With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a c...With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.展开更多
Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison e...Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).展开更多
Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additiona...Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additionally,single Cre-lox P recombination systems have limitations in tracing specific cell populations.This study proposes three reporting systems utilizing Cre,Dre,and Dre+Cre mediated recombination.These systems incorporate td Tomato expression on the cell membrane and Phi YFP expression within the nucleus,allowing for clear observation of the nucleus and membrane.The efficacy of these systems is successfully demonstrated by labeling cardiomyocytes and hepatocytes.The potential for dynamic visualization of the cell membrane is showcased using intravital imaging microscopy or threedimensional imaging.Furthermore,by combining this dual recombinase system with the Pro Tracer system,hepatocyte proliferation is traced with enhanced precision.This reporting system holds significant importance for advancing the understanding of cell fate studies in development,homeostasis,and diseases.展开更多
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 int...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.展开更多
Hepatitis C virus(HCV)infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20%of the total population are infected.Initial infection is usually asym...Hepatitis C virus(HCV)infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20%of the total population are infected.Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma.The management of HCV infection should not only be focus on therapy,but also to screen carrier individuals in order to prevent transmission.In the present,molecular detection and quantification of HCV genome by real time polymerase chain reaction(PCR)represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens.However,real time PCR is a complicated approach and of limited distribution.On the other hand,isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care.In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.展开更多
Astrocytes are the most abundant cell type in the central nervous system(CNS).They provide trophic support for neurons,modulate synaptic transmission and plasticity,and contribute to neuronal dysfunction.Many transgen...Astrocytes are the most abundant cell type in the central nervous system(CNS).They provide trophic support for neurons,modulate synaptic transmission and plasticity,and contribute to neuronal dysfunction.Many transgenic mouse lines have been generated to obtain astrocyte-specific expression of inducible Cre recombinase for functional studies;however,the expression patterns of inducible Cre recombinase in these lines have not been systematically characterized.We generated a new astrocyte-specific Aldh1 l1-CreER^(T2)knock-in mouse line and compared the expression pattern of Cre recombinase between this and five widely-used transgenic lines(hGfap-CreER^(T2)from The Jackson Laboratory and The Mutant Mouse Resource and Research Center,Glast-CreER^(T2),Cx30-CreER^(T2),and Fgfr3-iCreER^(T2))by crossing with Ai14 mice,which express tdTomato fluorescence following Cre-mediated recombination.In adult Aldh1 l1-CreER^(T2):Ai 14 transgenic mice,tdTomato was detected throughout the CNS,and five novel morphologicallydefined types of astrocyte were described.Among the six evaluated lines,the specificity of Cre-mediated recombination was highest when driven by Aldh1 l1 and lowest when driven by hGfap;in the latter mice,co-staining between tdTomato and NeuN was observed in the hippocampus and cortex.Notably,evident leakage was noted in Fgfr3-iCreER^(T2)mice,and the expression level of tdTomato was low in the thalamus when Cre recombinase expression was driven by Glast and in the capsular part of the central amygdaloid nucleus when driven by Cx30.Furthermore,tdTomato was clearly expressed in peripheral organs in four of the lines.Our results emphasize that the astrocyte-specific CreER^(T2)transgenic lines used in functional studies should be carefully selected.展开更多
Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we ge...Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H^＋-, K^＋-ATPase gene （Atp4b）. In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles （Smad4Ca/Co）. Multiple-tissue PCR of Atp4b-Cre;Smad4Co/＋ mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.展开更多
creening of foodborne pathogens is important to prevent contaminated foods from their supply chains.n this study, a portable detection device was developed for rapid, sensitive and simple detection of viable almonella...creening of foodborne pathogens is important to prevent contaminated foods from their supply chains.n this study, a portable detection device was developed for rapid, sensitive and simple detection of viable almonella using a finger-actuated microfluidic chip and an improved recombinase aided amplification (RAA) assay. Improved propidium monoazide(PMAxx) was combined with RAA to enable this device to distinguish viable bacteria from dead ones. The modification of PMAxx into dead bacteria, the magnetic xtraction of nucleic acids from viable bacteria and the RAA detection of extracted nucleic acids were performed using the microfluidic chip on its supporting device by finger press-release operations. The fluorescent signal resulting from RAA amplification of the nucleic acids was collected using a USB camera nd analyzed using a self-developed smartphone App to quantitatively determine the bacterial concenration. This device could detect Salmonella typhimurium in spiked chicken meats from 1.3 × 10^(2) CFU/m L o 1.3 × 10^(7) CFU/m L in 2 h with a lower detection limit of 130 CFU/m L, and has shown its potential for on-site detection of foodborne pathogens.展开更多
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 st...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.展开更多
Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse lin...Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlcd （Collα1） promoter （Collα1-Cre）. Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Coll αl-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles （Smad4^Co/Co）. Multiple tissue PCR of Collα1-Cre;Smad4^Co/＋ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Collα1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Collα1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.展开更多
[Objective]The paper was to develop a rapid method for the detection of spring Viremia of carp virus(SVCV).[Method]The specific primers were designed by targeting the G gene of SVCV.The recombinase polymerase amplific...[Objective]The paper was to develop a rapid method for the detection of spring Viremia of carp virus(SVCV).[Method]The specific primers were designed by targeting the G gene of SVCV.The recombinase polymerase amplification(RPA)assay for detecting SVCV was estab-lished by optimizing the reaction conditions.The optimal amplification temperature of RPA assay was 30℃,and the test could be finished within 20 min.[Result]The method was specific with no cross-reaction with other common fish infectious viruses.Sensitivity test showed that the lowest detection limit of the method was 89.2 copies/μL,higher than that of traditional RT-PCR.Moreover,a total of 80 clinical samples were detected by RPA and RT-PCR,respectively.The weak positive samples tested by RT-PCR could be detectable with RPA,indicating that RPA assay could be used in clinical detection.[Conclusion]The method established is rapid,simple,specific and sensitive for testing SVCV,and it will be widely used in grassroots laboratory and on-site inspection.展开更多
Background:Malaria remains a global health challenge,with 249 million cases and 608,000 deaths reported in 2022.While China achieved malaria elimination,imported cases surged by 194.4% in 2023,underscoring the need fo...Background:Malaria remains a global health challenge,with 249 million cases and 608,000 deaths reported in 2022.While China achieved malaria elimination,imported cases surged by 194.4% in 2023,underscoring the need for rapid diagnostics.Traditional methods like microscopy and rapid diagnostic tests(RDTs)face limitations in sensitivity and infrastructure requirements.This study aimed to establish and optimize a“one-pot”enzymatic recombinase amplification(ERA)assay for the molecular detection of Plasmodium falciparum and Plasmodium vivax,and to evaluate the efficacy of this assay through methodological verification and clinical performance.Methods:We designed a specific ERA assay targeting the conserved regions of P.falciparum and P.vivax genetic material.We evaluated the sensitivity and specificity of this assay using synthetic plasmids and genomic material.Additionally,we tested the stability of the reaction by incorporating potential interfering substances into the reaction system.Finally,we analyzed the detection performance of the ERA method against real-time fluorescent quantitative PCR and rapid diagnostic tests using clinical samples.Results:The detection process could be completed within 25 min at 35℃-40℃,and the results could be interpreted either under UV light or using a GeneScope instrument.The detection limit of the ERA assay was 250 copies/mL,which was 40 times more sensitive than fluorescent quantitative PCR.When evaluating the clinical performance using 75 clinical specimens,the detection rate of the ERA method was 94.54% compared with 89.09% for fluorescent quantitative PCR.The ERA assay and fluorescent quantitative PCR can achieve positive detection when blood samples were diluted 1024 times or even 4096 times.Comparatively,the detection capabilities of rapid diagnostic tests were significantly lower than that of the ERA assay.Conclusion:The ERA method shows good performance in the detection of P.falciparum and P.vivax,and can be used as a complementary tool for malaria screening and clinical diagnosis.展开更多
Introduction:Fluorescent probe-based recombinase aided amplification(RAA)offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design,restricting its widespre...Introduction:Fluorescent probe-based recombinase aided amplification(RAA)offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design,restricting its widespread application.Methods:A novel EvaGreen dye-based RAA(EvaGreen-RAA)assay utilizing self-avoiding molecular recognition system(SAMRS)primers was developed for the detection of Pseudomonas fluorescens(PF)and Bacillus cereus(BC)in milk.Conventional RAA was used as a reference method.Sensitivity was evaluated using nucleic acids from recombinant plasmids and simulated milk specimens.Additionally,a dual EvaGreen-RAA assay was investigated for simultaneous detection of mixed BC and PF in simulated milk specimens.Results:The EvaGreen-RAA demonstrated superior sensitivity compared to conventional RAA,with detection limits of 1 copy/μL versus 10 copies/μL for both BC and PF plasmids,respectively.In simulated milk specimens,EvaGreen-RAA detected BC and PF at concentrations of 100 CFU/mL and 200 CFU/mL,respectively,compared to 400 CFU/mL and 600 CFU/mL for conventional RAA.The dual EvaGreen-RAA assay successfully detected mixed BC and PF in simulated milk specimens at concentrations of 200 CFU/mL for each pathogen.Conclusion:The EvaGreen-RAA assay demonstrated significant advantages in terms of simplicity and enhanced sensitivity compared to fluorescent probe-based RAA,offering a novel approach for developing multiplex pathogen detection systems using melting curve analysis.展开更多
