Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three t...Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.展开更多
A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in...A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 ~C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV.展开更多
Objective Tick-borne encephalitis virus(TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis(TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease....Objective Tick-borne encephalitis virus(TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis(TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. Methods A reverse-transcription recombinase-aided amplification(RT-RAA) assay was developed. This assay can be completed in one closed tube at 39℃ within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type(WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. Results The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units(pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. Conclusion A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.展开更多
Objective To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use o...Objective To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.展开更多
Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the ep...Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT). Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR). Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies展开更多
West Nile virus(WNV)causes West Nile fever and West Nile encephalitis.Because infection by WNV creates serious public health problems,its simple,rapid,and visual detection is very important in clinical practice,especi...West Nile virus(WNV)causes West Nile fever and West Nile encephalitis.Because infection by WNV creates serious public health problems,its simple,rapid,and visual detection is very important in clinical practice,especially in resource-limited laboratories.We have developed a rapid,specific,and highly sensitive internally controlled reverse transcription recombinase-aided amplification(RTRAA)assay to detect WNV,using both real-timefluoresce nee and the lateral flow dipstick(LFD)at39.0°C for 30 min.The analytical sensitivity of theRT-RAA assay was 10 plasmid copies and 1.6 pfu perreacti on with real-time fluoresce nee,and 1,000plasmid copies per reaction with the LFD.No crossreactionwith other control viruses was observed.Compared with the RT-qPCR assay,the RT-RAA assaydemonstrated 100%sensitivity and 100%specificityfor WNV.展开更多
Objective People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus(YFV) is the only pathogen under surveillance in Sierra Leone owing to the...Objective People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus(YFV) is the only pathogen under surveillance in Sierra Leone owing to the undeveloped medical and public health system there. Most of the results of YFV identification are negative. Elucidation of the pathogen spectrum is required to reduce the prevalence of febrile jaundice. Methods In the present study, we used Ion Torrent semiconductor sequencing to profile the pathogen spectrum in archived YFV‐negative sera from 96 patients in Sierra Leone who presented with unexplained febrile jaundice. Results The most frequently identified sequencing reads belonged to the following pathogens: cytomegalovirus(89.58%), Epstein‐Barr virus(55.21%), hepatitis C virus(34.38%), rhinovirus(28.13%), hepatitis A virus(20.83%), coxsackievirus(10.42%), Ebola virus(8.33%), hepatitis E virus(8.33%), lyssavirus(4.17%), leptospirosis(4.17%), chikungunya virus(2.08%), Crimean‐Congo hemorrhagic fever virus(1.04%), and hepatitis B virus(1.04%). Conclusion The distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.展开更多
Epstein-Barr virus(EBV)and cytomegalovirus(CMV),two of the most prevalent human herpesviruses,cause a wide spectrum of diseases and symptoms and are associated with serious health problem.In this study,we developed an...Epstein-Barr virus(EBV)and cytomegalovirus(CMV),two of the most prevalent human herpesviruses,cause a wide spectrum of diseases and symptoms and are associated with serious health problem.In this study,we developed an internal control reference recombinase-aided amplification(ICR-RAA)assay for the rapid detection of EBV and CMV within 30 min.The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV,respectively,with no cross reaction with other pathogens.In comparison with those of the commercial quantitative polymerase chain reaction(qPCR),the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33%and 84.84%,respectively;the specificity was 98.75%and 100.00%,respectively;and the Kappa values were 0.930 and 0.892(P<0.05),respectively.In comparison with those of qPCR,the sensitivity of the EBV and CMV ICR-RAAs using the DNA by thermal lysis was 72.22%and 80.00%,respectively;the specificity was 100.00%。展开更多
Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherich...Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella, and 5higella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2). Methods A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System {Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9x102 CFU/mLfor S. aureus, 4.4x102 CFU/mL for L. monocytogenes, 3.0x102 CFU/mL for Salmonella, 2.5x102 CFU/mL for Shigella, 2.1x102 CFU/mL for V. parahaemolyticus, and 1.2x102 CFU/mL for E. coll. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10s CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).展开更多
Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly ...Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.展开更多
Objective Unbiased next generation sequencing(NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clin...Objective Unbiased next generation sequencing(NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. Methods A viral Sequence Independent Targeted Amplification(VSITA) approach using a set of non-ribosomal and virus-enriched octamers(V8) was developed and compared with traditionally used random hexamers(N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR(qP CR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. Results A minimum 30 hexamers matching to viral reference sequences(sense and antisense) were selected from a dataset of random 4,096(4~6) hexamers(N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480(30 × 4~2) octamers(V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted c DNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. Conclusion The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.展开更多
基金funded by the National Key R&D Program of China[2021YFC2301102]National Natural Science Foundation of China[82202593]Key R&D Program of Hebei Province[223777100D].
文摘Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.
基金supported by the Scientific Research Project of General Administration of Quality Supervision,Inspection and Quarantine of P.R.C(2011IK132)the China Mega-Project for Infectious Disease(2013ZX10004-001)
文摘A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 ~C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV.
基金supported by the National key research and development project [2017YFC1200505]the National Science and Technology Major Project of China [2018ZX10711001,2018ZX10101-002]the Development Grant of State Key Laboratory of Infectious Disease Prevention and Control [2015SKLID505,2014SKLID103]
文摘Objective Tick-borne encephalitis virus(TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis(TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. Methods A reverse-transcription recombinase-aided amplification(RT-RAA) assay was developed. This assay can be completed in one closed tube at 39℃ within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type(WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. Results The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units(pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. Conclusion A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.
