A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was perfor...A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.展开更多
Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods We designed,developed,and manufactured an integrated disposab...Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods We designed,developed,and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection.The precision of the liquid transfer and temperature control was tested.A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction(RT-PCR).The entire process,from SARS-CoV-2 nucleic acid extraction to amplification,was evaluated.Results The precision of the syringe transfer volume was 19.2±1.9μL(set value was 20),32.2±1.6(set value was 30),and 57.2±3.5(set value was 60).Temperature control in the amplification tube was measured at 60.0±0.0℃(set value was 60)and 95.1±0.2℃(set value was 95)respectively.SARS-Cov-2 nucleic acid extraction yield through the device was 7.10×10^(6) copies/mL,while a commercial kit yielded 2.98×10^(6) copies/mL.The mean time to complete the entire assay,from SARS-CoV-2 nucleic acid extraction to amplification detection,was 36 min and 45 s.The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL.Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test(POCT).展开更多
In this paper,we propose a new algorithm to handle massive data sets,which are modelled by modal regression models.Differing from the existing methods regarding distributed modal regression,the proposed method combine...In this paper,we propose a new algorithm to handle massive data sets,which are modelled by modal regression models.Differing from the existing methods regarding distributed modal regression,the proposed method combines the divide-and-conquer idea and a linear approximation algorithm.It is computationally fast and statistically efficient to implement.Theoretical analysis for the resultant distributed estimator under some regularity conditions is presented.Simulation studies are conducted to assess the effectiveness and flexibility of the proposed method with a finite sample size.Finally,an empirical application to the chemical sensors data is analysed for further illustration.展开更多
Coal/biomass combustion is a major source of submicron particulate matter(sub-PM),with mineral substances in the fuels playing a key role in the formation and growth of these particles.In this study,the temporal evolu...Coal/biomass combustion is a major source of submicron particulate matter(sub-PM),with mineral substances in the fuels playing a key role in the formation and growth of these particles.In this study,the temporal evolution of sub-PM is predicted by simulating coal/biomass combustion under different temperature,atmosphere,species,particle size and density conditions by using nucleation,condensation,coagulation and deposition sub-models.Compared with experimental data,the results show that the amount of sub-PM generated from pulverized coal combustion increases with higher temperatures and oxygen concentrations,and lignin(LN)produces the highest emission of sub-PM among different biomass types.The peak particle size distribution(PSD)of sub-PM across different experimental conditions is mainly centered around 0.1–0.2μm.The values of relative error are below 20%and even below 10%,indicating that the model is in good agreement with the experimental data.Subsequently,the effects of pulverized coal size and coal density on the PSD of sub-PM are predictively simulated by the verified model,the findings indicate that both of the peak PSD are among 0.08–0.23μm,the emission amount of sub-PM negatively relate to coal size and coal density.展开更多
Methotrexate,etoposide,dexamethasone,and pegaspargase(MESA)with sandwiched radiotherapy is known to be effective for early-stage extranodal natural killer/T-cell lymphoma,nasal type(NKTCL).We explored the efficacy and...Methotrexate,etoposide,dexamethasone,and pegaspargase(MESA)with sandwiched radiotherapy is known to be effective for early-stage extranodal natural killer/T-cell lymphoma,nasal type(NKTCL).We explored the efficacy and safety of reduced-intensity,non-intravenous etoposide,dexamethasone,and pegaspargase(ESA)with sandwiched radiotherapy.This multicenter,randomized,phase III trial enrolled patients aged between 14 and 70 years with newly diagnosed early-stage nasal NKTCL from 27 centers in China.Patients were randomly assigned(1:1)to receive ESA(pegaspargase 2,500 IU/m^(2)intramuscularly on day 1,etoposide 200 mg orally,and dexamethasone 40 mg orally on days 2–4)or MESA(methotrexate 1 g/m^(2)intravenously on day 1,etoposide 200 mg orally,and dexamethasone 40 mg orally on days 2–4,and pegaspargase 2,500 IU/m^(2)intramuscularly on day 5)regimen(four cycles),combined with sandwiched radiotherapy.展开更多
Here,we report the identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge,Guyana.In March 2019,14 employees of Chongqing Bosai Mining Company,China,working in a manganese mining of Guya...Here,we report the identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge,Guyana.In March 2019,14 employees of Chongqing Bosai Mining Company,China,working in a manganese mining of Guyana,had unexplained fever,and two of them died.We obtained lung and brain tissues as well as the blood samples from the two deceased cases(patient No.1 and 2),and bronchoscopy lavages and cerebrospinal fluid samples from one severe case(patient No.3),respectively.All samples were tested by pathological examination,high-throughput sequencing,and real-time PCR.Pathological detection showed the presence of spore-like structures in the lung tissue of patient No.1,indicating a fungal infection in this patient.Nanopore sequencing identified the existing of H.capsulatum in the lung tissue sample within 13 h.Next-generation sequencing identified specific fragments of H.capsulatum in all of the samples tested(lung,brain and blood serum from the deceased cases,and plasma from the severe case).Real-time PCR assays did not reveal any viral infection related to transmission from bat feces.We conclude that H.capsulatum was the causative pathogen of this disease cluster based on epidemiologic,clinical,pathological and nucleic acid evidence.展开更多