Background:Fluorescent recombinase-aided amplification(RAA)assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and s...Background:Fluorescent recombinase-aided amplification(RAA)assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity,compared with real-time PCR(qPCR)assays,but they require a complex probe design.To eliminate the addition of fluorescent probes for RAA,an EvaGreen dye-based recombinase-aided amplification(EvaGreen-RAA)assay using self-avoiding molecular recognition system(SAMRS)primers was developed.Methods:The SAMRS primers effectively avoided the production of primer dimers,thus improving the detection sensitivity,while EvaGreen dye was used to quantitatively measure the amplified products in real time.Using Staphy-lococcus aureus(SA)and Listeria monocytogenes(LM)as examples,EvaGreen-RAA with SAMRS primers was developed.As a reference and comparison,a traditional fluorescence probe RAA method and a RAA with SAMRS primers(SAMRS-RAA)for detecting SA and LM were also investigated.Serial di-lutions of recombinant plasmids were used to evaluate the sensitivity of the assays.Unenriched and enriched simulated milk samples were used to eval-uate the limits of detection(LOD)of these methods.Using high-resolution melting(HRM)was used to explore the sensitivity of the dual EvaGreen-RAA assay.Results:The sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS-RAA and EvaGreen-RAA for detecting SA and LM plasmids was 1 copies/μL.The LOD values of the EvaGreen-RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL,respectively,and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL.EvaGreen-RAA had linear amplification in real time in the range of 1-10^(5)copies/μL of the plasmids of SA and LM.The sensitivity of the dual EvaGreen-RAA assay for SA and LM was estimated to be 10^(2)CFU/mL.Conclusion:A real-time quantitative EvaGreen-RAA method for detecting SA and LM was developed,which eliminates the need to design complex RAA probes.This dye-based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens,which has many potential applications.展开更多
Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cann...Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cannot provide fast mobile on-site systems to detect Salmonella in food.Materials and Methods:A visual method was established based on multiple recombinase polymerase amplification(RPA)coupled with lateral flow dipsticks(LFD)for the simultaneous detection of Salmonella spp.,Salmonella Enteritidis and Salmonella Typhimurium in vitro and food.Results:The optimal volume and temperature for the multiplex RPA-LFD method were determined to be 25μL and 38°C,respectively.The reaction process was completed within 25 min and the results were observed visually.The limits of detection(LODs)were 2.8×10^(2),5.9×10^(2),and 7.6×10^(2) CFU/mL for Salmonella spp.,S.Enteritidis and S.Typhimurium,respectively.Meanwhile,the results of the established method showed no cross-reactivity between the Salmonella cells and other common foodborne bacteria,which was highly specific for Salmonella.More importantly,the developed method exhibited good performance in artificially contaminated chicken samples with the LODs of 2.8×10^(3),5.9×10^(3),and 7.6×10^(3) CFU/mL for Salmonella spp.,S.Enteritidis,and S.Typhimurium,respectively.Finally,the application of the multiple RPA-LFD methods in retailed food samples displayed that this method was effective and practical for the detection of Salmonella spp.in food.Conclusion:The developed multiplex RPA-LFD method provides a new sensitive and rapid alternative for the specific detection of Salmonella spp.and its important serovars in food.展开更多
Aeromonas salmonicida is a common pathogen of salmonid fishes that poses a significant threat to the fresh water and marine culture industry,potentially resulting in huge economic losses.To prevent and control fish di...Aeromonas salmonicida is a common pathogen of salmonid fishes that poses a significant threat to the fresh water and marine culture industry,potentially resulting in huge economic losses.To prevent and control fish diseases caused by A.salmonicida,rapid and effective diagnostic approaches must be developed,and which are important for routine monitoring and clinical care.By combining recombinase polymerase amplification(RPA)technology with a visible lateral flow strip(RPA-LF),we have enhanced both the precision of RPA detection and the convenience of real-time monitoring.In this study,we introduce a robust method for detecting A.salmonicida using RPA-LF.This assay specifically targets the ASA_1441 gene of A.salmonicida,ensuring high specificity,without cross-reactivity with other prevalent fresh water or marine pathogens.The optimal amplification temperature of the RPA assay was 39℃.Its sensitivity extends to as low as 100 fg of purified DNA,representing more than 1000-fold higher sensitivity than conventional PCR methods.Furthermore,to enhance the usability of the RPA-LF assay,we developed a rapid sample preparation method using cellulose dipsticks for nucleic acid extraction.This method achieves a limit of detection(LOD)as low as 1.67 CFU/μL and completes the entire process within 20 min.In conclusion,our findings present a rapid and precise tool for monitoring A.salmonicida infection in aquaculture and marine culture.This advancement offers valuable insights for effective disease prevention and control strategies.展开更多
Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are st...Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are still lacking.This study established a rapid soil DNA extraction(RSDE)method that can be used in field detection.In this method,we first utilized the optimized lysate to isolate DNA from soil and then used a filtration membrane and a DNA adsorption membrane to purify the DNA via the column method.Moreover,we used the pressure from the syringe instead of the conventional centrifugal force of the centrifuge to assist the sample filtration,resulting in very low requirements for this method,with an extraction time of less than 20 min.Furthermore,we demonstrated that the RSDE method was applicable for DNA extraction from different types of soils,with the demand for soil samples as low as 0.1 g and that the amount of obtained DNA was,to some extent,greater than that obtained by a commercial kit.Further analysis revealed that this extracted genomic DNA can be used directly for polymerase chain reaction(PCR)analysis,including ordinary PCR,real-time fluorescent quantitative PCR,and recombinase polymerase amplification(RPA)-CRISPR/Cas12a visual assays.In addition,we demonstrated that this method can be used to extract DNA from residual plant roots in addition to soil microbes,which lays a foundation for the comprehensive analysis of soil plants and microorganisms.In summary,the RSDE method proposed in this study may have wide application prospects.展开更多
Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease inclu...Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease include microscopy examination,quantitative real-time PCR,and loop-mediated isothermal amplification-lateral flow dipstick(LAMP-LFD).In this study,a recombinase polymerase amplification-lateral flow dipstick(RPA-LFD)method,targeting the beta-tubulin gene,was developed to detect G.plecoglossi,three sets of primers and probes were designed and screened,after which the initial reaction system was established.The RPA-LFD method for G.plecoglossi could complete nucleic acid amplification at 39℃ for 10 min,after which the amplification product was dropped on the LFD strip,and the results could then be observed within 5 min.A specificity assay revealed that there was no cross reactivity with other protozoa except G.plecoglossi.A sensitivity assay revealed that the detection limit was 9.38×10^(-6) ng/μL,which was more sensitive than that of conventional PCR.Compared with conventional detection methods,the novel RPA-LFD method has the advantages of simple operation,short operation time,high sensitivity,and high specificity for G.plecoglossi detection,indicating its potential use in rapid field detection of G.plecoglossi.展开更多
Fowl adenovirus(FAdV)serotype 4,recognized as the causative agent of hydropericardium syndrome(HPS)in chickens,causes substantial economic losses in poultry farming.To develop a simple,rapid,and reliable diagnostic me...Fowl adenovirus(FAdV)serotype 4,recognized as the causative agent of hydropericardium syndrome(HPS)in chickens,causes substantial economic losses in poultry farming.To develop a simple,rapid,and reliable diagnostic method for the timely detection of FAdV-4 nucleic acid,we integrated the CRISPR/Cas12a system with recombinase-aided amplification(RAA).This approach enables visual detection of FAdV-4 with a sensitivity of one genome copy.The results can be obtained within 40 to 50 min without the need for complex instrumentation,making it ideal for remote field applications.Using this method,we investigated the prevalence of FAdV-4 in both common farm poultry and wild birds.Our results indicated that the FAdV-4-positive rate in wild birds was 51.19%,suggesting that wild birds may serve as specific reservoirs for this virus.In summary,we present a sensitive,swift,accurate,and inexpensive detection method for FAdV-4,along with an investigation of its epidemic situation in birds.Our study advances the detection and epidemiological understanding of FAdV-4 transmission among farm poultry and wild birds.展开更多
Wheat powdery mildew caused by Blumeria graminis f.sp.tritici(Bgt)is an important disease worldwide.Detection of latent infection of leaves by the pathogen in late autumn is valuable for estimating the inoculum potent...Wheat powdery mildew caused by Blumeria graminis f.sp.tritici(Bgt)is an important disease worldwide.Detection of latent infection of leaves by the pathogen in late autumn is valuable for estimating the inoculum potential to assess disease risks in the spring.We developed a new tool for rapid detection and quantification of latent infection of seedlings by the pathogen.The method was based on recombinase polymerase amplification(RPA)coupled with an end-point detection via lateral flow device(LFD).The limit of detection is 100 agμL^(-1)of Bgt DNA,without noticeable interference from either other common wheat pathogens or wheat material(Triticum aestivum).It was evaluated on wheat seedlings for this accuracy and sensitivity in detecting latent infection of Bgt.We further extended this RPALFD assay to estimate the level of latent infection by Bgt based on imaging analysis.There was a strong correlation between the image-based and real-time PCR assay estimates of Bgt DNA.The present results suggested that this new tool can provide rapid and accurate quantification of Bgt in latently infected leaves and can be further development as an on-site monitoring tool.展开更多
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195)。
文摘With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.
基金supported by the University-Industry Col aborative Education Program,China(220904860093831)。
文摘Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).
基金supported by the National Key Research&Development Program of China(2021YFA0805100,2023YFA1800700,2019YFA0110403,2019YFA0802000)the National Science Foundation of China(82088101,32370885,92368103,32370897)the Westlake Education Foundation,and the Benyuan Charity Fund,Research Funds of Hangzhou Institute for Advanced Study(2022ZZ01015 and B04006C01600515)。
文摘Genetic lineage tracing has been widely employed to investigate cell lineages and fate.However,conventional reporting systems often label the entire cytoplasm,making it challenging to discern cell boundaries.Additionally,single Cre-lox P recombination systems have limitations in tracing specific cell populations.This study proposes three reporting systems utilizing Cre,Dre,and Dre+Cre mediated recombination.These systems incorporate td Tomato expression on the cell membrane and Phi YFP expression within the nucleus,allowing for clear observation of the nucleus and membrane.The efficacy of these systems is successfully demonstrated by labeling cardiomyocytes and hepatocytes.The potential for dynamic visualization of the cell membrane is showcased using intravital imaging microscopy or threedimensional imaging.Furthermore,by combining this dual recombinase system with the Pro Tracer system,hepatocyte proliferation is traced with enhanced precision.This reporting system holds significant importance for advancing the understanding of cell fate studies in development,homeostasis,and diseases.
