Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology.

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

Diagnostic value of IFN-gamma in tuberculous pleural effusion.

BACKGROUND: Simple, rapid, and accurate diagnosis of tuberculous pleural effusion (TPE) remains challenging. This study aimed to determine the accuracy of IFN-γ in diagnosing TPE. METHODS: We quantified the expression of interferon-gamma (IFN-γ) in blood (B), adenosine deaminase (ADA), and IFN-γ in pleural effusions (PE) from 25 TPE patients and 31 non-TPE patients using a combination of immunological assays and flow cytometric analysis. The diagnostic performance of these three biomarkers was evaluated using receiver operating characteristic (ROC) curves. RESULTS: We found that IFN-γ levels in blood and pleural fluid were higher in the TPE group than in the non-TPE group. The mean concentration of IFN-γ in pleural fluid of the TPE group was 3140.90 (1817.94, 6611.05) pg/mL, while that of the non-TPE group was 4.91 (0.69, 8.6) pg/mL), and the difference was statistically significant (z = 6.39, P < 0.001). The mean blood IFN-γ was 40.19 (16.45, 59.08) pg/mL in the TPE group and 2.76 (1.96, 6.02) pg/mL in the non-TPE group, which was statistically different (z = 5.12, P < 0.001). The area under the ROC curve (AUC) for pleural fluid IFN-γ, blood IFN-γ, and ADA were 0.999 (95 % CI: 0.994-1.00), 0.901 (95 % CI: 0.798-1.00) and 0.996 (95 % CI: 0.987-1.00), respectively. CONCLUSION: This study confirms that IFN-γ has high diagnostic validity in patients with TPE and can potentially be an excellent biomarker.

Methodological Evaluation of Carbapenemase Detection by Different Methods.

The global proliferation of carbapenemase-producing bacteria (CPB) has garnered significant attention worldwide. Early diagnosis of CPB and accurate identification of carbapenemases are crucial for preventing the spread of CPB and ensuring targeted antibiotic therapy. Therefore, efficient and accurate identification of carbapenemases is paramount in clinically treating diseases associated with CPB. In this study, 58 CPB strains were collected and detected using the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method, a rapid detection platform based on CRISPR-Cas12a gene editing and isothermal amplification. Additionally, four conventional methods (the APB/EDTA method, PCR, NG-test Carba 5, and GeneXpert Carba-R) were employed and compared against whole genome sequencing (WGS) results, considered the gold standard, to evaluate their efficacy in detecting carbapenemases. Detection by the APB/EDTA method revealed that 29 strains were positive for Class A serine endopeptidases, while 29 strains were positive for Class B metalloenzymes. The classification of these zymotypes was consistent with the sequencing result. All target carbapenemases for KPC were identified with 100% sensitivity using NG-test Carba 5, PCR, DETECTR, and GeneXpert Carba-R. In the case of NDM, both Xpert Carba-R and DETECTR showed a sensitivity of 100%. In contrast, NG-test Carba 5 and PCR had a slightly lower sensitivity of 96.7%, each missing one target carbapenemase. n this study, the APB/EDTA method is capable of identifying the zymotype classification but not the specific resistant genes, while Xpert Carba-R and DETECTR are able to detect all target carbapenemases.

Establishment and methodological evaluation of a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes based on CRISPR-Cas12a technology.

PURPOSE: The opportunistic pathogens causing Cryptococcal meningitis are Cryptococcus neoformans and Cryptococcus gattii species complexes. At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of Cryptococcus. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method. METHODS: Recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis. RESULTS: This study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 102 copies/µL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %. CONCLUSION: In this study, a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.

AcrAB-TolC efflux pump overexpression and tet(A) gene mutation increase tigecycline resistance in Klebsiella pneumoniae.

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-ß-naphthylamide dihydrochloride (PaßN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

A Method for Detecting Five Carbapenemases in Bacteria Based on CRISPR-Cas12a Multiple RPA Rapid Detection Technology.

