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1.
PLoS One ; 16(1): e0245164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33406112

RESUMO

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/µL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.


Assuntos
/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , /genética , /genética , Humanos , Malásia/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/genética , Recombinases/metabolismo , Transcrição Reversa/genética , Sensibilidade e Especificidade
2.
Food Chem ; 334: 127608, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32711280

RESUMO

Food analysis to ensure food safety and quality are relevant to all countries. This study aimed to develop a detection technique by combining recombinase polymerase amplification with CRISPR-Cas12a for food safety (termed RPA-Cas12a-FS). Our data showed that this novel method could be detected via fluorescence intensity for the molecular identification of foodborne pathogenic bacteria, genetically modified crops, and meat adulteration. After optimization, the sensitivity and stability of RPA-Cas12a-FS was further enhanced. The RPA-Cas12a-FS system could specifically detect target gene levels as low as 10 copies in 45 min at 37 °C. The RPA-Cas12a-FS system was sensitive both using standard samples in the lab and using samples from the field, which indicated that this detection method was practical. In conclusion, a simple, rapid, and highly sensitive detection method based on CRISPR-Cas12a was developed for molecular identification in the food safety field without requiring technical expertise or ancillary equipment.


Assuntos
Sistemas CRISPR-Cas , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Microbiologia de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Produtos Agrícolas/genética , Endodesoxirribonucleases/genética , Fluorescência , Inocuidade dos Alimentos , Carne , Plantas Geneticamente Modificadas/genética , RNA Guia , Recombinases/genética , Sensibilidade e Especificidade
3.
Nat Commun ; 11(1): 4758, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958811

RESUMO

Genetic programs operating in a history-dependent fashion are ubiquitous in nature and govern sophisticated processes such as development and differentiation. The ability to systematically and predictably encode such programs would advance the engineering of synthetic organisms and ecosystems with rich signal processing abilities. Here we implement robust, scalable history-dependent programs by distributing the computational labor across a cellular population. Our design is based on standardized recombinase-driven DNA scaffolds expressing different genes according to the order of occurrence of inputs. These multicellular computing systems are highly modular, do not require cell-cell communication channels, and any program can be built by differential composition of strains containing well-characterized logic scaffolds. We developed automated workflows that researchers can use to streamline program design and optimization. We anticipate that the history-dependent programs presented here will support many applications using cellular populations for material engineering, biomanufacturing and healthcare.


Assuntos
Modelos Genéticos , Biologia Sintética/métodos , Fenômenos Fisiológicos Celulares/genética , DNA/genética , DNA/metabolismo , Lógica , Recombinases/genética , Recombinases/metabolismo , Software , Fluxo de Trabalho
4.
Plant Dis ; 104(11): 2774-2778, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32924873

RESUMO

Late blight, caused by the oomycete Phytophthora infestans, is a major constraint on the production of potatoes and tomatoes as well as a constant threat to global food security. An early diagnostic tool is important for the effective management of late blight in the field. Here, in combination with a simplified DNA extraction method, we developed a lateral flow strip-based recombinase polymerase amplification (LF-RPA) assay for the rapid, equipment-free detection of P. infestans. This assay targets the Ras-related protein (Ypt1) gene and can be performed over a wide range of temperatures (25 to 45°C). All 12 P. infestans isolates yielded positive detection results using the LF-RPA assay, and no cross-reaction occurred with related oomycetes or fungal species. With this assay, the detection limit was 500 fg of genomic DNA in optimized conditions. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire LF-RPA assay enabled the detection of P. infestans within 30 min with no specialized equipment. When applied to field samples, it successfully detected P. infestans in naturally diseased potato plants from eight different fields in China. Therefore, the LF-RPA assay is simple, rapid, and cost-effective and has potential for further development as a kit for diagnosing late blight in resource-limited settings or even on-site.


Assuntos
Phytophthora infestans , China , Primers do DNA , Técnicas de Amplificação de Ácido Nucleico , Phytophthora infestans/genética , Recombinases/genética
5.
Am J Physiol Renal Physiol ; 319(3): F423-F435, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32657158

RESUMO

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.