基金supported by the National Key Research and Development Plan of China[2018YFC1602500]the Natural Science Foundation of Tianjin[19JCZDJC39900]
文摘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.
文摘Hepatitis C virus(HCV)infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20%of the total population are infected.Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma.The management of HCV infection should not only be focus on therapy,but also to screen carrier individuals in order to prevent transmission.In the present,molecular detection and quantification of HCV genome by real time polymerase chain reaction(PCR)represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens.However,real time PCR is a complicated approach and of limited distribution.On the other hand,isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care.In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.
基金supported by Grants from the National Natural Science Foundation of China(31430032,31830033,81971080,and 81671356)the Program for Changjiang Scholars and Innovative Research Teams in University(IRT_16R37)+1 种基金the Science and Technology Program of Guangdong(20188030334001)the Guangzhou Science and Technology Project(201707020027,201704020116)。
文摘Astrocytes are the most abundant cell type in the central nervous system(CNS).They provide trophic support for neurons,modulate synaptic transmission and plasticity,and contribute to neuronal dysfunction.Many transgenic mouse lines have been generated to obtain astrocyte-specific expression of inducible Cre recombinase for functional studies;however,the expression patterns of inducible Cre recombinase in these lines have not been systematically characterized.We generated a new astrocyte-specific Aldh1 l1-CreER^(T2)knock-in mouse line and compared the expression pattern of Cre recombinase between this and five widely-used transgenic lines(hGfap-CreER^(T2)from The Jackson Laboratory and The Mutant Mouse Resource and Research Center,Glast-CreER^(T2),Cx30-CreER^(T2),and Fgfr3-iCreER^(T2))by crossing with Ai14 mice,which express tdTomato fluorescence following Cre-mediated recombination.In adult Aldh1 l1-CreER^(T2):Ai 14 transgenic mice,tdTomato was detected throughout the CNS,and five novel morphologicallydefined types of astrocyte were described.Among the six evaluated lines,the specificity of Cre-mediated recombination was highest when driven by Aldh1 l1 and lowest when driven by hGfap;in the latter mice,co-staining between tdTomato and NeuN was observed in the hippocampus and cortex.Notably,evident leakage was noted in Fgfr3-iCreER^(T2)mice,and the expression level of tdTomato was low in the thalamus when Cre recombinase expression was driven by Glast and in the capsular part of the central amygdaloid nucleus when driven by Cx30.Furthermore,tdTomato was clearly expressed in peripheral organs in four of the lines.Our results emphasize that the astrocyte-specific CreER^(T2)transgenic lines used in functional studies should be carefully selected.
基金supported by Chinese National Key Program on Basic Research (Nos. 2006CB943501 and 2006BAI23B01-3)Key Project for Drug Discovery and Development in China (No. 2009ZX09501-027)+1 种基金Key Project for Infectious Diseases in China (No. 2008ZX10002-016)E-Institutes of Shanghai Municipal Education Commission (E03003)
文摘Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H^＋-, K^＋-ATPase gene （Atp4b）. In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles （Smad4Ca/Co）. Multiple-tissue PCR of Atp4b-Cre;Smad4Co/＋ mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.
基金funded by National Natural Science Foundation of China (No. 32071899)Walmart Foundation (No. UA2020– 154)。
文摘creening of foodborne pathogens is important to prevent contaminated foods from their supply chains.n this study, a portable detection device was developed for rapid, sensitive and simple detection of viable almonella using a finger-actuated microfluidic chip and an improved recombinase aided amplification (RAA) assay. Improved propidium monoazide(PMAxx) was combined with RAA to enable this device to distinguish viable bacteria from dead ones. The modification of PMAxx into dead bacteria, the magnetic xtraction of nucleic acids from viable bacteria and the RAA detection of extracted nucleic acids were performed using the microfluidic chip on its supporting device by finger press-release operations. The fluorescent signal resulting from RAA amplification of the nucleic acids was collected using a USB camera nd analyzed using a self-developed smartphone App to quantitatively determine the bacterial concenration. This device could detect Salmonella typhimurium in spiked chicken meats from 1.3 × 10^(2) CFU/m L o 1.3 × 10^(7) CFU/m L in 2 h with a lower detection limit of 130 CFU/m L, and has shown its potential for on-site detection of foodborne pathogens.
基金supported by National Key R&D Program of China[2017YFC200503]National Natural Science Foundation of China[No.42077399].
文摘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.
基金the National Natural Sci-ence Foundation of China (No. 30430350)the National Science Supporting Program (No. 2006BAI23B01-3).
