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
Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSP...Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay,named“Dual-RPA-LFD”,to visualize the dual detection of genetically modified(GM)crops.In which,the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits.Gradient diluted plasmids,transgenic standards,and actual samples were used as templates to conduct sensitivity,specificity,and practicality assays,respectively.The constructed method achieved the visual detection of plasmid at levels as low as 100 copies,demonstrating its high sensitivity.In addition,good applicability to transgenic samples was observed,with no cross-interference between two test lines and no influence from other genes.In conclusion,this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37°C in a rapid,equipmentfree field manner,providing a new alternative for rapid screening for transgenic assays in the field.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
Ureaplasma urealyticum(UU),is one of the most vital pathogens causing genitourinary tract infections of the body,and it can result in poor maternal and perinatal outcomes.The aim of this study was to establish a metho...Ureaplasma urealyticum(UU),is one of the most vital pathogens causing genitourinary tract infections of the body,and it can result in poor maternal and perinatal outcomes.The aim of this study was to establish a method to detect Ureaplasma urealyticum based on recombinant polymerase amplification(RPA)technique.Specific primers and probes were designed according to the 16sRNA gene sequence of Ureaplasma urealyticum.Six pathogens were detected for real-time fluorescence RPA specificity verification,including Mycoplasma hominis(MH),Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),Staphylococcus aureus,Escherichia coli,and Lactobacillus vaginalis.The sensitivity of the method was performed by gradient dilution of the extracted template.A total of 60 clinical samples were detected by the established real-time fluorescence RPA.Detection of Ureaplasma urealyticum can be completed within 20 minutes at 39°C using established RPA method.The minimum detection limit of Ureaplasma urealyticum by real-time fluorescence RPA was 3 pg.The evaluation of 60 clinical samples proved that RPA method was feasible.A high specificity,sensitivity,simplicity and rapidity method for Ureaplasma urealyticum detection was successfully established based on the real-time fluorescence RPA method.展开更多
Human bocavirus(HBoV)1 is considered an important pathogen that mainly affects infants aged 6–24 months,but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of ...Human bocavirus(HBoV)1 is considered an important pathogen that mainly affects infants aged 6–24 months,but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging.Herein,we present a novel faster,lower cost,reliable method for the detection of HBoV1,which integrates a recombinase polymerase amplification(RPA)assay with the CRISPR/Cas12a system,designated the RPA-Cas12a-fluorescence assay.The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37℃without the need for sophisticated instruments.The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens.Furthermore,the method was appraised using 28 clinical samples,and displayed high accuracy with positive and negative predictive agreement of 90.9%and 100%,respectively.Therefore,our proposed rapid and sensitive HBoV1 detection method,the RPA-Cas12a-fluorescence assay,shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care.The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection.The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.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
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.
基金supported by the Scientific and Innovative Action Plan of Shanghai(21N31900800)Shanghai Rising-Star Program(23QB1403500)+4 种基金the Shanghai Sailing Program(20YF1443000)Shanghai Science and Technology Commission,the Belt and Road Project(20310750500)Talent Project of SAAS(2023-2025)Runup Plan of SAAS(ZP22211)the SAAS Program for Excellent Research Team(2022(B-16))。
文摘Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient.In this study,a one-tube dual recombinase polymerase amplification(RPA)reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay,named“Dual-RPA-LFD”,to visualize the dual detection of genetically modified(GM)crops.In which,the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits.Gradient diluted plasmids,transgenic standards,and actual samples were used as templates to conduct sensitivity,specificity,and practicality assays,respectively.The constructed method achieved the visual detection of plasmid at levels as low as 100 copies,demonstrating its high sensitivity.In addition,good applicability to transgenic samples was observed,with no cross-interference between two test lines and no influence from other genes.In conclusion,this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37°C in a rapid,equipmentfree field manner,providing a new alternative for rapid screening for transgenic assays in the field.
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.
文摘Ureaplasma urealyticum(UU),is one of the most vital pathogens causing genitourinary tract infections of the body,and it can result in poor maternal and perinatal outcomes.The aim of this study was to establish a method to detect Ureaplasma urealyticum based on recombinant polymerase amplification(RPA)technique.Specific primers and probes were designed according to the 16sRNA gene sequence of Ureaplasma urealyticum.Six pathogens were detected for real-time fluorescence RPA specificity verification,including Mycoplasma hominis(MH),Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),Staphylococcus aureus,Escherichia coli,and Lactobacillus vaginalis.The sensitivity of the method was performed by gradient dilution of the extracted template.A total of 60 clinical samples were detected by the established real-time fluorescence RPA.Detection of Ureaplasma urealyticum can be completed within 20 minutes at 39°C using established RPA method.The minimum detection limit of Ureaplasma urealyticum by real-time fluorescence RPA was 3 pg.The evaluation of 60 clinical samples proved that RPA method was feasible.A high specificity,sensitivity,simplicity and rapidity method for Ureaplasma urealyticum detection was successfully established based on the real-time fluorescence RPA method.
基金Natural Science Foundation of ChinaGrant/Award Number:81973531+9 种基金Science and Technology Plan Project of Xi’anGrant/Award Number:22GXFW0007Shenzhen Science and Technology Innovation CommissionGrant/Award Number:20200812211704001Medical Scientific Research Foundation of Guangdong ProvinceGrant/Award Number:A2019502Nanshan District Science and Technology Plan ProjectGrant/Award Number:NS2022022Scientific Research Program Funded by Shaanxi Provincial Education DepartmentGrant/Award Number:22JC010
文摘Human bocavirus(HBoV)1 is considered an important pathogen that mainly affects infants aged 6–24 months,but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging.Herein,we present a novel faster,lower cost,reliable method for the detection of HBoV1,which integrates a recombinase polymerase amplification(RPA)assay with the CRISPR/Cas12a system,designated the RPA-Cas12a-fluorescence assay.The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37℃without the need for sophisticated instruments.The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens.Furthermore,the method was appraised using 28 clinical samples,and displayed high accuracy with positive and negative predictive agreement of 90.9%and 100%,respectively.Therefore,our proposed rapid and sensitive HBoV1 detection method,the RPA-Cas12a-fluorescence assay,shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care.The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection.The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.
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.
