[Objectives]This study aimed to evaluate the detection sensitivity of Staphylococcus aureus in dairy products utilizing the chip digital PCR(cdPCR)technique.[Methods]Specific primers and probes were designed and synth...[Objectives]This study aimed to evaluate the detection sensitivity of Staphylococcus aureus in dairy products utilizing the chip digital PCR(cdPCR)technique.[Methods]Specific primers and probes were designed and synthesized based on the conserved sequence of the heat-resistant nuclease gene nuc of S.aureus.cdPCR was employed to detect S.aureus,and the sensitivity of this technique was systematically assessed in samples exhibiting low levels of contamination.[Results]cdPCR demonstrated precise quantification when the initial concentration of the sample enrichment solution was equal to or greater than 50 CFU/mL.The detection dynamic range extended across at least five orders of magnitude,with a minimum DNA detection limit of 0.2304 pg/μL.In artificially contaminated cheese samples,the method s lower limit of quantification for detecting S.aureus was 8×10^(2) CFU/g.Regression analysis demonstrated that the gene copy number concentration measured by cdPCR exhibited a strong linear correlation with bacterial contamination concentration across a broad range.[Conclusions]The cdPCR method developed in this study demonstrates high sensitivity and robust quantitative capabilities,offering a reliable technical approach for the precise detection of low-level S.aureus contamination in dairy products.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
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
Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a ma...Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.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
Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Dro...Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.展开更多
Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has cert...Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher Scientific<sup>TM</sup> has recently commercialized the Applied Biosystems<sup>TM</sup> QuantStudio<sup>TM</sup> 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.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
Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantifica...Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantification technology, was used to identify single-copy homozygous transgenic lines from a batch of T0 transgenic rapeseed harboring 11 exogenous elements. Copy number of exogenous gene was evaluated in T0 generation based on calculated ratio between transgene and reference CruA gene, single-copy transformants were selected for selfing followed by subsequent zygosity analysis. Single-copy homozygous transgenic plants were successfully screened out in T1 generation by ddPCR.Segregation analysis with T2 seedlings verified that identification results of ddPCR were accurate and reliable. This study provides a novel rapid and accurate method for copy number and zygosity determination in transgenic rapeseed which overcomes disadvantages of traditional Southern analysis and recently developed real-time quantitative method.展开更多
BACKGROUND The Streptococcus salivarius(S.salivarius)group,which produces the enzyme urease has been identified as a potential contributor to ammonia production in the gut.Researchers have reported that patients with ...BACKGROUND The Streptococcus salivarius(S.salivarius)group,which produces the enzyme urease has been identified as a potential contributor to ammonia production in the gut.Researchers have reported that patients with minimal HE had an increased abundance of the S.salivarius group,which is a specific change in the gut microbiota that distinguishes them from healthy individuals.The correlation between the aggregation of specific bacterial species and fibrosis progression in chronic liver disease(CLD)is yet to be fully elucidated.AIM To quantify S.salivarius using digital PCR(dPCR)as a liver fibrosis marker of CLD.METHODS This study retrospectively analysed 52 patients with CLD.To quantify S.salivarius in patients with CLD using dPCR,we evaluated the specificity and sensitivity of S.salivarius bacterial load using dPCR for a type strain.Next,we evaluated the clinical usefulness of dPCR for S.salivarius load quantification for detecting liver fibrosis in patients with CLD.The liver fibrosis stage was categorized into mild and advanced fibrosis based on pathological findings.RESULTS The dPCR assay revealed that S.salivarius was highly positive for the tnpA gene.The lower limit of quantification for dPCR using the tnpA gene with a 1μL template comprising 1.28×102 CFU/mL was 4.3 copies.After considering the detection range in dPCR,we adjusted the extracted DNA concentration to 5.0×10-4 ng/μL from 200 mg stool samples.The median bacterial loads of S.salivarius in stool sample from patients with mild and advanced fibrosis were 1.9 and 7.4 copies/μL,respectively.The quantification of S.salivarius load was observed more frequently in patients with advanced fibrosis than in those with mild fibrosis(P=0.032).CONCLUSION Quantifying of S.salivarius load using digital PCR is a useful biomarker for liver fibrosis in patients with CLD.展开更多
A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of...A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.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
基金Supported by Science and Technology Program of Inner Mongolia Autonomous Region"Research and Demonstration of Novel Molecular Biological Identification Technology for Multiple Source Components in Milk and Dairy Products"(2025YFSH0029).