文摘Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlcd （Collα1） promoter （Collα1-Cre）. Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Coll αl-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles （Smad4^Co/Co）. Multiple tissue PCR of Collα1-Cre;Smad4^Co/＋ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Collα1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Collα1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.
基金Supported by National Key Research and Development Program (2017YFF0211103)Scientific Research Project of General Administration of Quality Supervision,Inspection and Quarantine (2017IK232)
文摘[Objective]The paper was to develop a rapid method for the detection of spring Viremia of carp virus(SVCV).[Method]The specific primers were designed by targeting the G gene of SVCV.The recombinase polymerase amplification(RPA)assay for detecting SVCV was estab-lished by optimizing the reaction conditions.The optimal amplification temperature of RPA assay was 30℃,and the test could be finished within 20 min.[Result]The method was specific with no cross-reaction with other common fish infectious viruses.Sensitivity test showed that the lowest detection limit of the method was 89.2 copies/μL,higher than that of traditional RT-PCR.Moreover,a total of 80 clinical samples were detected by RPA and RT-PCR,respectively.The weak positive samples tested by RT-PCR could be detectable with RPA,indicating that RPA assay could be used in clinical detection.[Conclusion]The method established is rapid,simple,specific and sensitive for testing SVCV,and it will be widely used in grassroots laboratory and on-site inspection.
基金supported by a National Key Research and Development Plan Project(2021YFC2301801).
文摘Background:Malaria remains a global health challenge,with 249 million cases and 608,000 deaths reported in 2022.While China achieved malaria elimination,imported cases surged by 194.4% in 2023,underscoring the need for rapid diagnostics.Traditional methods like microscopy and rapid diagnostic tests(RDTs)face limitations in sensitivity and infrastructure requirements.This study aimed to establish and optimize a“one-pot”enzymatic recombinase amplification(ERA)assay for the molecular detection of Plasmodium falciparum and Plasmodium vivax,and to evaluate the efficacy of this assay through methodological verification and clinical performance.Methods:We designed a specific ERA assay targeting the conserved regions of P.falciparum and P.vivax genetic material.We evaluated the sensitivity and specificity of this assay using synthetic plasmids and genomic material.Additionally,we tested the stability of the reaction by incorporating potential interfering substances into the reaction system.Finally,we analyzed the detection performance of the ERA method against real-time fluorescent quantitative PCR and rapid diagnostic tests using clinical samples.Results:The detection process could be completed within 25 min at 35℃-40℃,and the results could be interpreted either under UV light or using a GeneScope instrument.The detection limit of the ERA assay was 250 copies/mL,which was 40 times more sensitive than fluorescent quantitative PCR.When evaluating the clinical performance using 75 clinical specimens,the detection rate of the ERA method was 94.54% compared with 89.09% for fluorescent quantitative PCR.The ERA assay and fluorescent quantitative PCR can achieve positive detection when blood samples were diluted 1024 times or even 4096 times.Comparatively,the detection capabilities of rapid diagnostic tests were significantly lower than that of the ERA assay.Conclusion:The ERA method shows good performance in the detection of P.falciparum and P.vivax,and can be used as a complementary tool for malaria screening and clinical diagnosis.
基金Supported by Shandong Province Key Research and Development Plan(Major Scientific and Technological Innovation Project,2023CXGC010711)the National Key R&D Program of China(2021YFC2301102)the National Natural Science Foundation of China(82202593,U23A20106).
文摘Introduction:Fluorescent probe-based recombinase aided amplification(RAA)offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design,restricting its widespread application.Methods:A novel EvaGreen dye-based RAA(EvaGreen-RAA)assay utilizing self-avoiding molecular recognition system(SAMRS)primers was developed for the detection of Pseudomonas fluorescens(PF)and Bacillus cereus(BC)in milk.Conventional RAA was used as a reference method.Sensitivity was evaluated using nucleic acids from recombinant plasmids and simulated milk specimens.Additionally,a dual EvaGreen-RAA assay was investigated for simultaneous detection of mixed BC and PF in simulated milk specimens.Results:The EvaGreen-RAA demonstrated superior sensitivity compared to conventional RAA,with detection limits of 1 copy/μL versus 10 copies/μL for both BC and PF plasmids,respectively.In simulated milk specimens,EvaGreen-RAA detected BC and PF at concentrations of 100 CFU/mL and 200 CFU/mL,respectively,compared to 400 CFU/mL and 600 CFU/mL for conventional RAA.The dual EvaGreen-RAA assay successfully detected mixed BC and PF in simulated milk specimens at concentrations of 200 CFU/mL for each pathogen.Conclusion:The EvaGreen-RAA assay demonstrated significant advantages in terms of simplicity and enhanced sensitivity compared to fluorescent probe-based RAA,offering a novel approach for developing multiplex pathogen detection systems using melting curve analysis.