Introduction:After the epidemic in Wuhan City was brought under control in 2020,local outbreaks of coronavirus disease 2019(COVID-19)in the mainland of China were mainly due to imported COVID-19 cases.The ongoing evol...Introduction:After the epidemic in Wuhan City was brought under control in 2020,local outbreaks of coronavirus disease 2019(COVID-19)in the mainland of China were mainly due to imported COVID-19 cases.The ongoing evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has continued to generate new variants.Some have been designated as variants of concern(VOCs)by the World Health Organization(WHO).To better assess the role of imported SARS-CoV-2 surveillance and the prevalence of VOCs in 2021,the genomic surveillance data of SARS-CoV-2 from imported COVID-19 cases of 2021 in the mainland of China were analyzed.Methods:The analyses included the number of sequence submissions,time of sequence deposition,and time of detection of the VOCs in order to determine the timeliness and sensitivity of the surveillance.The proportions of VOCs were analyzed and compared with data from the Global Initiative of Sharing All Influenza Data(GISAID).Results:A total of 3,355 sequences of imported cases were submitted from 29 provincial-level administrative divisions,with differences in the number of sequence submissions and median time of sequence deposition.A total of 2,388 sequences with more than 90%genomic coverage were used for lineage analysis.The epidemic trend from Alpha to Delta to Omicron in imported cases was consistent with that in the GISAID.In addition,VOCs from imported cases were usually identified after WHO designation and before causing local outbreaks.Conclusions:The global distribution of SARSCoV-2 VOCs changed rapidly in 2021.Robust genomic surveillance of the imported SARS-CoV-2 in the mainland of China is of great significance.Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the etiological agent of coronavirus disease 2019(COVID-19),is constantly mutating under the different circumstances of global transmission(1).The emerging SARS-CoV-2 variants may have potential adverse impacts on epidemic traits and severity.To some extent,it is also capable of escaping natural and vaccine-induced immunity(2–3).Some of them were designated as variants of concern(VOCs)by the World Health Organization(WHO)(4).Therefore,robust surveillance is essential to assess the evolution of viruses in real time.After the epidemic in Wuhan City was brought under control in 2020,several COVID-19 outbreaks in the mainland of China have been proven to relate to SARS-CoV-2 contaminated cold-chain products(5–7),while most were caused by transmission through imported cases on flights,at isolation facilities,or in designated hospitals(8–9).Therefore,genomic surveillance for SARS-CoV-2 from imported cases is of great significance for monitoring the risk of different variants that were imported into the mainland of China,assessing the risk of importation-associated domestic spread,and helping guide public health interventions.On March 17,2020,the China CDC released a notice and launched genomic surveillance for SARS-CoV-2 from imported COVID-19 cases nationwide.The laboratories of provincial CDCs were required to conduct SARS-CoV-2 whole-genome sequencing for samples from imported cases and submit the genomic sequences to the China CDC in time.This study includes the analysis of genomic surveillance data of imported SARS-CoV-2 cases of 2021 from the mainland of China.展开更多
Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real...Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),the gold-standard method,still has shortfalls in diagnostic sensitivity and timeliness.Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay(RT-RAP)to detect HEV fragment within an hour.The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains.Among 15 types of HEV(species A-C),the sensitivity of RT-RAP was approximately 2-8 folds lower than that of the qRT-PCR in 9 types,and no-cross reaction with other viruses was observed.RT-RAP was further applied to analyze CSF and fecal specimens;the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results.These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.展开更多
1.Introduction The first wave of coronavirus disease 2019(COVID-19)outbreak in China's Mainland was declared controlled after April 2020.Since then,the origins of all local outbreaks were imported cases of COVID-1...1.Introduction The first wave of coronavirus disease 2019(COVID-19)outbreak in China's Mainland was declared controlled after April 2020.Since then,the origins of all local outbreaks were imported cases of COVID-19 infection or imported articles contaminated with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)[1],[2],[3].The volume of import and export trade in China is enormous,and the entry of SARS-CoV-2-contaminated articles into China is inevitable.Additionally,SARS-CoV-2 can survive on the surface of noncold-chain products for prolonged periods in cold environments during fall and winter[4],[5],[6].展开更多
The epidemiological characteristics of emerging infectious disease outbreaks in recent years have underscored the critical importance of controlling imported infectious diseases.In this study,we implemented dynamic tr...The epidemiological characteristics of emerging infectious disease outbreaks in recent years have underscored the critical importance of controlling imported infectious diseases.In this study,we implemented dynamic tracking of microbial invasions by monitoring environmental microbes at the customs and ports.From July to September 2024,a total of 126 environmental samples were collected from three ports of entry in Shenzhen,China.Metagenomic analysis detected 55 non-viral microbial communities and 12 viral taxa.Among these,26.8%of the bacteria,100%of the fungi,71.4%of the protists,and none of the archaea exhibited potential pathogenic properties.Viruses were the most prevalent,including bacteriophages(100%),unclassified viruses(96.8%),giant viruses(27.8%),fungal viruses(4.8%),and vertebrate viruses(1.6%).No statistical differences were observed