Brucellosis is a global public health issue that severely affects human health,Brucella melitensis is currently the predominant species in China.Brucella spondylitis is the primary cause of the debilitating and disabl...Brucellosis is a global public health issue that severely affects human health,Brucella melitensis is currently the predominant species in China.Brucella spondylitis is the primary cause of the debilitating and disabling complications[1].The lumbar vertebra was the most commonly affected site,followed by the thoracic,cervical,thoracolumbar,and lumbosacral segments,and back pain,fever,sweating,and fatigue were the most common symptoms[2].However,the diagnosis of Brucella spondylitis is challenging owing to its wide spectrum of clinical presentations,cross-reactions with other bacteria,and low strain isolation rate.Therefore,a timely and accurate diagnosis of spinal brucellosis is crucial for implementing an effective therapeutic plan and improving treatment outcomes.Droplet digital polymerase chain reaction(ddPCR)is widely used for low-abundance nucleic acid detection and is useful for diagnosing infectious diseases[3].Therefore,this study aimed to evaluate the ddPCR approach for the diagnosis of brucellosis with spondylitis to improve its clinical diagnostic capacity.展开更多
Droplet digital PCR(ddPCR),as the third-generation PCR technology,demonstrates significant advantages in the etiological diagnosis of infectious diseases due to its absolute quantification,ultra-high sensitivity,and m...Droplet digital PCR(ddPCR),as the third-generation PCR technology,demonstrates significant advantages in the etiological diagnosis of infectious diseases due to its absolute quantification,ultra-high sensitivity,and multiplex detection capabilities.This article reports a case of a patient with fever of unknown origin,where ddPCR rapidly confirmed a drug-resistant bacterial infection and dynamically monitored treatment efficacy.Combining literature evidence,this paper systematically elaborates on the technical principles,clinical performance,and practical value of ddPCR in febrile patients.展开更多
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in dise...Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.展开更多
Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid det...Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus＇（Las）, the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR（qPCR） for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples（46.15%） with high Ct value（〉35） were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.展开更多
AIMTo assess KRAS G12D mutation detection by droplet digital PCR(ddPCR)in stool-derived DNA from colorectal cancer(CRC)patients.METHODSIn this study,tumor tissue and stool samples were collected from 70 patients with ...AIMTo assess KRAS G12D mutation detection by droplet digital PCR(ddPCR)in stool-derived DNA from colorectal cancer(CRC)patients.METHODSIn this study,tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy.KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded(FFPE)tumor tissues.The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation.Wild-type(WT)tumors,as determined by pyrosequencing,were included as controls;analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.RESULTSAmong the total 70 patients included,KRAS mutations were detected by pyrosequencing in 32(45.71%),whereas 38(54.29%)had WT tumors.The frequency of KRAS mutations was higher in left-sided tumors(11 located in the right colon,15 in the left,and 6 in the rectum).The predominant point mutation was KRAS G12D(14.29%,n=10),which was more frequent in early-stage tumors(I-IIA,n=7).In agreement with pyrosequencing results,the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA,and only a residual number of mutated copies was found in WT controls.The KRAS G12D mutation was also detected in stool-derived DNA in 80%of all fecal samples from CRC patients with this point mutation.CONCLUSIONddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients,especially at early stages.This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.展开更多
Background:The present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses,explicitly focusing on norovirus genogroup II(GII),astrovirus,rotavirus,Aichi virus,sapovirus,hepatitis A and...Background:The present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses,explicitly focusing on norovirus genogroup II(GII),astrovirus,rotavirus,Aichi virus,sapovirus,hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR(ddPCR).Enteric viruses are of significant public health concern,as they are the leading cause of diseases like gastroenteritis.Regular monitoring of environmental samples,particularly from wastewater treatment plants,is crucial for early detection and control of these viruses.This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions.Our protocol's objective is to establish a novel ddPCRbased methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal,India.Our assay is capable of accurately quantifying virus concentrations without standard curves,minimizing extensive optimization,and enhancing sensitivity and precision,especially for low-abundance targets.Methods:The study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years,ensuring comprehensive coverage and consistent data.Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater,a more advanced technique.Additionally,we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses.Conclusion:This study will aid in understanding these viruses’genetic diversity and mutation rates,which is crucial for developing tailored intervention strategies.The findings will be instrumental in shaping public health responses and improving epidemiological surveillance,especially in localities heaving sewage networks.展开更多
[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-s...[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.展开更多
