process can be completed in approximately an hour, and the identification results can be determined by the naked eye using lateral flow strips or a portable mini-UV torch. A method was developed to extract DNA from P. marginatus within 5 min. This method was combined with the RPA-CRISPR/Cas12a assay to achieve rapid and simple detection. In addition, two portable thermos cups with temperature displays were used to maintain the reagents and assay reactions in the field. CONCLUSION: This assay represents the first application of portable and easily available items (mini-UV torch and thermos cup) based on the combination of RPA and CRISPR/Cas12a for rapid pest detection. This method is rapid, highly specific, and instrument-flexible, allowing for the early monitoring of P. marginatus in the field. This study provides guidance for the development of suitable management strategies. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.