Assuntos
Túbulos Renais Distais/enzimologia , Recombinases/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Recombinases/genética , Simportadores de Cloreto de Sódio/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Tamoxifeno/farmacologia
6.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
7.
BMC Vet Res ; 16(1): 208, 2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32571305

RESUMO

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), an intestinal coronavirus that causes acute diarrhea and high mortality in suckling piglets, can result in high economic losses in the swine industry. In recent years, despite the use of China's current vaccine immunization strategy, multiple types of PEDV strains were still found in immunized swine herds. Our research aims to explore a new rapid differentiation method to distinguish the different types of PEDV strains and assess the safety evaluation of classical attenuated vaccine strains in swine herds. RESULTS: In the study, a differential one-step quantitative real-time fluorescent reverse transcription recombinase polymerase amplification (real-time RT-RPA) method based on the PEDV universal real-time RT-RPA assay was established according to the ORF1 deletion sequences of three classical attenuated vaccine strains (PEDV attenuated vaccine KC189944, attenuated CV777 and DR13) and five Vero cell-adapted isolates (JS2008, SDM, SQ2014, SC1402, HLJBY), which could effectively differentiate PEDV classical attenuated vaccine strains from wild-type strains (PEDV classical wild strains and variant strains). The detection limits of PEDV RNA in the both PEDV real-time RT-RPA assays were 300 copies within 20 min at 39 °C, and the detection limits of classical attenuated vaccine strain CV777, Vero-cell-adapted isolate JS2008, and PEDV wild-type strain DX were 100.5 TCID50/100 µL, 101.1 TCID50/100 µL, and 101.2 TCID50/100 µL, respectively. Both assays were highly specific for PEDV, showing no cross-reactivity with other enteral viruses. CONCLUSION: This RPA method we developed is simple, time-effective, and safe and provides a reliable technical tool for the differential diagnosis and clinical epidemic surveillance of PEDV classical attenuated vaccine strains and wild-type strains.


Assuntos
Infecções por Coronavirus/veterinária , Técnicas de Amplificação de Ácido Nucleico/veterinária , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Recombinases/isolamento & purificação , Vacinas Virais/imunologia , Animais , Infecções por Coronavirus/virologia , Recombinases/genética , Suínos , Vacinas Atenuadas
8.
Nat Biotechnol ; 38(7): 824-844, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572269

RESUMO

The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma/genética , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Edição de Genes/métodos , Humanos , Recombinases/genética , Transposases/genética
9.
Food Chem ; 324: 126821, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32361093

RESUMO

As large-scale planting of genetically modified (GM) crops increases, the development of a rapid and convenient method for on-site detection of GM crops is important. We combined the advantages of recombinase polymerase amplification (RPA) and fluorescence detection to establish a rapid, sensitive, specific, and simple detection platform for on-site detection of MON863 maize. Test samples were added directly to the platform after simple pre-treatment with a DNA extraction-free method. Results were obtained through real-time monitoring with a portable instrument, which facilitated sample-in/answer-out on-site detection. The entire detection process, including sample preparation, RPA and identification of amplification results, was accomplished in approximately 10 min. Furthermore, the detection was achieved with a simple and inexpensive portable device. This method has high potential for application in other fields requiring rapid detection of DNA targets, such as in field research, resource-limited areas, and science education.


Assuntos
Produtos Agrícolas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Primers do DNA/genética , Fluorescência , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/genética
10.
Nat Struct Mol Biol ; 27(5): 424-437, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32398827

RESUMO

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Genoma Humano/genética , Mitose , Animais , Linhagem Celular , DNA Helicases/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Instabilidade Genômica , Humanos , Imunoprecipitação , Camundongos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
11.
Nat Struct Mol Biol ; 27(5): 438-449, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32398829

RESUMO

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Recombinases/metabolismo , Transcrição Genética , DNA Helicases/genética , Disceratose Congênita/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Retardo do Crescimento Fetal/genética , Mutação em Linhagem Germinativa , Células HeLa , Humanos , Deficiência Intelectual/genética , Microcefalia/genética , Recombinases/genética
12.
Int J Mol Sci ; 21(8)2020 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-32290544