基金National Key R&D Program of China,Grant/Award Number:2021YFC2301102National Natural Science Foundation of China,Grant/Award Numbers:82202593,U23A20106。
文摘Background:Fluorescent recombinase-aided amplification(RAA)assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity,compared with real-time PCR(qPCR)assays,but they require a complex probe design.To eliminate the addition of fluorescent probes for RAA,an EvaGreen dye-based recombinase-aided amplification(EvaGreen-RAA)assay using self-avoiding molecular recognition system(SAMRS)primers was developed.Methods:The SAMRS primers effectively avoided the production of primer dimers,thus improving the detection sensitivity,while EvaGreen dye was used to quantitatively measure the amplified products in real time.Using Staphy-lococcus aureus(SA)and Listeria monocytogenes(LM)as examples,EvaGreen-RAA with SAMRS primers was developed.As a reference and comparison,a traditional fluorescence probe RAA method and a RAA with SAMRS primers(SAMRS-RAA)for detecting SA and LM were also investigated.Serial di-lutions of recombinant plasmids were used to evaluate the sensitivity of the assays.Unenriched and enriched simulated milk samples were used to eval-uate the limits of detection(LOD)of these methods.Using high-resolution melting(HRM)was used to explore the sensitivity of the dual EvaGreen-RAA assay.Results:The sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS-RAA and EvaGreen-RAA for detecting SA and LM plasmids was 1 copies/μL.The LOD values of the EvaGreen-RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL,respectively,and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL.EvaGreen-RAA had linear amplification in real time in the range of 1-10^(5)copies/μL of the plasmids of SA and LM.The sensitivity of the dual EvaGreen-RAA assay for SA and LM was estimated to be 10^(2)CFU/mL.Conclusion:A real-time quantitative EvaGreen-RAA method for detecting SA and LM was developed,which eliminates the need to design complex RAA probes.This dye-based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens,which has many potential applications.
基金supported by the National Key R&D Program of China(No.2019YFE0119700)the National Natural Science Foundation of China(No.32172316).
文摘Objectives:Salmonella spp.is a world-leading foodborne pathogen and its rapid detection is essential for ensuring food safety.Conventional methods require expensive instruments,considerable operational skills and cannot provide fast mobile on-site systems to detect Salmonella in food.Materials and Methods:A visual method was established based on multiple recombinase polymerase amplification(RPA)coupled with lateral flow dipsticks(LFD)for the simultaneous detection of Salmonella spp.,Salmonella Enteritidis and Salmonella Typhimurium in vitro and food.Results:The optimal volume and temperature for the multiplex RPA-LFD method were determined to be 25μL and 38°C,respectively.The reaction process was completed within 25 min and the results were observed visually.The limits of detection(LODs)were 2.8×10^(2),5.9×10^(2),and 7.6×10^(2) CFU/mL for Salmonella spp.,S.Enteritidis and S.Typhimurium,respectively.Meanwhile,the results of the established method showed no cross-reactivity between the Salmonella cells and other common foodborne bacteria,which was highly specific for Salmonella.More importantly,the developed method exhibited good performance in artificially contaminated chicken samples with the LODs of 2.8×10^(3),5.9×10^(3),and 7.6×10^(3) CFU/mL for Salmonella spp.,S.Enteritidis,and S.Typhimurium,respectively.Finally,the application of the multiple RPA-LFD methods in retailed food samples displayed that this method was effective and practical for the detection of Salmonella spp.in food.Conclusion:The developed multiplex RPA-LFD method provides a new sensitive and rapid alternative for the specific detection of Salmonella spp.and its important serovars in food.
基金financially supported by National Key Research and Development Program of China(2022YFE0101200)National Natural Science Foundation of China(32102850)Shanghai Agricultural Science and Technology Innovation Project(T2023328).
文摘Aeromonas salmonicida is a common pathogen of salmonid fishes that poses a significant threat to the fresh water and marine culture industry,potentially resulting in huge economic losses.To prevent and control fish diseases caused by A.salmonicida,rapid and effective diagnostic approaches must be developed,and which are important for routine monitoring and clinical care.By combining recombinase polymerase amplification(RPA)technology with a visible lateral flow strip(RPA-LF),we have enhanced both the precision of RPA detection and the convenience of real-time monitoring.In this study,we introduce a robust method for detecting A.salmonicida using RPA-LF.This assay specifically targets the ASA_1441 gene of A.salmonicida,ensuring high specificity,without cross-reactivity with other prevalent fresh water or marine pathogens.The optimal amplification temperature of the RPA assay was 39℃.Its sensitivity extends to as low as 100 fg of purified DNA,representing more than 1000-fold higher sensitivity than conventional PCR methods.Furthermore,to enhance the usability of the RPA-LF assay,we developed a rapid sample preparation method using cellulose dipsticks for nucleic acid extraction.This method achieves a limit of detection(LOD)as low as 1.67 CFU/μL and completes the entire process within 20 min.In conclusion,our findings present a rapid and precise tool for monitoring A.salmonicida infection in aquaculture and marine culture.This advancement offers valuable insights for effective disease prevention and control strategies.
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195).
