Multiplex detection of the big five carbapenemase genes using solid-phase recombinase polymerase amplification.

Five carbapenemase enzymes, coined the 'big five', have been identified as the biggest threat to worldwide antibiotic resistance based on their broad substrate affinity and global prevalence. Here we show the development of a molecular detection method for the gene sequences from the five carbapenemases utilising the isothermal amplification method of recombinase polymerase amplification (RPA). We demonstrate the successful detection of each of the big five carbapenemase genes with femtomolar detection limits using a spatially separated multiplex amplification strategy. The approach uses tailed oligonucleotides for hybridisation, reducing the complexity and cost of the assay compared to classical RPA detection strategies. The reporter probe, horseradish peroxidase, generates the measureable output on a benchtop microplate reader, but more notably, our study leverages the power of a portable Raman spectrometer, enabling up to a 19-fold enhancement in the limit of detection. Significantly, the development approach employed a solid-phase RPA format, wherein the forward primers targeting each of the five carbapenemase genes are immobilised to a streptavidin-coated microplate. The adoption of this solid-phase methodology is pivotal for achieving a successful developmental pathway when employing this streamlined approach. The assay takes 2 hours until result, including a 40 minutes RPA amplification step at 37 °C. This is the first example of using solid-phase RPA for the detection of the big five and represents a milestone towards the developments of an automated point-of-care diagnostic for the big five using RPA.

Comprehensive Interactome Mapping of the DNA Repair Scaffold SLX4 Using Proximity Labeling and Affinity Purification.

The DNA repair scaffold SLX4 has pivotal roles in cellular processes that maintain genome stability, most notably homologous recombination. Germline mutations in SLX4 are associated with Fanconi anemia, a disease characterized by chromosome instability and cancer susceptibility. The role of mammalian SLX4 in homologous recombination depends critically on binding and activating structure-selective endonucleases, namely SLX1, MUS81-EME1, and XPF-ERCC1. Increasing evidence indicates that cells rely on distinct SLX4-dependent complexes to remove DNA lesions in specific regions of the genome. Despite our understanding of SLX4 as a scaffold for DNA repair proteins, a detailed repertoire of SLX4 interactors has never been reported. Here, we provide a comprehensive map of the human SLX4 interactome using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS). We identified 221 unique high-confidence interactors, of which the vast majority represent novel SLX4-binding proteins. Network analysis of these hits revealed pathways with known involvement of SLX4, such as DNA repair, and several emerging pathways of interest, including RNA metabolism and chromatin remodeling. In summary, the comprehensive SLX4 interactome we report here provides a deeper understanding of how SLX4 functions in DNA repair while revealing new cellular processes that may involve SLX4.

To unwind the biological knots: The DNA/RNA G-quadruplex resolvase RHAU (DHX36) in development and disease.

The G-quadruplex (G4) sequences are short fragments of 4-interval triple guanine (G) with frequent and ubiquitous distribution in the genome and RNA transcripts. The G4 sequences are usually folded into secondary "knot" structure via Hoogsteen hydrogen bond to exert negative regulation on a variety of biological processes, including DNA replication and transcription, mRNA translation, and telomere maintenance. Recent structural biological and mouse genetics studies have demonstrated that RHAU (DHX36) can bind and unwind the G4 "knots" to modulate embryonic development and postnatal organ function. Deficiency of RHAU gives rise to embryonic lethality, impaired organogenesis, and organ dysfunction. These studies uncovered the pivotal G4 resolvase function of RHAU to release the G4 barrier, which plays fundamental roles in development and physiological homeostasis. This review discusses the latest advancements and findings in deciphering RHAU functions using animal models.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus.

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION: A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Fabrication of Wearable PDMS Device for Rapid Detection of Nucleic Acids via Recombinase Polymerase Amplification Operated by Human Body Heat.

Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft-but tight-contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus / 生物医学与环境科学（英文）

OBJECTIVE@#To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene.@*METHODS@#A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay.@*RESULTS@#The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses.@*CONCLUSION@#A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology.

Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10-4 single cysts and single females, 4-3 single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.

Sensitive and semiquantitative detection of soil-transmitted helminth infection in stool using a recombinase polymerase amplification-based assay.