RESUMO

Homologous recombination is essential for chromosome segregation during meiosis I. Meiotic recombination is initiated by the introduction of double-strand breaks (DSBs) at specific genomic locations called hotspots, which are catalyzed by Spo11 and its partners. DSB hotspots during meiosis are marked with Set1-mediated histone H3K4 methylation. The Spo11 partner complex, Rec114-Mer2-Mei4, essential for the DSB formation, localizes to the chromosome axes. For efficient DSB formation, a hotspot with histone H3K4 methylation on the chromatin loops is tethered to the chromosome axis through the H3K4 methylation reader protein, Spp1, on the axes, which interacts with Mer2. In this study, we found genetic interaction of mutants in a histone modification protein complex called PAF1C with the REC114 and MER2 in the DSB formation in budding yeast Saccharomyces cerevisiae. Namely, the paf1c mutations rtf1 and cdc73 showed synthetic defects in meiotic DSB formation only when combined with a wild-type-like tagged allele of either the REC114 or MER2. The synthetic defect of the tagged REC114 allele in the DSB formation was seen also with the set1, but not with spp1 deletion. These results suggest a novel role of histone modification machinery in DSB formation during meiosis, which is independent of Spp1-mediated loop-axis tethering.


Assuntos
DNA Fúngico/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Meiose/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Alelos , Cromatina/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Mutação/genética , Recombinases/genética , Saccharomyces cerevisiae/genética
13.
Nat Methods ; 17(4): 422-429, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203389

RESUMO

Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.


Assuntos
Genômica/métodos , Optogenética , Recombinases/metabolismo , Animais , Encéfalo/citologia , Regulação da Expressão Gênica , Engenharia Genética , Camundongos , Neurônios/metabolismo , Recombinases/genética , Peixe-Zebra
14.
Nat Commun ; 11(1): 1417, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184398

RESUMO

Holliday junctions (HJs) are key DNA intermediates in genetic recombination and are eliminated by nuclease, termed resolvase, to ensure genome stability. HJ resolvases have been identified across all kingdoms of life, members of which exhibit sequence-dependent HJ resolution. However, the molecular basis of sequence selectivity remains largely unknown. Here, we present the chloroplast resolvase MOC1, which cleaves HJ in a cytosine-dependent manner. We determine the crystal structure of MOC1 with and without HJs. MOC1 exhibits an RNase H fold, belonging to the retroviral integrase family. MOC1 functions as a dimer, and the HJ is embedded into the basic cleft of the dimeric enzyme. We characterize a base recognition loop (BR loop) that protrudes into and opens the junction. Residues from the BR loop intercalate into the bases, disrupt the C-G base pairing at the crossover and recognize the cytosine, providing the molecular basis for sequence-dependent HJ resolution by a resolvase.


Assuntos
Arabidopsis/enzimologia , Cloroplastos/enzimologia , DNA Cruciforme/metabolismo , Oryza/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Recombinases/química , Recombinases/metabolismo , Soja/enzimologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Cloroplastos/química , Cloroplastos/genética , DNA Cruciforme/química , DNA Cruciforme/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Oryza/química , Oryza/genética , Oryza/metabolismo , Recombinases/genética , Soja/química , Soja/genética , Soja/metabolismo
15.
Genes (Basel) ; 11(1)2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936378

RESUMO

The execution of recombinational pathways during the repair of certain DNA lesions or in the meiotic program is associated to the formation of joint molecules that physically hold chromosomes together. These structures must be disengaged prior to the onset of chromosome segregation. Failure in the resolution of these linkages can lead to chromosome breakage and nondisjunction events that can alter the normal distribution of the genomic material to the progeny. To avoid this situation, cells have developed an arsenal of molecular complexes involving helicases, resolvases, and dissolvases that recognize and eliminate chromosome links. The correct orchestration of these enzymes promotes the timely removal of chromosomal connections ensuring the efficient segregation of the genome during cell division. In this review, we focus on the role of different DNA processing enzymes that collaborate in removing the linkages generated during the activation of the homologous recombination machinery as a consequence of the appearance of DNA breaks during the mitotic and meiotic programs. We will also discuss about the temporal regulation of these factors along the cell cycle, the consequences of their loss of function, and their specific role in the removal of chromosomal links to ensure the accurate segregation of the genomic material during cell division.