文摘Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are still lacking.This study established a rapid soil DNA extraction(RSDE)method that can be used in field detection.In this method,we first utilized the optimized lysate to isolate DNA from soil and then used a filtration membrane and a DNA adsorption membrane to purify the DNA via the column method.Moreover,we used the pressure from the syringe instead of the conventional centrifugal force of the centrifuge to assist the sample filtration,resulting in very low requirements for this method,with an extraction time of less than 20 min.Furthermore,we demonstrated that the RSDE method was applicable for DNA extraction from different types of soils,with the demand for soil samples as low as 0.1 g and that the amount of obtained DNA was,to some extent,greater than that obtained by a commercial kit.Further analysis revealed that this extracted genomic DNA can be used directly for polymerase chain reaction(PCR)analysis,including ordinary PCR,real-time fluorescent quantitative PCR,and recombinase polymerase amplification(RPA)-CRISPR/Cas12a visual assays.In addition,we demonstrated that this method can be used to extract DNA from residual plant roots in addition to soil microbes,which lays a foundation for the comprehensive analysis of soil plants and microorganisms.In summary,the RSDE method proposed in this study may have wide application prospects.
基金Supported by the Visiting and Training Foundation of Teachers in Ordinary Undergraduate Universities of Shandong Province,the Qingdao Agricultural University Doctoral Start-Up Fund(No.6631122030)the Advanced Talents Foundation of QAU(No.6651118016)+2 种基金the Fish Innovation Team of Shandong Agriculture Research System(No.SDAIT12-06)the Shandong Engineering Research Center for Prevention and Control of Aquatic Animal Disease,the“First Class Fishery Discipline”Program[(2020)3]of Shandong Provincethe Key R&D Program(Soft Science Project)of Shandong Province,China(No.2023 RKY 06004)。
文摘Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease include microscopy examination,quantitative real-time PCR,and loop-mediated isothermal amplification-lateral flow dipstick(LAMP-LFD).In this study,a recombinase polymerase amplification-lateral flow dipstick(RPA-LFD)method,targeting the beta-tubulin gene,was developed to detect G.plecoglossi,three sets of primers and probes were designed and screened,after which the initial reaction system was established.The RPA-LFD method for G.plecoglossi could complete nucleic acid amplification at 39℃ for 10 min,after which the amplification product was dropped on the LFD strip,and the results could then be observed within 5 min.A specificity assay revealed that there was no cross reactivity with other protozoa except G.plecoglossi.A sensitivity assay revealed that the detection limit was 9.38×10^(-6) ng/μL,which was more sensitive than that of conventional PCR.Compared with conventional detection methods,the novel RPA-LFD method has the advantages of simple operation,short operation time,high sensitivity,and high specificity for G.plecoglossi detection,indicating its potential use in rapid field detection of G.plecoglossi.
基金funded by the Seed Industry Vitalization Program of Guangdong Province,grant number 2022-XPY-06–001.
文摘Fowl adenovirus(FAdV)serotype 4,recognized as the causative agent of hydropericardium syndrome(HPS)in chickens,causes substantial economic losses in poultry farming.To develop a simple,rapid,and reliable diagnostic method for the timely detection of FAdV-4 nucleic acid,we integrated the CRISPR/Cas12a system with recombinase-aided amplification(RAA).This approach enables visual detection of FAdV-4 with a sensitivity of one genome copy.The results can be obtained within 40 to 50 min without the need for complex instrumentation,making it ideal for remote field applications.Using this method,we investigated the prevalence of FAdV-4 in both common farm poultry and wild birds.Our results indicated that the FAdV-4-positive rate in wild birds was 51.19%,suggesting that wild birds may serve as specific reservoirs for this virus.In summary,we present a sensitive,swift,accurate,and inexpensive detection method for FAdV-4,along with an investigation of its epidemic situation in birds.Our study advances the detection and epidemiological understanding of FAdV-4 transmission among farm poultry and wild birds.
基金supported by the funding from the National Natural Science Foundation of China(32072359)。
文摘Wheat powdery mildew caused by Blumeria graminis f.sp.tritici(Bgt)is an important disease worldwide.Detection of latent infection of leaves by the pathogen in late autumn is valuable for estimating the inoculum potential to assess disease risks in the spring.We developed a new tool for rapid detection and quantification of latent infection of seedlings by the pathogen.The method was based on recombinase polymerase amplification(RPA)coupled with an end-point detection via lateral flow device(LFD).The limit of detection is 100 agμL^(-1)of Bgt DNA,without noticeable interference from either other common wheat pathogens or wheat material(Triticum aestivum).It was evaluated on wheat seedlings for this accuracy and sensitivity in detecting latent infection of Bgt.We further extended this RPALFD assay to estimate the level of latent infection by Bgt based on imaging analysis.There was a strong correlation between the image-based and real-time PCR assay estimates of Bgt DNA.The present results suggested that this new tool can provide rapid and accurate quantification of Bgt in latently infected leaves and can be further development as an on-site monitoring tool.
