Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during which the type Ⅳ pili plays an important role. Type Ⅳ pili can adhere on the surfaces of host cell and other medium, facilitating formation of bacterial biofilms, bacterial aggregations and microcolonies, and is also a critical factor in liquid conjugation. PilV is an adhesin-type protein found on the tip of type Ⅳ pili encoded by plasmid R64, and can recognize the lipopolysaccharid (LPS) molecules that locate on bacterial membrane. The shufflon is a clustered inversion region that diversifies the PilV protein, which consequently affects the recipient recognition and conjugation frequency in liquid mating. The shufflon was firstly discovered on an IncI1 plasmid R64 and has been identified subsequently in plasmids IncI2, IncK and IncZ, as well as the pathogenicity island of Salmonella typhi. The shufflon consists of four segments including A, B, C, and D, and a specific recombination site named sfx. The shufflon is regulated by its downstream-located recombinase-encoding gene rci, and different rearrangements of the shufflon region in different plasmids were observed. Mobile colistin resistance gene mcr-1, which has attracted substantial attentions recently, is mainly located in IncI2 plasmid. The shufflon may be one of the contributors to fast spread of mcr-1. Herein, we reviewed the discovery, structure, function and prevalence of plasmid mediated shufflon, aiming to provide a theoretical basis on transmission mechanism and control strategy of drug-resistant plasmids.
Plasmids/genetics* ; Proteins/genetics* ; Bacteria/genetics* ; Recombinases ; Genes, Bacterial ; Anti-Bacterial Agents

OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ² = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ² = 11.20, P < 0.05) and fluorescent RAA assay (χ² = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.
Animals ; Clonorchis sinensis/genetics* ; Metacercariae/genetics* ; Recombinases ; Fresh Water ; Fishes ; DNA ; Fish Diseases/diagnosis*

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.
African Swine Fever/virology* ; African Swine Fever Virus/isolation & purification* ; Animals ; Nucleic Acid Amplification Techniques/methods* ; Recombinases/chemistry* ; Sensitivity and Specificity ; Swine ; Viral Proteins/genetics*

Objective: To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) . Methods: We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD). Results: CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 ^-18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 ² CFU/g. Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.
CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods* ; Recombinases/genetics* ; Vibrio parahaemolyticus/genetics*

Animals ; Biological Assay ; Nucleic Acid Amplification Techniques ; Phlebovirus/isolation & purification* ; Psychodidae/virology* ; Recombinases/metabolism* ; Reverse Transcription

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (
Adolescent ; Adult ; Child ; Child, Preschool ; Cytomegalovirus/genetics* ; Cytomegalovirus Infections/virology* ; DNA, Viral/analysis* ; Epstein-Barr Virus Infections/virology* ; Female ; Herpesvirus 4, Human/genetics* ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Nucleic Acid Amplification Techniques ; Recombinases/genetics* ; Young Adult

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.
Animals ; Diagnosis ; DNA ; Enterobacteriaceae ; Enzyme-Linked Immunosorbent Assay ; Genome ; Limit of Detection ; Methods ; Mycobacterium avium subsp. paratuberculosis ; Mycobacterium avium ; Mycobacterium ; Paratuberculosis ; Point-of-Care Testing ; Polymerase Chain Reaction ; Recombinases ; Ruminants ; Sensitivity and Specificity

Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.
Amygdala ; Animals ; Astrocytes* ; Brain* ; Calbindins ; Central Nervous System ; Cerebellum ; Dentate Gyrus ; Hippocampus ; Humans ; Hypothalamus ; Mice* ; Neurons ; Pars Compacta ; Recombinases ; Sensitivity and Specificity* ; Thalamus ; Tyrosine 3-Monooxygenase

Genetically engineered mouse models are commonly preferred for studying the human disease due to genetic and pathophysiological similarities between mice and humans. In particular, Cre-loxP system is widely used as an integral experimental tool for generating the conditional. This system has enabled researchers to investigate genes of interest in a tissue/cell (spatial control) and/or time (temporal control) specific manner. A various tissue-specific Cre-driver mouse lines have been generated to date, and new Cre lines are still being developed. This review provides a brief overview of Cre-loxP system and a few commonly used promoters for expression of tissue-specific Cre recombinase. Also, we finally introduce some available links to the Web sites that provides detailed information about Cre mouse lines including their characterization.
Animals ; Humans ; Mice* ; Recombinases

Various genetic switches have been developed to let engineered cells perform designed functions. However, a sustained input is often needed to maintain the on/off state, which is energy-consuming and sensitive to perturbation. Therefore, we developed a set of transcriptional switches for cell states control that were constructed by the inversion effect of site-specific recombinases on terminators. Such a switch could respond to a pulse signal and maintain the new state by itself until the next input. With a bottom-up design principle, we first characterized the terminators and recombinases. Then the mutual interference was studied to select compatible pairs, which were used to achieve one-time and two-time state transitions. Finally, we constructed a biological seven-segment display as a demonstration to prove such switch's immense potential for application.
Recombinases ; metabolism