文摘[Objectives]This study aimed to evaluate the detection sensitivity of Staphylococcus aureus in dairy products utilizing the chip digital PCR(cdPCR)technique.[Methods]Specific primers and probes were designed and synthesized based on the conserved sequence of the heat-resistant nuclease gene nuc of S.aureus.cdPCR was employed to detect S.aureus,and the sensitivity of this technique was systematically assessed in samples exhibiting low levels of contamination.[Results]cdPCR demonstrated precise quantification when the initial concentration of the sample enrichment solution was equal to or greater than 50 CFU/mL.The detection dynamic range extended across at least five orders of magnitude,with a minimum DNA detection limit of 0.2304 pg/μL.In artificially contaminated cheese samples,the method s lower limit of quantification for detecting S.aureus was 8×10^(2) CFU/g.Regression analysis demonstrated that the gene copy number concentration measured by cdPCR exhibited a strong linear correlation with bacterial contamination concentration across a broad range.[Conclusions]The cdPCR method developed in this study demonstrates high sensitivity and robust quantitative capabilities,offering a reliable technical approach for the precise detection of low-level S.aureus contamination in dairy products.
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 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Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.
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.
文摘Injectable drugs manufactured in E. coli must be tested for host residual DNA (hr DNA) impurity in ensuring drug purity and safety. Because of low allowable hr DNA as impurity, highly sensitive methods are needed. Droplet digital PCR (ddPCR) is a new method where the reaction is partitioned into about 20,000 nanoliter-sized droplets and each droplet acts as individual PCR reaction. After completion of end-point PCR, droplets are analyzed for fluorescence and categorized as positive or negative and DNA quantified using Poisson statistics. Here we describe development of a direct E. coli hr DNA dd PCR method where the drug is directly added to the ddPCR reaction. We show that the ddPCR method has acceptable precision and high accuracy, works with different biologic drugs, and compared to qPCR shows higher tolerance of drug matrices. The method does not require DNA extraction or standard curves for quantification of hr DNA in unknown samples.
文摘Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher Scientific<sup>TM</sup> has recently commercialized the Applied Biosystems<sup>TM</sup> QuantStudio<sup>TM</sup> 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.
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Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantification technology, was used to identify single-copy homozygous transgenic lines from a batch of T0 transgenic rapeseed harboring 11 exogenous elements. Copy number of exogenous gene was evaluated in T0 generation based on calculated ratio between transgene and reference CruA gene, single-copy transformants were selected for selfing followed by subsequent zygosity analysis. Single-copy homozygous transgenic plants were successfully screened out in T1 generation by ddPCR.Segregation analysis with T2 seedlings verified that identification results of ddPCR were accurate and reliable. This study provides a novel rapid and accurate method for copy number and zygosity determination in transgenic rapeseed which overcomes disadvantages of traditional Southern analysis and recently developed real-time quantitative method.
文摘BACKGROUND The Streptococcus salivarius(S.salivarius)group,which produces the enzyme urease has been identified as a potential contributor to ammonia production in the gut.Researchers have reported that patients with minimal HE had an increased abundance of the S.salivarius group,which is a specific change in the gut microbiota that distinguishes them from healthy individuals.The correlation between the aggregation of specific bacterial species and fibrosis progression in chronic liver disease(CLD)is yet to be fully elucidated.AIM To quantify S.salivarius using digital PCR(dPCR)as a liver fibrosis marker of CLD.METHODS This study retrospectively analysed 52 patients with CLD.To quantify S.salivarius in patients with CLD using dPCR,we evaluated the specificity and sensitivity of S.salivarius bacterial load using dPCR for a type strain.Next,we evaluated the clinical usefulness of dPCR for S.salivarius load quantification for detecting liver fibrosis in patients with CLD.The liver fibrosis stage was categorized into mild and advanced fibrosis based on pathological findings.RESULTS The dPCR assay revealed that S.salivarius was highly positive for the tnpA gene.The lower limit of quantification for dPCR using the tnpA gene with a 1μL template comprising 1.28×102 CFU/mL was 4.3 copies.After considering the detection range in dPCR,we adjusted the extracted DNA concentration to 5.0×10-4 ng/μL from 200 mg stool samples.The median bacterial loads of S.salivarius in stool sample from patients with mild and advanced fibrosis were 1.9 and 7.4 copies/μL,respectively.The quantification of S.salivarius load was observed more frequently in patients with advanced fibrosis than in those with mild fibrosis(P=0.032).CONCLUSION Quantifying of S.salivarius load using digital PCR is a useful biomarker for liver fibrosis in patients with CLD.
文摘A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus.
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.