Objectives:Cancer treatment relies heavily on accurate diagnosis and effective monitoring of the disease.These processes often involve invasive procedures,such as colonoscopy,to detect malignant tissues,followed by mo...Objectives:Cancer treatment relies heavily on accurate diagnosis and effective monitoring of the disease.These processes often involve invasive procedures,such as colonoscopy,to detect malignant tissues,followed by molecular analyses to determine relevant biomarkers.This study aimed to evaluate the clinical performance of droplet digital PCR(ddPCR)for detecting Kirsten Rat Sarcoma Viral Proto-Oncogene(KRAS),Neuroblastoma RAS Viral Oncogene Homolog(NRAS),and B-Raf Murine Sarcoma Viral Oncogene Homolog B(BRAF)mutations in circulating tumor DNA(ctDNA)from colorectal cancer patients using liquid biopsy.Methods:ctDNA was isolated from colorectal cancer(CRC)patients(n=110)and analyzed for KRAS,BRAF,and NRAS mutations.The ctDNA obtained through liquid biopsy was analyzed using ddPCR,and the findings were compared with sequencing data from tumor DNA archived in formalin-fixed paraffin-embedded(FFPE)blocks.Results:For KRAS mutations,ddPCR achieved a sensitivity of 72.0%and a specificity of 71.4%.However,when pooling all target mutations(KRAS,NRAS and BRAF),the overall sensitivity and specificity were lower,at 48.3%and 51.1%,respectively.Conclusion:The results of this study indicate that the ddPCR analysis of ctDNA may provide complementary information for the molecular diagnosis of CRC patients.展开更多
Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,i...Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.展开更多
Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition.Exosomes take part in cellular communication and receptor mediation and tra...Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition.Exosomes take part in cellular communication and receptor mediation and transfer their cargos(e.g.,proteins,m RNA and DNA).Quantitative analysis of tumor-related nucleic acid mutations can be a potential method to cancer diagnosis and prognosis in early stages.Here we present an integrated microfluidic system for exosome on-chip isolation and lung cancer RNA analysis through droplet digital PCR(dd PCR).Gradient dilution experiments show great linearity over a large concentration range with R^(2)=0.9998.Utilizing the system,four cell lines and two mutation targets were parallelly detected for mutation analysis.The experiments demonstrated mutation heterogeneity and the results were agree with cell researches.These results proved our integrated microfluidic system as a promising means for early cancer diagnosis and prognosis in the era of liquid biopsy.展开更多
Background:Zoonotic schistosomiasis,caused by Schistosoma japonicum,remains a major public health problem in the Philippines.This study aimed to evaluate the commercially available rapid diagnostic point-of-care circu...Background:Zoonotic schistosomiasis,caused by Schistosoma japonicum,remains a major public health problem in the Philippines.This study aimed to evaluate the commercially available rapid diagnostic point-of-care circulating cathodic antigen(POC-CCA)test in detecting individuals infected with S.japonicum in a human cohort from an endemic area for schistosomiasis japonica in the Philippines.Methods:Clinical samples were collectedin 18 barangays endemic for S.japonicum infection in Laoang and Palapag municipalities,Northern Samar,the Philippines,in 2015.The presence of CCA in flter-concentrated urine samples(n=412)was evaluated using the commercial kits and the results were converted to images,which were further analyzed by ImageJ software to calculate R values.The diagnostic performance of the immunochromatographic POCCCA test was compared using the Kato-Katz(KK)procedure,in-house enzyme-linked immunosorbent assays(ELISAs)and droplet digital(dd)PCR assays as reference.Results:The POC-CCA test was able to detect S.japonicum-infected individuals in the cohort with an eggs per gram of faeces(EPG)more than or equal to 10 with sensitivity/specifcity values of 63.3%/93.3%.However,the assay showed an inability to diagnose schistosomiasis japonica infections in all cohort KK-positive individuals,of which the majority had an extremely low egg burden(EPG:1–9).The prevalence of S.japonicum infection in the total cohort determined by the POC-CCA test was 12.4%,only half of that determined by the KK method(26.2%).When compared with the ELISAs and ddPCR assays as a reference,the POC-CCA assay was further shown to be a test with low sensitivity.Nevertheless,the assay exhibited signifcant positive correlations with egg burden determined by the KK technique and the target gene copy number index values determined by the ddPCR assays within the entire cohort.Conclusions:By using in silico image analysis,the POC-CCA cassette test could be converted to a quantitative assay to avoid reader-variability.Because of its low sensitivity,the commercially available POC-CCA assay had limited potential for determining the status of a S.japonicum infection in the target cohort.The assay should be applied with caution in populations where schistosome parasites(especially S.japonicum)are present at low infection intensity.展开更多
基金supported by the Key Research and Development Projects of the Ningxia Hui Autonomous Region(Grant no.2022BEG03161)。
文摘Brucellosis is a global public health issue that severely affects human health,Brucella melitensis is currently the predominant species in China.Brucella spondylitis is the primary cause of the debilitating and disabling complications[1].The lumbar vertebra was the most commonly affected site,followed by the thoracic,cervical,thoracolumbar,and lumbosacral segments,and back pain,fever,sweating,and fatigue were the most common symptoms[2].However,the diagnosis of Brucella spondylitis is challenging owing to its wide spectrum of clinical presentations,cross-reactions with other bacteria,and low strain isolation rate.Therefore,a timely and accurate diagnosis of spinal brucellosis is crucial for implementing an effective therapeutic plan and improving treatment outcomes.Droplet digital polymerase chain reaction(ddPCR)is widely used for low-abundance nucleic acid detection and is useful for diagnosing infectious diseases[3].Therefore,this study aimed to evaluate the ddPCR approach for the diagnosis of brucellosis with spondylitis to improve its clinical diagnostic capacity.
