[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
The microfluidic polymerase chain reaction(PCR)chips have undergone extensive development and nowadays have become an important domain of miniaturization technology application.Here,we review the advances of microflui...The microfluidic polymerase chain reaction(PCR)chips have undergone extensive development and nowadays have become an important domain of miniaturization technology application.Here,we review the advances of microfluidic PCR chips over the past years,from the first single chamber stationary PCR chip to the new SlipChip PCR.First,the three distinct types of microfluidic PCR chips are discussed,including chamber stationary PCR chips,flow-through PCR chips and convection PCR chips.Then we focus on droplet PCR chips and SlipChip PCR.Although they are at an early stage,they show the great potential for high-throughput PCR and robust chip.Finally,general discussions on integrated chips are given.The low cost,portable,high-throughout integrated PCR chips will certainly be further developed in spite of many challenges.展开更多
A novel continuous-flow PCR chip adopting self-heating, passive-cooling mode to realize the DNA fragments amplification was presented. Using the ANSYS finite element analysis, the temperature distribution of the chip ...A novel continuous-flow PCR chip adopting self-heating, passive-cooling mode to realize the DNA fragments amplification was presented. Using the ANSYS finite element analysis, the temperature distribution of the chip is simulated and analyzed.The optimal size of the chip is 30×22 mm2, the roundabout micro-channel is the 90 μm width, 40 μm depth. Two micro-heater with the nickel-chrome alloy material film are formed on the side of silicon belonging to denaturation and renaturation zones needed for PCR reaction, and two adiabatic structures with groove on side of silicon by anisotropy etching. By the mode of heating local zones at single side, three wider constant temperature zones could be formed, which are 60 ℃,72 ℃,95 ℃ and suitable for PCR,and the temperature-difference could be restricted in less than 5 ℃.展开更多
Objective To detect the Epstein-Barr virus(EBV)viral load of children after hematopoietic stem cell transplantation(HSCT)using chip digital PCR(cdPCR).Methods The sensitivity of cdPCR was determined using EBV plasmids...Objective To detect the Epstein-Barr virus(EBV)viral load of children after hematopoietic stem cell transplantation(HSCT)using chip digital PCR(cdPCR).Methods The sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 strain.The specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses(herpes simplex virus 1,herpes simplex virus 2,varicella zoster virus,human cytomegalovirus,human herpesvirus 6,and human herpesvirus 7).From May 2019 to September 2020,64 serum samples of children following HSCT were collected.EBV infection and the viral load of serum samples were detected by cdPCR.The epidemiological characteristics of EBV infections were analyzed in HSCT patients.Results The limit of detection of EBV cdPCR was 110 copies/mL,and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 plasmid.The result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain,and both were more sensitive than that of quantitative PCR.Using cdPCR,the incidence of EBV infection was 18.75%in 64 children after HSCT.The minimum EBV viral load was 140 copies/mL,and the maximum viral load was 3,209 copies/mL using cdPCR.The average hospital stay of children with EBV infection(184±91 days)was longer than that of children without EBV infection(125±79 days),P=0.026.Conclusion EBV cdPCR had good sensitivity and specificity.The incidence of EBV infection was 18.75%in 64 children after HSCT from May 2019 to September 2020.EBV cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.展开更多
Continuous flow PCR(polymerase chain reaction)chip holds impressive advantages compared to micro chamber PCR chip.In order to have better understanding of kinetic characteristics of continuous flow PCR chip,a comprehe...Continuous flow PCR(polymerase chain reaction)chip holds impressive advantages compared to micro chamber PCR chip.In order to have better understanding of kinetic characteristics of continuous flow PCR chip,a comprehensive mathematical model is presented in this paper,including melting,annealing and extension phases of a typical PCR process which has the essence of a convection-diffusion-reaction system.Using this model,we can simulate the PCR process in series of reaction cycles.Numerical results show that the average sample velocity plays a significant role in affecting the amplification efficiency.Also,appropriate combination of the PCR mixture is important for high-quality DNA amplification.Giving a large initial DNA concentration range,the continuous flow PCR scheme holds excellent real-time detection ability theoretically.The present numerical model bridges the temperature distribution to the real DNA amplification,and thereby is able to successfully predict continuous flow PCR properties which are important for the chip design.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.