BACKGROUND: Soil-transmitted helminths (STHs) are parasitic nematodes that inhabit the human intestine. They affect more than 1.5 billion people worldwide, causing physical and cognitive impairment in children. The global strategy to control STH infection includes periodic mass drug administration (MDA) based on the results of diagnostic testing among populations at risk, but the current microscopy method for detecting infection has diminished sensitivity as the intensity of infection decreases. Thus, improved diagnostic tools are needed to support decision-making for STH control programs. METHODOLOGY: We developed a nucleic acid amplification test based on recombinase polymerase amplification (RPA) technology to detect STH in stool. We designed primers and probes for each of the four STH species, optimized the assay, and then verified its performance using clinical stool samples. PRINCIPAL FINDINGS: Each RPA assay was as sensitive as a real-time polymerase chain reaction (PCR) assay in detecting copies of cloned target DNA sequences. The RPA assay amplified the target in DNA extracted from human stool samples that were positive for STH based on the Kato-Katz method, with no cross-reactivity of the non-target genomic DNA. When tested with clinical stool samples from patients with infections of light, moderate, and heavy intensity, the RPA assays demonstrated performance comparable to that of real-time PCR, with better results than Kato-Katz. This new rapid, sensitive and field-deployable method for detecting STH infections can help STH control programs achieve their goals. CONCLUSIONS: Semi-quantitation of target by RPA assay is possible and is comparable to real-time PCR. With proper instrumentation, RPA assays can provide robust, semi-quantification of STH DNA targets as an alternative field-deployable indicator to counts of helminth eggs for assessing infection intensity.

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations.

Isothermal DNA amplification, such as recombinase polymerase amplification (RPA), is well suited for point-of-care testing (POCT) as it does not require lengthy thermal cycling. By exploiting DNA amplification at low temperatures that do not denature heat-sensitive molecules such as proteins, we have developed a blocking RPA method to detect gene mutations and examine the epigenetic status of DNA. We found that both nucleic acid blockers and nuclease-dead clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins suppress RPA reactions by blocking elongation by DNA polymerases in a sequence-specific manner. By examining these suppression events, we are able to discriminate single-nucleotide mutations in cancer cells and evaluate genome-editing events. Methyl-CpG binding proteins similarly inhibit elongation by DNA polymerases on CpG-methylated template DNA in our RPA reactions, allowing for the detection of methylated CpG islands. Thus, the use of heat-sensitive molecules such as proteins and ribonucleoprotein complexes as blockers in low-temperature isothermal DNA amplification reactions markedly expands the utility and application of these methods.

Isothermal recombinase polymerase amplification-lateral flow detection of SARS-CoV-2, the etiological agent of COVID-19.

The rapid detection of novel pathogens including SARS-CoV-2 necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Delay in diagnosis has facilitated the rapid spread of this novel virus throughout the world resulting in global mortality that will surpass 2.5 million people. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral cDNA nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 nasopharyngeal samples using CDC's N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100 % concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

RNA diversification by a self-reproducing ribozyme revealed by deep sequencing and kinetic modelling.

We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by a kinetic model. This shows that key mechanisms can be integrated and controlled toward Darwinian evolution in RNA reaction networks.

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus.

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

An isothermal recombinase polymerase amplification and lateral flow strip combined method for rapid on-site detection of Vibrio vulnificus in raw seafood.

Vibrio vulnificus is an important foodborne pathogenic bacterium that mainly contaminates seafood. Rapid and accurate technologies that suitable for on-site detection are critical for effective control of its spreading. Conventional detection methods and polymerase chain reaction (PCR)-based and qPCR-based approaches have application limitations in on-site scenarios. Application of loop-mediated isothermal amplification (LAMP) technology was a good step towards the on-site detection. In this study, a recombinase polymerase amplification (RPA)-based detection method for V. vulnificus was developed combining with lateral flow strip (LFS) for visualized signal. The method targeted the conservative empV gene encoding the extracellular metalloproteinase, and finished detection in 35 min at a conveniently low temperature of 37 °C. It showed good specificity and an excellent sensitivity of 2 copies of the genome or 10-1 colony forming unit (CFU) per reaction, or 1 CFU/10 g in spiked food samples with enrichment. The method tolerated unpurified templates directly from sample boiling, which added the convenience of the overall procedure. Application of the RPA-LFS method for clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. This RPA-LFS combined method is well suited for on-site detection of V. vulnificus.

DNA-driven condensation assembles the meiotic DNA break machinery.

The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein1,2. The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures3-5, but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery2. Each subcomplex of Rec114-Mei4 (a 2:1 heterotrimer) or Mer2 (a coiled-coil-containing homotetramer) is monodispersed in solution, but they independently condense with DNA into reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive this condensation. Mutations that weaken protein-DNA interactions strongly disrupt both condensate formation and DSBs in vivo, and thus these processes are highly correlated. In vitro, condensates fuse into mixed RMM clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centres of DSB activity. We propose that multilayered control of Spo11 arises from the recruitment of regulatory components and modulation of the biophysical properties of the condensates.

Computational Methods for the Design of Recombinase Logic Circuits.