in viral distribution across areas(χ^(2)=18.70,P=0.541),sites(χ^(2)=14.02,P=0.597),or ports of entry(χ^(2)=10.27,P=0.247).However,viral distribution varied significantly across three sampling months(χ^(2)=21.06,P=0.002),with a higher proportion of giant viruses detected in July.Thirty-nine and forty microorganisms were identified across the six areas and five sites,respectively,with relatively few area/site-specific microorganisms.Four distinct disinfection level zones were categorized:relatively safe zone,less safe zone,general disinfection zone and key disinfection zone.Two strains of viruses with potential pathogenicity were identified:pigeon circovirus and Influenza A virus(H4N2).This study established a metagenomics-based surveillance framework for microbial risk assessment in high-risk port environments and proposed a four-tier disinfection strategy to prioritize high-contact zones.Our findings highlighted environmental metagenomics as a critical complement to traveler screening and provided early warning signals for the prevention and control of imported infectious diseases.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
Emerging and re-emerging pathogens are great challenges to the public health(1).A cluster of pneumonia cases with an unknown cause occurred in Wuhan starting on December 21,2019.As of January 20,2020,a total of 201 ca...Emerging and re-emerging pathogens are great challenges to the public health(1).A cluster of pneumonia cases with an unknown cause occurred in Wuhan starting on December 21,2019.As of January 20,2020,a total of 201 cases of pneumonia in China have been confirmed.A team of professionals from the National Health Commission and China CDC conducted epidemiological and etiological investigations.On January 3,2020,the first complete genome of the novelβgenus coronaviruses(2019-nCoVs)was identified in samples of bronchoalveolar lavage fluid(BALF)from a patient from Wuhan by scientists of the National Institute of Viral Disease Control and Prevention(IVDC)through a combination of Sanger sequencing,Illumina sequencing,and nanopore sequencing.Three distinct strains have been identified,the virus has been designated as 2019-nCoV,and the disease has been subsequently named novel coronavirus-infected pneumonia(NCIP).展开更多
Dear Editor,Polymerase chain reaction(PCR)is a molecular diagnostic technique that has been widely used for diagnosing viral diseases.Nested PCR(N-PCR)is a variation on the standard PCR technique that involves two amp...Dear Editor,Polymerase chain reaction(PCR)is a molecular diagnostic technique that has been widely used for diagnosing viral diseases.Nested PCR(N-PCR)is a variation on the standard PCR technique that involves two amplification steps,yielding greater sensitivity and specificity.But there are two main disadvantages:the protocol is more complex than conventional PCR,and the risk of cross-contamination is relatively high.One-step nested PCR(OSN-PCR)enables conventional N-PCR to be performed as a closed reaction in a single tube that contains both outer and inner primers.This strategy makes OSN-PCR more rapid than N-PCR,lowers the probability of cross-contamination,and requires a smaller amount of reagents.展开更多
In 2008,China launched a national surveillance system for hand‐foot‐and‐mouth disease(HFMD).Several million cases of HFMD are reported every year,coxsackievirus A16(CVA16)was the leading cause of HFMD epidemic in Y...In 2008,China launched a national surveillance system for hand‐foot‐and‐mouth disease(HFMD).Several million cases of HFMD are reported every year,coxsackievirus A16(CVA16)was the leading cause of HFMD epidemic in Yantai city,China in recent years,but the information of epidemiology and molecular characterization of CVA16 in Yantai is limited.The aim of this study is to investigate the epidemiological characteristics and pathogenic spectrum of HFMD,and most importantly,the molecular characterization of CVA16 in Yantai from 2018 to 2021.A total of 2,000 clinical samples were collected in Yantai city from 2018 to 2021 and the enterovirus typing was performed using real‐time reverse transcriptase–polymerase chain reaction(qRT‐PCR).VP1 coding regions of 41 CVA16 isolates were amplified and Sanger sequenced,and phylogenetic analysis was performed.During the study period,HFMD became prevalent from May to August each year.It peaked in June and declined in September.The incidence was highest in children aged 1 to 5 years,while more common in males than females.1,617 out of 2,000 clinical collection of samples were tested positive for enterovirus.Among them,614 were identified as CVA16,45 were enterovirus A71(EV A17),and 958 were other enterovirus serotypes.All 41 CVA16 strains belonged to the Bla and B1b genotypes.Homology analysis showed that 41 CVA16 isolates shared 83.2%–100%nucleotide and 93.7%–100%amino acid similarity among themselves.The results of this study update molecular epidemiology of CVA16 and provide a reference for HFMD prevention and control.展开更多
Introduction:The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron variant is the dominant circulating strain worldwide.To assess the importation of SARS-CoV-2 variants in the mainland of China during...Introduction:The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron variant is the dominant circulating strain worldwide.To assess the importation of SARS-CoV-2 variants in the mainland of China during the Omicron epidemic,the genomic surveillance data of SARS-CoV-2 from imported coronavirus disease 2019(COVID-19)cases in the mainland of China during the first half of 2022 were analyzed.Methods:Sequences submitted from January to July 2022,with a collection date before June 30,2022,were incorporated.The proportions of SARS-CoV-2 variants as well as the relationships between the origin and destination of each Omicron imported case were analyzed.Results:4,946 sequences of imported cases were submitted from 27 provincial-level administrative divisions(PLADs),and the median submission interval was within 1 month after collection.In 3,851 Omicron sequences with good quality,1 recombinant(XU)and 4 subvariants under monitoring(BA.4,BA.5,BA.2.12.1,and BA.2.13)were recorded,and 3 of them(BA.4,BA.5,and BA.2.12.1)caused local transmissions in the mainland of China later than that recorded in the surveillance.Omicron subvariants dominated in the first half of 2022 and shifted from BA.1 to BA.2 then to BA.4 and BA.5.The percentage of BA.2 in the imported SARS-CoV-2 surveillance data was far higher than that in the Global Initiative on Sharing All Influenza Data(GISAID).The imported cases from Hong Kong Special Administrative Region,China,accounted for 32.30%of Omicron cases sampled,and 98.71%of them were BA.2.Conclusions:The Omicron variant showed the intra-Omicron evolution in the first half of 2022,and all of the Omicron subvariants were introduced into the mainland of China multiple times from multiple different locations.展开更多