基金supported by Ministry of Science and Technology of China(2010CB933901)Science and Technology Innovation Fund of SJTU-University of Michigan。
文摘The microfluidic polymerase chain reaction(PCR)chips have undergone extensive development and nowadays have become an important domain of miniaturization technology application.Here,we review the advances of microfluidic PCR chips over the past years,from the first single chamber stationary PCR chip to the new SlipChip PCR.First,the three distinct types of microfluidic PCR chips are discussed,including chamber stationary PCR chips,flow-through PCR chips and convection PCR chips.Then we focus on droplet PCR chips and SlipChip PCR.Although they are at an early stage,they show the great potential for high-throughput PCR and robust chip.Finally,general discussions on integrated chips are given.The low cost,portable,high-throughout integrated PCR chips will certainly be further developed in spite of many challenges.
基金the National Natural Science Foundation of China(Grant No.60576047)
文摘A novel continuous-flow PCR chip adopting self-heating, passive-cooling mode to realize the DNA fragments amplification was presented. Using the ANSYS finite element analysis, the temperature distribution of the chip is simulated and analyzed.The optimal size of the chip is 30×22 mm2, the roundabout micro-channel is the 90 μm width, 40 μm depth. Two micro-heater with the nickel-chrome alloy material film are formed on the side of silicon belonging to denaturation and renaturation zones needed for PCR reaction, and two adiabatic structures with groove on side of silicon by anisotropy etching. By the mode of heating local zones at single side, three wider constant temperature zones could be formed, which are 60 ℃,72 ℃,95 ℃ and suitable for PCR,and the temperature-difference could be restricted in less than 5 ℃.
基金supported by the Research Project Supported by the China Mega-Project for Infectious Disease[2018ZX10102001,2018ZX10711001,and 2018ZX10734404]National Pathogen Resource Collection Center[NPRC-32]the SKLID Development Grant[2011SKLID104]。
文摘Objective To detect the Epstein-Barr virus(EBV)viral load of children after hematopoietic stem cell transplantation(HSCT)using chip digital PCR(cdPCR).Methods The sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 strain.The specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses(herpes simplex virus 1,herpes simplex virus 2,varicella zoster virus,human cytomegalovirus,human herpesvirus 6,and human herpesvirus 7).From May 2019 to September 2020,64 serum samples of children following HSCT were collected.EBV infection and the viral load of serum samples were detected by cdPCR.The epidemiological characteristics of EBV infections were analyzed in HSCT patients.Results The limit of detection of EBV cdPCR was 110 copies/mL,and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 plasmid.The result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain,and both were more sensitive than that of quantitative PCR.Using cdPCR,the incidence of EBV infection was 18.75%in 64 children after HSCT.The minimum EBV viral load was 140 copies/mL,and the maximum viral load was 3,209 copies/mL using cdPCR.The average hospital stay of children with EBV infection(184±91 days)was longer than that of children without EBV infection(125±79 days),P=0.026.Conclusion EBV cdPCR had good sensitivity and specificity.The incidence of EBV infection was 18.75%in 64 children after HSCT from May 2019 to September 2020.EBV cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.
基金supported by the National Natural Science Foundation of China(Grant No.60606014)Major State Basic Research Development Program(973 Program)(Grant No.2009CB320300)the President Foundation of Peking University
文摘Continuous flow PCR(polymerase chain reaction)chip holds impressive advantages compared to micro chamber PCR chip.In order to have better understanding of kinetic characteristics of continuous flow PCR chip,a comprehensive mathematical model is presented in this paper,including melting,annealing and extension phases of a typical PCR process which has the essence of a convection-diffusion-reaction system.Using this model,we can simulate the PCR process in series of reaction cycles.Numerical results show that the average sample velocity plays a significant role in affecting the amplification efficiency.Also,appropriate combination of the PCR mixture is important for high-quality DNA amplification.Giving a large initial DNA concentration range,the continuous flow PCR scheme holds excellent real-time detection ability theoretically.The present numerical model bridges the temperature distribution to the real DNA amplification,and thereby is able to successfully predict continuous flow PCR properties which are important for the chip design.