文摘Droplet digital PCR(ddPCR),as the third-generation PCR technology,demonstrates significant advantages in the etiological diagnosis of infectious diseases due to its absolute quantification,ultra-high sensitivity,and multiplex detection capabilities.This article reports a case of a patient with fever of unknown origin,where ddPCR rapidly confirmed a drug-resistant bacterial infection and dynamically monitored treatment efficacy.Combining literature evidence,this paper systematically elaborates on the technical principles,clinical performance,and practical value of ddPCR in febrile patients.
基金supported by the the National Key Research and Development Program of China (2017YFD0201602)the National Natural Science Foundation of China (31401704)the Beijing Academy of Agriculture and Forestry Foundation, China (KJCX20180203)
文摘Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.
基金funded by the National Natural Sciences Foundation of China (31671992, 31301635)the Chongqing Science and Technology Commission Project, China (cstc2016shms-ztzx80003)the Guangxi Key Laboratory of Citrus Biology, Guangxi Academy of Specialty Crops, China (SYS2015K004)
文摘Citrus Huanglongbing（HLB, yellow shoot disease） is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR（ddPCR） assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus＇（Las）, the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR（qPCR） for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples（46.15%） with high Ct value（〉35） were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.
基金Supported by“Fondo de Investigaciones Sanitarias(FIS)-FEDER”,Ministry of Health,Spain,No.PI13/01924 to García-Olmo DRETIC Program of ISCIII-FEDER,No.RD12/0019/0035 to Olmedillas-López S
文摘AIMTo assess KRAS G12D mutation detection by droplet digital PCR(ddPCR)in stool-derived DNA from colorectal cancer(CRC)patients.METHODSIn this study,tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy.KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded(FFPE)tumor tissues.The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation.Wild-type(WT)tumors,as determined by pyrosequencing,were included as controls;analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.RESULTSAmong the total 70 patients included,KRAS mutations were detected by pyrosequencing in 32(45.71%),whereas 38(54.29%)had WT tumors.The frequency of KRAS mutations was higher in left-sided tumors(11 located in the right colon,15 in the left,and 6 in the rectum).The predominant point mutation was KRAS G12D(14.29%,n=10),which was more frequent in early-stage tumors(I-IIA,n=7).In agreement with pyrosequencing results,the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA,and only a residual number of mutated copies was found in WT controls.The KRAS G12D mutation was also detected in stool-derived DNA in 80%of all fecal samples from CRC patients with this point mutation.CONCLUSIONddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients,especially at early stages.This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.
文摘Background:The present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses,explicitly focusing on norovirus genogroup II(GII),astrovirus,rotavirus,Aichi virus,sapovirus,hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR(ddPCR).Enteric viruses are of significant public health concern,as they are the leading cause of diseases like gastroenteritis.Regular monitoring of environmental samples,particularly from wastewater treatment plants,is crucial for early detection and control of these viruses.This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions.Our protocol's objective is to establish a novel ddPCRbased methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal,India.Our assay is capable of accurately quantifying virus concentrations without standard curves,minimizing extensive optimization,and enhancing sensitivity and precision,especially for low-abundance targets.Methods:The study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years,ensuring comprehensive coverage and consistent data.Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater,a more advanced technique.Additionally,we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses.Conclusion:This study will aid in understanding these viruses’genetic diversity and mutation rates,which is crucial for developing tailored intervention strategies.The findings will be instrumental in shaping public health responses and improving epidemiological surveillance,especially in localities heaving sewage networks.
基金Supported by Science and Technology Development Plan Fund Project of Jilin Province(20210202101NC)YDZJ202203C G Z H 050.
文摘[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.
基金funded by the Ministry of Health of the Czech Republic—conceptual development of research organization(MMCI,00209805)Czech Science Foundation(No.25-15990S)+1 种基金the project 7D241003 EUREKA EUROSTARS35897,project SALVAGE(P JAC,reg.No.CZ.02.01.01/00/22_008/0004644)—funded by the European Unionby the State Budget of the Czech Republic,and by the LRI project BBMRI.cz(Nos.LM2023033 and CZ.02.1.01/0.0/0.0/16_013/0001674.).