Introduction: As the last line of defense for clinical treatment, Carbapenem antibiotics are increasingly challenged by multi-drug resistant bacteria containing carbapenemases. The rapid spread of these multidrug-resistant bacteria is the greatest threat to severe global health problems. Methods: To solve the problem of rapid transmission of this multidrug-resistant bacteria, we have developed a rapid detection technology using CRPSPR-Cas12a gene editing based on multiple Recombinase polymerase amplification. This technical method can directly isolate the genes of carbapenemase-containing bacteria from samples, with a relatively short detection time of 30 minutes. The instrument used for the detection is relatively inexpensive. Only a water bath can complete the entire experiment of Recombinase polymerase amplification and trans cleavage. This reaction requires no lid during the entire process while reducing a large amount of aerosol pollution. Results: The detection sensitivity of this method is 1.5 CFU/mL, and the specificity is 100%. Discussion: This multi-scene detection method is suitable for screening populations in wild low-resource environments and large-scale indoor crowds. It can be widely used in hospital infection control and prevention and to provide theoretical insights for clinical diagnosis and treatment.

Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a.

Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 102 copies/µl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.

Establishing a pulmonary aspergillus fumigatus infection diagnostic platform based on RPA-CRISPR-Cas12a.

In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 102 copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.

Comprehensive Analysis of TICRR in Hepatocellular Carcinoma Based on Bioinformatics Analysis.

Hepatocellular carcinoma (HCC) is one of the leading cause of cancer-associated death in the world. However, due to the complexity of HCC, it is urgent for us to find a reliable and accurate biomarker for HCC gene therapy.TopBP1-interacting checkpoint and replication regulator (TICRR), known as Treslin in vertebrate and sld3 in yeast, is involved in the tumorigenesis, progression, matastasis, diagnosis, and predicting prognosis of HCC. Disappointingly, the mechanism of TICRR expression in HCC is still not described in detail and requires further analysis. In this study, TCGA ( www.tcga-data.nci.nih.gov/tcga/ ) datasets and GEO ( www.ncbi.nlm.nih.gov/geo ) datasets were used to analyze the expression of TICRR in HCC, the relevance of TICRR mRNA expression and clinicopathological characteristics in patients with HCC, and the relationship between TICRR expression and immune infiltration level in Patients with HCC. Based on MethSurv database, the impact of TICRR in patients with HCC was investigated. In addition, GO/KEGG enrichment analysis of TICRR co-expression was performed using the R package. TICRR was found drastically highly expressed in a variety of cancer types including HCC.ROC curve analysis showed that TICRR had higher accuracy in predicting HCC compared with AFP. The expression level of TICRR was marked positively correlated with tumor stage and prognosis in Patients with HCC.GO/KEGG enrichment analysis showed that TICRR was associated with cell division and cell cycle as well as p53 signaling pathway. In addition, patients with high TICRR methylation of cg05841809, cg09403165, and cg03312532 CpG sites were significantly correlated with poor prognosis of HCC. This study demonstrated that increased TICRR expression in HCC might play an important role in the tumorigenesis, progression, diagnosis, and predicting prognosis of HCC. Therefore, TICRR might be used as a promising diagnostic and prognostic biomarker for HCC gene therapy.

Combination of AS101 and Mefloquine Inhibits Carbapenem-Resistant Pseudomonas aeruginosa in vitro and in vivo.

Background: In recent years, carbapenem-resistant Pseudomonas aeruginosa (CRPA) has spread around the world, leading to a high mortality and close attention of medical community. In this study, we aim to find a new strategy of treatment for CRPA infections. Methods: Eight strains of CRPA were collected, and PCR detected the multi-locus sequence typing (MLST). The antimicrobial susceptibility test was conducted using the VITEK@2 compact system. The minimum inhibitory concentration (MIC) for AS101 and mefloquine was determined using the broth dilution method. Antibacterial activity was tested in vitro and in vivo through the chessboard assay, time killing assay, and a mouse model. The mechanism of AS101 combined with mefloquine against CRPA was assessed through the biofilm formation inhibition assay, electron microscopy, and detection of reactive oxygen species (ROS). Results: The results demonstrated that all tested CRPA strains exhibited multidrug resistance. Moreover, our investigation revealed a substantial synergistic antibacterial effect of AS101-mefloquine in vitro. The assay for inhibiting biofilm formation indicated that AS101-mefloquine effectively suppressed the biofilm formation of CRPA-5 and CRPA-6. Furthermore, AS101-mefloquine were observed to disrupt the bacterial cell wall and enhance the permeability of the cell membrane. This effect was achieved by stimulating the production of ROS, which in turn hindered the growth of CRPA-3. To evaluate the therapeutic potential, a murine model of CRPA-3 peritoneal infection was established. Notably, AS101-mefloquine administration resulted in a significant reduction in bacterial load within the liver, kidney, and spleen of mice after 72 hours of treatment. Conclusion: The present study showed that the combination of AS101 and mefloquine yielded a notable synergistic bacteriostatic effect both in vitro and in vivo, suggesting a potential clinical application of this combination in the treatment of CRPA.