基金supported by the China Mega-Project for Infectious Disease(2016ZX10004-101,2016ZX10004-215)Beijing Municipal Science&Technology Commission Project(D151100002115003)Guangzhou Municipal Science&Technology Commission Project(2015B2150820)
文摘Objective To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.
基金supported by research grants from the Diagnosis of Infectious Pathogens and Combination of Diagnostic Technologies (2008ZX10004-002)Prevention and Control of Major Infectious Disease such as AIDS and Viral Hepatitis,State Eleventh Five-Year Plan
文摘Objective To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test. Methods Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT). Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR). Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies
基金supported by grants from the China Mega-Projects for Infectious Disease [2017ZX10302301-004-002,2018ZX10711001,2017ZX10104001,2018ZX10713-002]IVDC [2019HYDQNJJ03]
文摘West Nile virus(WNV)causes West Nile fever and West Nile encephalitis.Because infection by WNV creates serious public health problems,its simple,rapid,and visual detection is very important in clinical practice,especially in resource-limited laboratories.We have developed a rapid,specific,and highly sensitive internally controlled reverse transcription recombinase-aided amplification(RTRAA)assay to detect WNV,using both real-timefluoresce nee and the lateral flow dipstick(LFD)at39.0°C for 30 min.The analytical sensitivity of theRT-RAA assay was 10 plasmid copies and 1.6 pfu perreacti on with real-time fluoresce nee,and 1,000plasmid copies per reaction with the LFD.No crossreactionwith other control viruses was observed.Compared with the RT-qPCR assay,the RT-RAA assaydemonstrated 100%sensitivity and 100%specificityfor WNV.
基金supported by grants from the National Key Research and Development Plan of China [2016TFC1202700,2016YFC1200903,and 2017YFC1200503]the China Mega‐Project for Infectious Disease [2017ZX10302301‐004,2017ZX100101,and 2017ZX10104001]
文摘Objective People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus(YFV) is the only pathogen under surveillance in Sierra Leone owing to the undeveloped medical and public health system there. Most of the results of YFV identification are negative. Elucidation of the pathogen spectrum is required to reduce the prevalence of febrile jaundice. Methods In the present study, we used Ion Torrent semiconductor sequencing to profile the pathogen spectrum in archived YFV‐negative sera from 96 patients in Sierra Leone who presented with unexplained febrile jaundice. Results The most frequently identified sequencing reads belonged to the following pathogens: cytomegalovirus(89.58%), Epstein‐Barr virus(55.21%), hepatitis C virus(34.38%), rhinovirus(28.13%), hepatitis A virus(20.83%), coxsackievirus(10.42%), Ebola virus(8.33%), hepatitis E virus(8.33%), lyssavirus(4.17%), leptospirosis(4.17%), chikungunya virus(2.08%), Crimean‐Congo hemorrhagic fever virus(1.04%), and hepatitis B virus(1.04%). Conclusion The distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.
基金the China Mega-Projects for Infectious Disease[grant no.2018ZX10711001,2017ZX10104001 and 2018ZX10713-002]the National Institute for Viral Disease Control and Prevention[grant no.2019HYDQNJJ03].
文摘Epstein-Barr virus(EBV)and cytomegalovirus(CMV),two of the most prevalent human herpesviruses,cause a wide spectrum of diseases and symptoms and are associated with serious health problem.In this study,we developed an internal control reference recombinase-aided amplification(ICR-RAA)assay for the rapid detection of EBV and CMV within 30 min.The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV,respectively,with no cross reaction with other pathogens.In comparison with those of the commercial quantitative polymerase chain reaction(qPCR),the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33%and 84.84%,respectively;the specificity was 98.75%and 100.00%,respectively;and the Kappa values were 0.930 and 0.892(P<0.05),respectively.In comparison with those of qPCR,the sensitivity of the EBV and CMV ICR-RAAs using the DNA by thermal lysis was 72.22%and 80.00%,respectively;the specificity was 100.00%。
基金supported by the China Mega Project for Infectious Disease(2013ZX10004-001,2012ZX10004-215,2013ZX10004-202,and 2013ZX10004804-007)
文摘Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella, and 5higella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2). Methods A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System {Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9x102 CFU/mLfor S. aureus, 4.4x102 CFU/mL for L. monocytogenes, 3.0x102 CFU/mL for Salmonella, 2.5x102 CFU/mL for Shigella, 2.1x102 CFU/mL for V. parahaemolyticus, and 1.2x102 CFU/mL for E. coll. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10s CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).
基金supported by the National key research and development plan(2016TFC1202700,2016YFC1200900)Beijing Municipal Science&Technology Commission project(grant numbers D151100002115003)Guangzhou Municipal Science&Technology Commission project(grant numbers 2015B2150820)
文摘Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.
基金supported by grants from the National key research and development plan of China[2016TFC1202700,2016YFC1200903,and 2017YFC1200503]China Mega-Project for Infectious Disease[2017ZX10302301-004,2017ZX100101,and 2017ZX10104001]
文摘Objective Unbiased next generation sequencing(NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. Methods A viral Sequence Independent Targeted Amplification(VSITA) approach using a set of non-ribosomal and virus-enriched octamers(V8) was developed and compared with traditionally used random hexamers(N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR(qP CR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. Results A minimum 30 hexamers matching to viral reference sequences(sense and antisense) were selected from a dataset of random 4,096(4~6) hexamers(N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480(30 × 4~2) octamers(V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted c DNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. Conclusion The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.