Synthetic biology aims at engineering new biological systems and functions that can be used to provide new technological solutions to worldwide challenges. Detection and processing of multiple signals are crucial for many synthetic biology applications. A variety of logic circuits operating in living cells have been implemented. One particular class of logic circuits uses site-specific recombinases mediating specific DNA inversion or excision. Recombinase logic offers many interesting features, including single-layer architectures, memory, low metabolic footprint, and portability in many species. Here, we present two automated design strategies for recombinase-based logic circuits, one based on the distribution of computation within a multicellular consortia and the other one being a single-cell design. The two design strategies are complementary and are both adapted for none expert as a design web-interface exits for each strategy, the CALIN and RECOMBINATOR web-interface for respectively the multicellular and single-cell design strategy. In this book chapter, we are guiding the reader step by step through recombinase-logic circuit design from selecting the design strategy fitting to his/her final system of interest to obtaining the final design using one of our design web-interface.

Structural basis for the activation and suppression of transposition during evolution of the RAG recombinase.

Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with methionine at residue 848 and cryo-electron microscopy, we determined structures that capture RAG engaged with transposon ends and U-shaped target DNA prior to integration (the target capture complex) and two forms of the RAG strand transfer complex that differ based on whether target site DNA is annealed or dynamic. Target site DNA base unstacking, flipping, and melting by RAG1 methionine 848 explain how this residue activates transposition, how RAG can stabilize sharp bends in target DNA, and why replacement of residue 848 by arginine during RAG domestication led to suppression of transposition activity. RAG2 extends a jawed vertebrate-specific loop to interact with target site DNA, and functional assays demonstrate that this loop represents another evolutionary adaptation acquired during RAG domestication to inhibit transposition. Our findings identify mechanistic principles of the final step in cut-and-paste transposition and the molecular and structural logic underlying the transformation of RAG from transposase to recombinase.

A recombinase polymerase amplification-based lateral flow strip assay for rapid detection of genogroup II noroviruses in the field.

Human norovirus is the leading cause of viral gastroenteritis worldwide. Rapid detection facilitates management of disease outbreaks, but field diagnosis is difficult to achieve due to the lack of reliable and portable methods. Recombinase polymerase amplification (RPA) is a robust isothermal amplification method that is capable of rapidly amplifying and detecting nucleic acids using simple equipment. In this study, RPA combined with lateral flow (LF) strips specific for human genogroup II (GII) noroviruses was established and evaluated. The assay specifically detects purified GII noroviruses as well as RNA in boiled human stool samples, with a sensitivity of 50 norovirus genome copies per reaction. The whole detection procedure of the one-step RT-RPA-LF is completed within 20 min, which is eight times faster than that of the standard real-time RT-PCR. The RT-RPA-LF method described here is suitable for rapid field diagnosis of all GII noroviruses in human stool samples.

Electron Acceptors Co-Regulated Self-Powered Photoelectrochemical Strategy and Its Application for Circulating Tumor Nucleic Acid Detection Coupled with Recombinase Polymerase Amplification.

Biosensor working in a self-powered mode has been widely concerned because it produces a signal when the bias potential is 0 V. However, the self-powered mode is used only when the materials have self-powered properties. Conversion of non-self-powered to self-powered through molecular regulation can solve this problem effectively. Here, we fabricated a self-powered photoelectrochemical mode based on co-regulation of electron acceptors methylene blue (MB) and p-nitrophenol (p-NP). AuNPs@ZnSe nanosheet-modified gold electrode (AuNPs@ZnSeNSs/GE) gave a small photocurrent at 0 V. In the presence of MB and p-NP, AuNPs@ZnSeNSs/GE gave the strongest photocurrent at 0 V. Accordingly, an electron acceptor co-regulated self-powered photoelectrochemical assay was fabricated. As proof-of-concept demonstrations, this assay was applied for prostate cancer circulating tumor nucleic acid biomarker, KLK2 and PCA3, detection combined with in situ recombinase polymerase amplification strategy. This assay generated a strong photocurrent and was sensitive to the variation of KLK2 and PCA3 concentration. The limits of detection were 30 and 32 aM, respectively. We anticipate this electron acceptor co-regulated self-powered photoelectrochemical mode to pave a new way for the development of self-powered sensing.

Characterization of an archaeal recombinase paralog that exhibits novel anti-recombinase activity.

The process of homologous recombination is heavily dependent on the RecA family of recombinases for repair of DNA double-strand breaks. These recombinases are responsible for identifying homologies and forming heteroduplex DNA between substrate ssDNA and dsDNA templates, activities that are modified by various accessory factors. In this work we describe the biochemical functions of the SsoRal2 recombinase paralog from the crenarchaeon Sulfolobus solfataricus. We found that the SsoRal2 protein is a DNA-independent ATPase that, unlike the other S. solfataricus paralogs, does not bind either ss- or dsDNA. Instead, SsoRal2 alters the ssDNA binding activity of the SsoRadA recombinase in conjunction with another paralog, SsoRal1. In the presence of SsoRal1, SsoRal2 has a modest effect on strand invasion but effectively abrogates strand exchange activity. Taken together, these results indicate that SsoRal2 assists in nucleoprotein filament modulation and control of strand exchange in S. solfataricus.