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection.

The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.

A PCR-Reverse Dot Blot Hybridization Based Microfluidics Detection System for the Rapid Identification of 13 Fungal Pathogens Directly After Blood Cultures Over a Period of Time.

Introduction: It is time-consuming to identify fungal pathogens from positive blood cultures using the standard culture-based method. And delayed diagnosis of bloodstream infection leads to significantly increased mortality. Methods: We developed a PCR-reverse dot blot hybridization combined with microfluidic chip techniques to rapidly identify 13 fungal pathogens within 3-4 h using the sample of blood cultured over a period of time. Results: We performed clinical validation using 43 blood culture-positive samples with a sensitivity of 96.7%, a specificity of 100%, and a concordance rate of 97.7%. Samples with different culture durations were evaluated using our approach, showing a detection rate of 85.2% at 16 h and 96.3% at 24 h; the platform could reach a detection limit of 103cfu/mL for the Candida spp. and 103 copies/mL for Aspergillus spp. Discussion: The detection rate of the platform is much higher than the positive rates of concurrent blood cultures. This method bears substantial clinical application potential as it incorporates the microfluidic platform with low reagent consumption, automation, and low cost (about 10 dollars).

A microfluidic chip-based multiplex PCR-reverse dot blot hybridization technique for rapid detection of enteropathogenic bacteria.

Diarrhea caused by enteropathogenic bacteria is a major public health issue worldwide, especially in developing countries. In this study, a microfluidic chip-based multiplex polymerase chain reaction (PCR)-reverse dot blot hybridization technology for the rapid and simultaneous detection of 11 enteropathogenic bacteria was developed and the entire process was completed within 3-4 h. The specificity of this method was analyzed using 11 types of pure target bacterial colonies and another 7 types of pure bacterial colonies, and its sensitivity was evaluated with the serial 10-fold dilution of 11 types of pure target bacterial colonies. The detection limit of this method was as low as 103-102 CFU/mL, and it exhibited high specificity for enteropathogenic bacteria. A total of 60 clinical diarrheal fecal samples were detected using this method, the results of which were compared with those of the conventional reference method, which resulted in a positive coincident rate of 100% and a negative coincident rate of 93.75%. Based on the findings, it could be concluded that multiplex PCR-reverse dot blot hybridization based on the microfluidic chip is a rapid, economical, sensitive, specific, and high-throughput method for detecting enteropathogenic bacteria.

Creating an ultra-sensitive detection platform for monkeypox virus DNA based on CRISPR technology.

The recent major worldwide outbreak of monkeypox virus (MPXV) has highlighted the urgent need for accurate MPXV detection methods. Although quantitative PCR (qPCR) technique is currently the gold standard for MPXV diagnosis, the high costs associated with the technique and the need for complex instrumentation, limits its application in resource-poor settings. CRISPR technology has developed rapidly in recent years and provides an effective tool for point-of-care testing pathogen identification. Here, we exploited the cleavage properties of the Cas12a enzyme and Cas13a enzyme, to detect the MPXV specific genes, F3L gene and B6R gene, respectively. We developed two detection protocols: a 2-step method in which the CRISPR Dual System reaction and the multiplex recombinase polymerase amplification reaction were carried out in separate tubes and a single-tube method in which both reactions were carried out in one tube. Evaluation of the two methods showed that our protocol can detect the MPXV genome down to 10° copies/µL with good specificity and no cross-reactivity with other poxviruses pseudoviruses, and bacteria. Mock positive samples were used to assess clinical applicability, with the results showing satisfactory concordance with the qPCR method for parallel testing. In conclusion, our study provides a reliable molecular diagnostic strategy for detection of MPXV.

Establishment of a Novel Detection Platform for Clostridioides difficile Toxin Genes Based on Orthogonal CRISPR.