Introduction:Recently,a local cluster epidemic has occurred in Shijiazhuang City,Hebei Province.Failure to promptly identify patients with fever in rural areas was the major reason for this epidemic.Methods:We present...Introduction:Recently,a local cluster epidemic has occurred in Shijiazhuang City,Hebei Province.Failure to promptly identify patients with fever in rural areas was the major reason for this epidemic.Methods:We presented the field evaluation of a new real-time reverse transcription recombinase-aided amplification(RT-RAA)kit incorporating an endogenous internal control in a single-tube format,completed at the Hebei CDC from January 17,2021 to January 27,2021.Results:We evaluated the diagnostic performance of RT-RAA assay using automatic extracted RNA of 808 clinical samples.Compared with reverse transcriptase real-time quantitative PCR(qRT-PCR),RT-RAA kit achieved 92.41%sensitivity,98.78%specificity and a 96.29%coincidence rate,demonstrating an excellent agreement between the RTRAA assay and qRT-PCR assay.Furthermore,58 samples were extracted using a manual extraction method within 5 minutes,but only samples with high nucleic acid concentration(cycle threshold value not higher than 32)could be stably detected.Discussion:The RT-RAA is more suitable to meet the needs of rapid,sensitive,and accurate detection in community-level medical institutions.展开更多
Background:Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification(RAA)assay were studied for the first time,and amplification of a long-fragment(509 bp)was initially explored.Method...Background:Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification(RAA)assay were studied for the first time,and amplification of a long-fragment(509 bp)was initially explored.Methods:Using recombinant plasmids and clinical samples,RAA fluorescence and basic methods were used to evaluate the efficacy.The fluorescence method was evaluated by threshold time and fluorescence value,and the basic method was characterized by 2%agarose gel electrophoresis.Results:Taking a previously established RAA assay for HPV18 as an example,we demonstrated that the addition of 0.2 M,0.4 M,and 0.6 M betaine and 10%pullulan could enhance the RAA.The new RAA assays with betaine and pullulan were named B-RAA and P-RAA,respectively.Using the B-RAA and P-RAA fluorescence methods,the threshold time values could be shortened by 1.72-2.32 minutes and 2.60 minutes,respectively,and the fluorescence values could be enhanced by 8847.25-9094.37 mv and 5250 mv,respectively.Using the basic method,the sensitivity could be increased 10-fold.We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 102 copies/μL(compared with 103 copies/μL in the RAA assay).Conclusions:Thus,we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.展开更多
ABSTRACT Background:COVID-19 infection is a major public health problem worldwide,and the D614G mutation enhances the infectivity of COVID-19.Methods:A probe-directed recombinase amplification(PDRA)assay was discussed...ABSTRACT Background:COVID-19 infection is a major public health problem worldwide,and the D614G mutation enhances the infectivity of COVID-19.Methods:A probe-directed recombinase amplification(PDRA)assay was discussed to detect the D614G mutation at 39℃for 30 min.The sensitivity,specificity,and reproducibility of the PDRA were evaluated by D614 and G614 recombinant plasmids.The clinical performance of PDRA assay was validated by testing of 53 previously confirmed COVID-19 positive RNAs and 10 negative samples.Direct sequencing was carried out in parallel for comparison.Result:With good reproducibility and specificity,the PDRA assay worked well with the concentration in the range of 103–107 copies/reaction.Compared with direct sequencing as a reference,the recombinase-aided amplification(RAA)assay obtained 100%sensitivity and 100%specificity using clinical samples.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
文摘A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for human enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) infection was further evaluated. The one step reaction was performed in a single tube at 65?C for 45 min for EV71 and 35 min for CVA16. The detection limits of RT-LAMP assays for both EV71 and CVA16 were 0.1 of a 50% tissue culture infective dose (TCID50) per reaction, based on 10—Fold dilutions of a titrated EV71 or CVA16 strain. The specific assay showed there were no cross-reactions with Coxsackievirus A (CVA) viruses (CVA 2, 4, 5, 7, 9, 10, 14, and 25), Coxsackievirus B (CVB) viruses (CVB 1, 2, 3, 4, and 5) or ECHO viruses (ECHO 3, 6, 11, and 19). In parallel with commercial quantitative real-time polymerase chain reaction (qRT-PCR) diagnostic kits for EV71 and CVA16, the RT-LAMP assay was evaluated with 515 clinical specimens, the results showed the RT-LAMP assay and the qRT-PCR assay were in complete agreement for 513/515 (99.6%) of the specimens. Two samples with discrepant results from two methods were further verified by nested reverse transcription polymerase chain reaction (nRT-PCR) assay and sequencing to be true positives for CVA16. In conclusion, RT-LAMP assay is demonstrated to be a sensitive and specific assay and have a great potential for the rapid and visual screening of EV71 and CVA16 in China, especially in those resource-limited hospitals and rural clinics of provincial and municipal regions.