文摘Objectives:Cancer treatment relies heavily on accurate diagnosis and effective monitoring of the disease.These processes often involve invasive procedures,such as colonoscopy,to detect malignant tissues,followed by molecular analyses to determine relevant biomarkers.This study aimed to evaluate the clinical performance of droplet digital PCR(ddPCR)for detecting Kirsten Rat Sarcoma Viral Proto-Oncogene(KRAS),Neuroblastoma RAS Viral Oncogene Homolog(NRAS),and B-Raf Murine Sarcoma Viral Oncogene Homolog B(BRAF)mutations in circulating tumor DNA(ctDNA)from colorectal cancer patients using liquid biopsy.Methods:ctDNA was isolated from colorectal cancer(CRC)patients(n=110)and analyzed for KRAS,BRAF,and NRAS mutations.The ctDNA obtained through liquid biopsy was analyzed using ddPCR,and the findings were compared with sequencing data from tumor DNA archived in formalin-fixed paraffin-embedded(FFPE)blocks.Results:For KRAS mutations,ddPCR achieved a sensitivity of 72.0%and a specificity of 71.4%.However,when pooling all target mutations(KRAS,NRAS and BRAF),the overall sensitivity and specificity were lower,at 48.3%and 51.1%,respectively.Conclusion:The results of this study indicate that the ddPCR analysis of ctDNA may provide complementary information for the molecular diagnosis of CRC patients.
基金financially supported by the Science and Technology Commission of Shanghai Municipality(21ZR1427200).
文摘Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.
基金National Natural Science Foundation of China(Nos.61971410,and 62001458)Shanghai Sailing Program(No.20YF1457100)。
文摘Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition.Exosomes take part in cellular communication and receptor mediation and transfer their cargos(e.g.,proteins,m RNA and DNA).Quantitative analysis of tumor-related nucleic acid mutations can be a potential method to cancer diagnosis and prognosis in early stages.Here we present an integrated microfluidic system for exosome on-chip isolation and lung cancer RNA analysis through droplet digital PCR(dd PCR).Gradient dilution experiments show great linearity over a large concentration range with R^(2)=0.9998.Utilizing the system,four cell lines and two mutation targets were parallelly detected for mutation analysis.The experiments demonstrated mutation heterogeneity and the results were agree with cell researches.These results proved our integrated microfluidic system as a promising means for early cancer diagnosis and prognosis in the era of liquid biopsy.
基金funded by the National Health and Medical Research Council(NHMRC)of Australia(ID:APP1160046,APP1102926,APP1037304,APP1098244,and APP1194462)DPM is a NHMRC Leadership Fellow and Senior Scientist at QIMRB.
文摘Background:Zoonotic schistosomiasis,caused by Schistosoma japonicum,remains a major public health problem in the Philippines.This study aimed to evaluate the commercially available rapid diagnostic point-of-care circulating cathodic antigen(POC-CCA)test in detecting individuals infected with S.japonicum in a human cohort from an endemic area for schistosomiasis japonica in the Philippines.Methods:Clinical samples were collectedin 18 barangays endemic for S.japonicum infection in Laoang and Palapag municipalities,Northern Samar,the Philippines,in 2015.The presence of CCA in flter-concentrated urine samples(n=412)was evaluated using the commercial kits and the results were converted to images,which were further analyzed by ImageJ software to calculate R values.The diagnostic performance of the immunochromatographic POCCCA test was compared using the Kato-Katz(KK)procedure,in-house enzyme-linked immunosorbent assays(ELISAs)and droplet digital(dd)PCR assays as reference.Results:The POC-CCA test was able to detect S.japonicum-infected individuals in the cohort with an eggs per gram of faeces(EPG)more than or equal to 10 with sensitivity/specifcity values of 63.3%/93.3%.However,the assay showed an inability to diagnose schistosomiasis japonica infections in all cohort KK-positive individuals,of which the majority had an extremely low egg burden(EPG:1–9).The prevalence of S.japonicum infection in the total cohort determined by the POC-CCA test was 12.4%,only half of that determined by the KK method(26.2%).When compared with the ELISAs and ddPCR assays as a reference,the POC-CCA assay was further shown to be a test with low sensitivity.Nevertheless,the assay exhibited signifcant positive correlations with egg burden determined by the KK technique and the target gene copy number index values determined by the ddPCR assays within the entire cohort.Conclusions:By using in silico image analysis,the POC-CCA cassette test could be converted to a quantitative assay to avoid reader-variability.Because of its low sensitivity,the commercially available POC-CCA assay had limited potential for determining the status of a S.japonicum infection in the target cohort.The assay should be applied with caution in populations where schistosome parasites(especially S.japonicum)are present at low infection intensity.