Clostridioides difficile is one of the leading pathogens causing nosocomial infection. The infection can range from mild to severe, and rapid identification is pivotal for early clinical diagnosis and appropriate treatment. Here, a genetic testing platform for toxins, referred to as OC-MAB (orthogonal CRISPR system combined with multiple recombinase polymerase amplification [RPA]), was developed to detect the C. difficile toxin genes tcdA and tcdB. While recognizing the amplified products of the tcdA gene and the tcdB gene, Cas13a and Cas12a could activate their cleavage activities to cut labeled RNA and DNA probes, respectively. The cleaved products were subsequently identified by dual-channel fluorescence using a quantitative PCR (qPCR) instrument. Finally, they could also be combined with labeled antibodies on immunochromatographic test strips to achieve visual detection. The OC-MAB platform exhibited ultrahigh sensitivity in detecting the tcdA and tcdB genes at levels of as low as 102 to 101 copies/mL. When testing 72 clinical stool samples, the sensitivity (95% confidence interval [CI], 0.90, 1) and specificity (95% CI, 0.84, 1) of the single-tube method based on the fluorescence readout was 100%, with a positive predictive value (PPA) value of 100% (95% CI, 0.90, 1) and a negative predictive value (NPA) value of 100% (95% CI, 0.84, 1), compared to the results of qPCR. Likewise, the sensitivity of the 2-step method based on the test strip readout was 100% (95% CI, 0.90, 1), while the specificity was 96.3% (95% CI, 0.79, 0.99), with a PPA of 98% (95% CI, 0.87, 0.99) and an NPA of 100% (95% CI, 0.90, 1). In short, orthogonal CRISPR technology is a promising tool for the detection of C. difficile toxin genes. IMPORTANCE C. difficile is currently the primary causative agent of hospital-acquired antibiotic-induced diarrhea, and timely and accurate diagnosis is crucial for hospital-acquired infection control and epidemiological investigation. Here, a new method for the identification of C. difficile was developed based on the recently popular CRISPR technology, and an orthogonal CRISPR dual system was utilized for the simultaneous detection of toxin genes A and B. It also uses a currently rare CRISPR dual-target lateral flow strip with powerful color-changing capabilities, which is appropriate for point-of-care testing (POCT).

Comprehensive Study of Untargeted Metabolomics and 16S rRNA Reveals the Mechanism of Fecal Microbiota Transplantation in Improving a Mouse Model of T2D.

Background: Fecal microbiota transplantation (FMT) has emerged as a new therapy targeting gastrointestinal microbiota for the treatment of a growing number of diseases in recent years. Previous studies have suggested that FMT may be a potential therapy for type 2 diabetes (T2D), but the underlying mechanism remains unclear. Therefore, in the present study, we aimed to investigate the role of FMT in T2D and its underlying mechanisms. Methods: To induce T2D, mice were fed a high-fat diet and injected with low-dose streptozotocin (STZ) for four weeks. The mice were then randomly divided into four groups: control group (n = 7), T2D group (n = 7), metformin (MET)-treated group (n = 7), and FMT group (n = 7). The MET group was orally administered 0.2 g/kg MET, the FMT group was orally administered 0.3 mL of bacterial solution, and the other two groups were orally administered the same volume of saline for four weeks. Serum and fecal samples were collected for non-targeted metabolomics, biochemical indicators, and 16S rRNA sequencing, respectively. Results: Our results demonstrated that FMT had a curative effect on T2D by ameliorating hyperlipidemia and hyperglycemia. Using 16S rRNA sequencing and serum untargeted metabolomic analysis, we found that FMT could restore the disorders of gastrointestinal microbiota in T2D mice. Moreover, corticosterone, progesterone, L-urobilin, and other molecules were identified as biomarkers after FMT treatment. Our bioinformatics analysis suggested that steroid hormone biosynthesis, arginine, proline metabolism, and unsaturated fatty acid biosynthesis could be potential regulatory mechanisms of FMT. Conclusion: In summary, our study provides comprehensive evidence for the role of FMT in the treatment of T2D. FMT has the potential to become a promising strategy for the treatment of metabolic disorders, T2D, and diabetes-related complications.

The Characteristics of Extended-Spectrum ß-Lactamases (ESBLs)-Producing Escherichia coli in Bloodstream Infection.