基金supported by National Key R&D Program of China[2021YFC2301103 and 2022YFE0202600]Shenzhen Science and Technology Program[JSGG20220606142605011].
文摘Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods We designed,developed,and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection.The precision of the liquid transfer and temperature control was tested.A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction(RT-PCR).The entire process,from SARS-CoV-2 nucleic acid extraction to amplification,was evaluated.Results The precision of the syringe transfer volume was 19.2±1.9μL(set value was 20),32.2±1.6(set value was 30),and 57.2±3.5(set value was 60).Temperature control in the amplification tube was measured at 60.0±0.0℃(set value was 60)and 95.1±0.2℃(set value was 95)respectively.SARS-Cov-2 nucleic acid extraction yield through the device was 7.10×10^(6) copies/mL,while a commercial kit yielded 2.98×10^(6) copies/mL.The mean time to complete the entire assay,from SARS-CoV-2 nucleic acid extraction to amplification detection,was 36 min and 45 s.The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL.Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test(POCT).
基金supported by the National Natural Science Foundation of China(grant number 12101439)supported by Fundamental Research Funds for the Central Universities[grant number 2021CDJQY-047]National Natural Science Foundation of China[grant number 11801202].
文摘In this paper,we propose a new algorithm to handle massive data sets,which are modelled by modal regression models.Differing from the existing methods regarding distributed modal regression,the proposed method combines the divide-and-conquer idea and a linear approximation algorithm.It is computationally fast and statistically efficient to implement.Theoretical analysis for the resultant distributed estimator under some regularity conditions is presented.Simulation studies are conducted to assess the effectiveness and flexibility of the proposed method with a finite sample size.Finally,an empirical application to the chemical sensors data is analysed for further illustration.
基金supported by the National Natural Science Foundation of China(grant No.51376063).
文摘Coal/biomass combustion is a major source of submicron particulate matter(sub-PM),with mineral substances in the fuels playing a key role in the formation and growth of these particles.In this study,the temporal evolution of sub-PM is predicted by simulating coal/biomass combustion under different temperature,atmosphere,species,particle size and density conditions by using nucleation,condensation,coagulation and deposition sub-models.Compared with experimental data,the results show that the amount of sub-PM generated from pulverized coal combustion increases with higher temperatures and oxygen concentrations,and lignin(LN)produces the highest emission of sub-PM among different biomass types.The peak particle size distribution(PSD)of sub-PM across different experimental conditions is mainly centered around 0.1–0.2μm.The values of relative error are below 20%and even below 10%,indicating that the model is in good agreement with the experimental data.Subsequently,the effects of pulverized coal size and coal density on the PSD of sub-PM are predictively simulated by the verified model,the findings indicate that both of the peak PSD are among 0.08–0.23μm,the emission amount of sub-PM negatively relate to coal size and coal density.
基金supported by the Union for China Lymphoma Investigators,and funded by the Multicenter Clinical Research Project by Shanghai Jiao Tong University School of Medicine(DLY201601)the National Key R&D Program of China(2022YFC 2502600)+3 种基金the National Natural Science Foundation of China(82130004,81830007,82070204,and 81670176)Chang Jiang Scholars Program,Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support(20152206 and 20152208)the Clinical Research Plan of Shanghai Hospital Development Center(SHDC2020CR1032B)the Collaborative Innovation Center of Systems Biomedicine,and the Samuel Waxman Cancer Research Foundation.
文摘Methotrexate,etoposide,dexamethasone,and pegaspargase(MESA)with sandwiched radiotherapy is known to be effective for early-stage extranodal natural killer/T-cell lymphoma,nasal type(NKTCL).We explored the efficacy and safety of reduced-intensity,non-intravenous etoposide,dexamethasone,and pegaspargase(ESA)with sandwiched radiotherapy.This multicenter,randomized,phase III trial enrolled patients aged between 14 and 70 years with newly diagnosed early-stage nasal NKTCL from 27 centers in China.Patients were randomly assigned(1:1)to receive ESA(pegaspargase 2,500 IU/m^(2)intramuscularly on day 1,etoposide 200 mg orally,and dexamethasone 40 mg orally on days 2–4)or MESA(methotrexate 1 g/m^(2)intravenously on day 1,etoposide 200 mg orally,and dexamethasone 40 mg orally on days 2–4,and pegaspargase 2,500 IU/m^(2)intramuscularly on day 5)regimen(four cycles),combined with sandwiched radiotherapy.
基金This work was supported by grants from the China MegaProjects for Infectious Disease(2018ZX10713-002,2018ZX10711001,2017ZX10104001,and 2017ZX10302301-004-002).