Assuntos
Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Recombinação Homóloga/genética , Humanos , Meiose/fisiologia , Mitose/fisiologia , Recombinases/genética , Recombinases/metabolismo
16.
BMC Infect Dis ; 20(1): 79, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992210

RESUMO

BACKGROUND: Mycoplasma pneumoniae is one of the most common causative pathogens of community-acquired pneumonia (CAP), accounting for as many as 30-50% of CAP during peak years. An early and rapid diagnostic method is key for guiding clinicians in their choice of antibiotics. METHODS: The recombinase-aided amplification (RAA) assay is a recently developed, rapid detection method that has been used for the detection of several pathogens. The assays were performed in a one-step single tube reaction at 39° Celsius within 15-30 min. In this study, we established an RAA assay for M. pneumoniae using clinical specimens for validation and commercial real-time PCR as the reference method. RESULTS: The analytical sensitivity of the RAA assay was 2.23 copies per reaction, and no cross-reactions with any of the other 15 related respiratory bacterial pathogens were observed. Compared with the commercial real-time PCR assay used when testing 311 respiratory specimens, the RAA assay obtained 100% sensitivity and 100% specificity with a kappa value of 1. CONCLUSIONS: These results demonstrate that the proposed RAA assay will be of benefit as a faster, sensitive, and specific alternative tool for the detection of M. pneumoniae.


Assuntos
Mycoplasma pneumoniae/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia por Mycoplasma/microbiologia , Recombinases/genética , Adolescente , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/microbiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Nucleic Acids Res ; 48(1): e1, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31612958

RESUMO

Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.


Assuntos
Bioensaio , Biblioteca Gênica , Plasmídeos/química , Transgenes , Animais , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HT29 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Células NIH 3T3 , Oncogenes , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinases/genética , Recombinases/metabolismo , Recombinação Genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
18.
Environ Microbiol ; 22(1): 158-169, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31715642

RESUMO

Cell to cell DNA transfer between Thermus thermophilus, or transjugation, requires the natural competence apparatus (NCA) of the recipient cell and a DNA donation machinery in the donor. In T. thermophilus HB27, two mobile genetic elements with functional similarities to Integrative and Conjugative Elements (ICEs) coexist, ICETh1 encoding the DNA transfer apparatus and ICETh2, encoding a putative replication module. Here, we demonstrate that excision and integration of both elements depend on a single tyrosine recombinase encoded by ICETh2, and that excision is not required but improves the transfer of these elements to a recipient cell. These findings along with previous results suggest that ICETh1 and ICETh2 depend on each other for spreading among T. thermophilus by transjugation.


Assuntos
Conjugação Genética , Sequências Repetitivas Dispersas , Thermus thermophilus/genética , Recombinases/genética
19.
J Biol Chem ; 295(3): 690-700, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31771978

RESUMO

Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter (R26-TLR), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre+Dre-, Cre-Dre+, and Cre+Dre+ cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity.


Assuntos
Linhagem da Célula/genética , Integrases/genética , Recombinases/genética , Células-Tronco/citologia , Alelos , Animais , Diferenciação Celular/genética , Fluorescência , Marcação de Genes , Genes Reporter/genética , Integrases/química , Camundongos , Camundongos Transgênicos/genética , Células-Tronco/química
20.
Nat Commun ; 10(1): 4845, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649244

RESUMO

Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.


Assuntos
Temperatura Baixa , DNA/metabolismo , Engenharia Genética/métodos , Luz , Recombinases/genética , Animais , DNA Nucleotidiltransferases , Redes Reguladoras de Genes , Células HEK293 , Humanos , Integrases , Camundongos , Recombinases/metabolismo
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