Background: Bloodstream infection (BSI) is a common type of infection frequently diagnosed in clinics. The emergence and spread of ESBLs-producing Escherichia coli (E. coli) has emerged as one of the biggest challenges in global community health. Methods: The production of ESBLs was determined by the composite disk diffusion method. The expression of the various resistance and virulence genes were detected by PCR and sequencing. Multi-locus sequence typing (MLST) and phylogenetic groups were used for the classification. The transfer of resistant plasmids was determined by conjugation assay. The statistical differences were analyzed using Statistical Product and Service Solutions (SPSS) version 23.0. Results: A total of 60 strains of ESBLs-producing E. coli were collected. The resistance genes that were identified included bla CTX-M, bla TEM, bla SHV, bla OXA-1 and mcr-1. The most common one was the bla CTX-M including bla CTX-M-27 (n = 16), bla CTX-M-14 (n = 15), bla CTX-M-15 (n = 11), bla CTX-M-55 (n = 14) and bla CTX-M-65 (n = 5). A total of 31 STs were detected, and the most abundant among which was ST131 (n = 16, 26.7%). Most of the E. coli (n = 46, 76.7%) belonged to the groups B2 and D. And some virulence genes were related to the classification of the E. coli. Among them, the detection rates of hek/hra, kpsMII and papGII-III in groups B2 and D were higher than those in groups A and B1. The detection rates of cnf1, iucC and papGII-III in ST131 were higher than those in non-ST131. And the distributions of hek/hra, iroN, iucC, kpsMII and papGII-III were related to the bla CTX-M subtypes. Finally, most bacterial (n = 32, 53.3%) resistance genes could be transferred between the bacteria by plasmids, especially IncFIB. Conclusion: ESBLs-producing E. coli in BSI exhibited had high resistance rates and carried a variety of virulence factors (VFs). This is necessary to strengthen the monitoring of ESBLs-producing isolates in the medical environment.

Rapid visualization of Clostridioides difficile toxins A and B by multiplex RPA combined with CRISPR-Cas12a.

Purpose: Clostridioides difficile (C. difficile) infection is the most common cause of nosocomial infection, which is a severe challenge in modern medical care. Currently, many laboratory diagnostic methods for C. difficile are available, such as PCR, culture-based tests, and antigen-based tests. However, these methods are not suitable for rapid point-of-care testing (POCT). Therefore, it is of great significance to develop a rapid, sensitive, and cost-effective method to detect C. difficile toxin genes. Methods: Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for rapid POCT. In this study, we developed a rapid and specific detection platform for dual C. difficile toxins by combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a. Results: The platform includes multiplex RPA-cas12a-fluorescence assay and multiplex RPA-cas12a-LFS (Lateral flow strip) assay, through which the detection limit for tcdA and tcdB was 10 copies/µL and 1 copy/µL, respectively. The results can be more clearly distinguished using a violet flashlight, which realized a portable visual readout. The platform can be tested within 50 min. Furthermore, our method did not cross-react with other pathogens that cause intestinal diarrhea. The results of 10 clinical samples using our method was 100% consistent with those from real-time PCR detection. Conclusion: In conclusion, the CRISPR-based double toxin gene detection platform for C. difficile is an effective, specific, and sensitive detection method, which can be used as a powerful on-site detection tool for POCT in the future.

The role of HBV cccDNA in occult hepatitis B virus infection.

Occult hepatitis B virus (HBV) infection (OBI) refers to the presence of replication-competent HBV DNA in the liver, with or without HBV DNA in the blood, in individuals who tested negative for HBV surface antigen (HBsAg). In this peculiar phase of HBV infection, the covalently closed circular DNA (cccDNA) is in a low state of replication. Several advances have been made toward clarifying the mechanisms involved in such a suppression of viral activity, which seems to be mainly related to the host's immune control and epigenetic factors. Although the underlying mechanisms describing the genesis of OBI are not completely known, the presence of viral cccDNA, which remains in a low state of replication due to the host's strong immune suppression of HBV replication and gene expression, appears to be the causative factor. Through this review, we have provided an updated account on the role of HBV cccDNA in regulating OBI. We have comprehensively described the HBV cell cycle, cccDNA kinetics, current regulatory mechanisms, and the therapeutic methods of cccDNA in OBI-related diseases.

Establishment and Methodological Evaluation of a Method for Rapid Detection of Helicobacter pylori and Virulence Genes Based on CRISPR-Cas12a.

Introduction: More than half of the world's people are infected or have been infected with Helicobacter pylori. This infection is related to many diseases, with its pathogenicity related to virulence factors. Therefore, the rapid diagnosis of H. pylori and genotyping of virulence genes play an extremely important role in the clinical treatment and control of transmission. Methods: To this end, we developed a molecular detection method based on RPA- CRISPR-Cas12a technology for the specific genes 16S rDNA gene, cytotoxin associated gene A(cagA), and vacuolating cytotoxin A (vacA) of H. pylori. Results: The results of which were displayed by lateral flow strips. Macroscopic observation takes only about 25 minutes and the sensitivity is 2ng/microliter. Discussion: The method is simple, convenient to operate and has low costs, and can therefore be applied widely to the detection and typing of H. pylori in various environments such as primary hospitals, community clinics, outdoors, and large medical institutions.