文摘Here,we report the identification of Histoplasma causing an unexplained disease cluster in Matthews Ridge,Guyana.In March 2019,14 employees of Chongqing Bosai Mining Company,China,working in a manganese mining of Guyana,had unexplained fever,and two of them died.We obtained lung and brain tissues as well as the blood samples from the two deceased cases(patient No.1 and 2),and bronchoscopy lavages and cerebrospinal fluid samples from one severe case(patient No.3),respectively.All samples were tested by pathological examination,high-throughput sequencing,and real-time PCR.Pathological detection showed the presence of spore-like structures in the lung tissue of patient No.1,indicating a fungal infection in this patient.Nanopore sequencing identified the existing of H.capsulatum in the lung tissue sample within 13 h.Next-generation sequencing identified specific fragments of H.capsulatum in all of the samples tested(lung,brain and blood serum from the deceased cases,and plasma from the severe case).Real-time PCR assays did not reveal any viral infection related to transmission from bat feces.We conclude that H.capsulatum was the causative pathogen of this disease cluster based on epidemiologic,clinical,pathological and nucleic acid evidence.
基金National Key Research and Development Program of China(2021YFC0863000).
文摘Introduction:After the epidemic in Wuhan City was brought under control in 2020,local outbreaks of coronavirus disease 2019(COVID-19)in the mainland of China were mainly due to imported COVID-19 cases.The ongoing evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has continued to generate new variants.Some have been designated as variants of concern(VOCs)by the World Health Organization(WHO).To better assess the role of imported SARS-CoV-2 surveillance and the prevalence of VOCs in 2021,the genomic surveillance data of SARS-CoV-2 from imported COVID-19 cases of 2021 in the mainland of China were analyzed.Methods:The analyses included the number of sequence submissions,time of sequence deposition,and time of detection of the VOCs in order to determine the timeliness and sensitivity of the surveillance.The proportions of VOCs were analyzed and compared with data from the Global Initiative of Sharing All Influenza Data(GISAID).Results:A total of 3,355 sequences of imported cases were submitted from 29 provincial-level administrative divisions,with differences in the number of sequence submissions and median time of sequence deposition.A total of 2,388 sequences with more than 90%genomic coverage were used for lineage analysis.The epidemic trend from Alpha to Delta to Omicron in imported cases was consistent with that in the GISAID.In addition,VOCs from imported cases were usually identified after WHO designation and before causing local outbreaks.Conclusions:The global distribution of SARSCoV-2 VOCs changed rapidly in 2021.Robust genomic surveillance of the imported SARS-CoV-2 in the mainland of China is of great significance.Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the etiological agent of coronavirus disease 2019(COVID-19),is constantly mutating under the different circumstances of global transmission(1).The emerging SARS-CoV-2 variants may have potential adverse impacts on epidemic traits and severity.To some extent,it is also capable of escaping natural and vaccine-induced immunity(2–3).Some of them were designated as variants of concern(VOCs)by the World Health Organization(WHO)(4).Therefore,robust surveillance is essential to assess the evolution of viruses in real time.After the epidemic in Wuhan City was brought under control in 2020,several COVID-19 outbreaks in the mainland of China have been proven to relate to SARS-CoV-2 contaminated cold-chain products(5–7),while most were caused by transmission through imported cases on flights,at isolation facilities,or in designated hospitals(8–9).Therefore,genomic surveillance for SARS-CoV-2 from imported cases is of great significance for monitoring the risk of different variants that were imported into the mainland of China,assessing the risk of importation-associated domestic spread,and helping guide public health interventions.On March 17,2020,the China CDC released a notice and launched genomic surveillance for SARS-CoV-2 from imported COVID-19 cases nationwide.The laboratories of provincial CDCs were required to conduct SARS-CoV-2 whole-genome sequencing for samples from imported cases and submit the genomic sequences to the China CDC in time.This study includes the analysis of genomic surveillance data of imported SARS-CoV-2 cases of 2021 from the mainland of China.
基金This work was supported by the National Key R&D Program of China(2021YFC2301102)National Natural Science Foundation of China(82202593)the Central Guidance on Local Science and Technology Development Fund of Hebei Province(216Z7713G).
文摘Human enteroviruses(HEVs)include many different types that cause a wide range of diseases,and an effective method of genus-level identification has therefore significant clinical implications.However,quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR),the gold-standard method,still has shortfalls in diagnostic sensitivity and timeliness.Here we established a one-step real-time reverse-transcription recombinase-aided PCR assay(RT-RAP)to detect HEV fragment within an hour.The RT-RAP assay showed a detection limit of 5 copies/μL using recombinant plasmids and was extensively verified using 15 HEV strains.Among 15 types of HEV(species A-C),the sensitivity of RT-RAP was approximately 2-8 folds lower than that of the qRT-PCR in 9 types,and no-cross reaction with other viruses was observed.RT-RAP was further applied to analyze CSF and fecal specimens;the clinical performance demonstrated that the RT-RAP and the commercial qRT-PCR kit provided consistent results.These results indicated that RT-RAP assay may be a promising approach for rapid and sensitive detection of HEV.
基金This work was supported by the National Natural Science Foundation of China(42177418)the National Key Research and Development Program of China(2021YFC0863000,2021YFC2301100).
文摘1.Introduction The first wave of coronavirus disease 2019(COVID-19)outbreak in China's Mainland was declared controlled after April 2020.Since then,the origins of all local outbreaks were imported cases of COVID-19 infection or imported articles contaminated with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)[1],[2],[3].The volume of import and export trade in China is enormous,and the entry of SARS-CoV-2-contaminated articles into China is inevitable.Additionally,SARS-CoV-2 can survive on the surface of noncold-chain products for prolonged periods in cold environments during fall and winter[4],[5],[6].
基金supported by the National Key R&D Program of China(2023YFC2605300&2024YFC2311500).
文摘The epidemiological characteristics of emerging infectious disease outbreaks in recent years have underscored the critical importance of controlling imported infectious diseases.In this study,we implemented dynamic tracking of microbial invasions by monitoring environmental microbes at the customs and ports.From July to September 2024,a total of 126 environmental samples were collected from three ports of entry in Shenzhen,China.Metagenomic analysis detected 55 non-viral microbial communities and 12 viral taxa.Among these,26.8%of the bacteria,100%of the fungi,71.4%of the protists,and none of the archaea exhibited potential pathogenic properties.Viruses were the most prevalent,including bacteriophages(100%),unclassified viruses(96.8%),giant viruses(27.8%),fungal viruses(4.8%),and vertebrate viruses(1.6%).No statistical differences were observed in viral distribution across areas(χ^(2)=18.70,P=0.541),sites(χ^(2)=14.02,P=0.597),or ports of entry(χ^(2)=10.27,P=0.247).However,viral distribution varied significantly across three sampling months(χ^(2)=21.06,P=0.002),with a higher proportion of giant viruses detected in July.Thirty-nine and forty microorganisms were identified across the six areas and five sites,respectively,with relatively few area/site-specific microorganisms.Four distinct disinfection level zones were categorized:relatively safe zone,less safe zone,general disinfection zone and key disinfection zone.Two strains of viruses with potential pathogenicity were identified:pigeon circovirus and Influenza A virus(H4N2).This study established a metagenomics-based surveillance framework for microbial risk assessment in high-risk port environments and proposed a four-tier disinfection strategy to prioritize high-contact zones.Our findings highlighted environmental metagenomics as a critical complement to traveler screening and provided early warning signals for the prevention and control of imported infectious diseases.
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.
文摘Emerging and re-emerging pathogens are great challenges to the public health(1).A cluster of pneumonia cases with an unknown cause occurred in Wuhan starting on December 21,2019.As of January 20,2020,a total of 201 cases of pneumonia in China have been confirmed.A team of professionals from the National Health Commission and China CDC conducted epidemiological and etiological investigations.On January 3,2020,the first complete genome of the novelβgenus coronaviruses(2019-nCoVs)was identified in samples of bronchoalveolar lavage fluid(BALF)from a patient from Wuhan by scientists of the National Institute of Viral Disease Control and Prevention(IVDC)through a combination of Sanger sequencing,Illumina sequencing,and nanopore sequencing.Three distinct strains have been identified,the virus has been designated as 2019-nCoV,and the disease has been subsequently named novel coronavirus-infected pneumonia(NCIP).
基金supported by the National Key Research and Development Plan (2016TFC1202700, 2016YFC1200900)Beijing Municipal Science & Technology Commission Project (D151100002115003)Guangzhou Municipal Science & Technology Commission project (2015B2150820)
文摘Dear Editor,Polymerase chain reaction(PCR)is a molecular diagnostic technique that has been widely used for diagnosing viral diseases.Nested PCR(N-PCR)is a variation on the standard PCR technique that involves two amplification steps,yielding greater sensitivity and specificity.But there are two main disadvantages:the protocol is more complex than conventional PCR,and the risk of cross-contamination is relatively high.One-step nested PCR(OSN-PCR)enables conventional N-PCR to be performed as a closed reaction in a single tube that contains both outer and inner primers.This strategy makes OSN-PCR more rapid than N-PCR,lowers the probability of cross-contamination,and requires a smaller amount of reagents.
基金supported by Shandong Provincial Preventive Medicine Association Project(LYH 2017‐26).
文摘In 2008,China launched a national surveillance system for hand‐foot‐and‐mouth disease(HFMD).Several million cases of HFMD are reported every year,coxsackievirus A16(CVA16)was the leading cause of HFMD epidemic in Yantai city,China in recent years,but the information of epidemiology and molecular characterization of CVA16 in Yantai is limited.The aim of this study is to investigate the epidemiological characteristics and pathogenic spectrum of HFMD,and most importantly,the molecular characterization of CVA16 in Yantai from 2018 to 2021.A total of 2,000 clinical samples were collected in Yantai city from 2018 to 2021 and the enterovirus typing was performed using real‐time reverse transcriptase–polymerase chain reaction(qRT‐PCR).VP1 coding regions of 41 CVA16 isolates were amplified and Sanger sequenced,and phylogenetic analysis was performed.During the study period,HFMD became prevalent from May to August each year.It peaked in June and declined in September.The incidence was highest in children aged 1 to 5 years,while more common in males than females.1,617 out of 2,000 clinical collection of samples were tested positive for enterovirus.Among them,614 were identified as CVA16,45 were enterovirus A71(EV A17),and 958 were other enterovirus serotypes.All 41 CVA16 strains belonged to the Bla and B1b genotypes.Homology analysis showed that 41 CVA16 isolates shared 83.2%–100%nucleotide and 93.7%–100%amino acid similarity among themselves.The results of this study update molecular epidemiology of CVA16 and provide a reference for HFMD prevention and control.
文摘Introduction:The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron variant is the dominant circulating strain worldwide.To assess the importation of SARS-CoV-2 variants in the mainland of China during the Omicron epidemic,the genomic surveillance data of SARS-CoV-2 from imported coronavirus disease 2019(COVID-19)cases in the mainland of China during the first half of 2022 were analyzed.Methods:Sequences submitted from January to July 2022,with a collection date before June 30,2022,were incorporated.The proportions of SARS-CoV-2 variants as well as the relationships between the origin and destination of each Omicron imported case were analyzed.Results:4,946 sequences of imported cases were submitted from 27 provincial-level administrative divisions(PLADs),and the median submission interval was within 1 month after collection.In 3,851 Omicron sequences with good quality,1 recombinant(XU)and 4 subvariants under monitoring(BA.4,BA.5,BA.2.12.1,and BA.2.13)were recorded,and 3 of them(BA.4,BA.5,and BA.2.12.1)caused local transmissions in the mainland of China later than that recorded in the surveillance.Omicron subvariants dominated in the first half of 2022 and shifted from BA.1 to BA.2 then to BA.4 and BA.5.The percentage of BA.2 in the imported SARS-CoV-2 surveillance data was far higher than that in the Global Initiative on Sharing All Influenza Data(GISAID).The imported cases from Hong Kong Special Administrative Region,China,accounted for 32.30%of Omicron cases sampled,and 98.71%of them were BA.2.Conclusions:The Omicron variant showed the intra-Omicron evolution in the first half of 2022,and all of the Omicron subvariants were introduced into the mainland of China multiple times from multiple different locations.
基金Supported by grants from National institute for viral disease control and prevention,China CDC(2019HYDQNJJ03)the key R&D projects in Zibo City(2020kj100011).
文摘Introduction:Recently,a local cluster epidemic has occurred in Shijiazhuang City,Hebei Province.Failure to promptly identify patients with fever in rural areas was the major reason for this epidemic.Methods:We presented the field evaluation of a new real-time reverse transcription recombinase-aided amplification(RT-RAA)kit incorporating an endogenous internal control in a single-tube format,completed at the Hebei CDC from January 17,2021 to January 27,2021.Results:We evaluated the diagnostic performance of RT-RAA assay using automatic extracted RNA of 808 clinical samples.Compared with reverse transcriptase real-time quantitative PCR(qRT-PCR),RT-RAA kit achieved 92.41%sensitivity,98.78%specificity and a 96.29%coincidence rate,demonstrating an excellent agreement between the RTRAA assay and qRT-PCR assay.Furthermore,58 samples were extracted using a manual extraction method within 5 minutes,but only samples with high nucleic acid concentration(cycle threshold value not higher than 32)could be stably detected.Discussion:The RT-RAA is more suitable to meet the needs of rapid,sensitive,and accurate detection in community-level medical institutions.
基金supported by grants from the Youth Foundation of Academician Hou Yunde[grant number 2019HYDQNJJ03]China Mega-Projects for Infec-tious Disease[grant number 2017ZX10302301-004-002].
文摘Background:Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification(RAA)assay were studied for the first time,and amplification of a long-fragment(509 bp)was initially explored.Methods:Using recombinant plasmids and clinical samples,RAA fluorescence and basic methods were used to evaluate the efficacy.The fluorescence method was evaluated by threshold time and fluorescence value,and the basic method was characterized by 2%agarose gel electrophoresis.Results:Taking a previously established RAA assay for HPV18 as an example,we demonstrated that the addition of 0.2 M,0.4 M,and 0.6 M betaine and 10%pullulan could enhance the RAA.The new RAA assays with betaine and pullulan were named B-RAA and P-RAA,respectively.Using the B-RAA and P-RAA fluorescence methods,the threshold time values could be shortened by 1.72-2.32 minutes and 2.60 minutes,respectively,and the fluorescence values could be enhanced by 8847.25-9094.37 mv and 5250 mv,respectively.Using the basic method,the sensitivity could be increased 10-fold.We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 102 copies/μL(compared with 103 copies/μL in the RAA assay).Conclusions:Thus,we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.
文摘ABSTRACT Background:COVID-19 infection is a major public health problem worldwide,and the D614G mutation enhances the infectivity of COVID-19.Methods:A probe-directed recombinase amplification(PDRA)assay was discussed to detect the D614G mutation at 39℃for 30 min.The sensitivity,specificity,and reproducibility of the PDRA were evaluated by D614 and G614 recombinant plasmids.The clinical performance of PDRA assay was validated by testing of 53 previously confirmed COVID-19 positive RNAs and 10 negative samples.Direct sequencing was carried out in parallel for comparison.Result:With good reproducibility and specificity,the PDRA assay worked well with the concentration in the range of 103–107 copies/reaction.Compared with direct sequencing as a reference,the recombinase-aided amplification(RAA)assay obtained 100%sensitivity and 100%specificity using clinical samples.
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.
