Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further ...Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.展开更多
Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus...Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.展开更多
Objective: To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma (NPC) tissues. Methods: The total RNA was extracted from N...Objective: To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma (NPC) tissues. Methods: The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients' nasopharynx tissues treated as control group. Results: The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients' nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients' nasopharynx tissues, the difference was significant (P 〈 0.01). The expression of Survivin mRNA could be detected both in stage Ⅰ + Ⅱ and stage Ⅲ + Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups (P 〉 0.05). There was no relationship between Survivin mRNA expression and age and sex of NPC patients (P 〉 0.05). Conclusion: Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.展开更多
This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from s...This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.展开更多
Objective: Multidrug resistance(MDR) is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia(ALL...Objective: Multidrug resistance(MDR) is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia(ALL) which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction(FQ-RT-PCR), and combine minimal residual desease(MRD) detection by flow cytometry(FCM) and to study their relationship with treatment response and prognosis of ALL. Methods:The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission(CR), 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results:Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group(P 〉 0.05), but significantly higher in the recurrence group than that in newly diagnosed disease group and control group(0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05). 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration(0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01). For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group(P〉 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group(0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05). Conclusion:The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration(within 3 years). Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.展开更多
[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel rea...[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses (WSSV, IHHNV, TSV and YHV ). [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies (E = 1.06, 1.07, 0.92 and 0.92, respectively), good hnear relationship ( r = 1 ), uniform repeatability ( standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ) and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR (average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ). The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.展开更多
Porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus(PEDV)with symptoms of vomit,diarrhea,loss of appetite of suckling pig,has led to serious economic ...Porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus(PEDV)with symptoms of vomit,diarrhea,loss of appetite of suckling pig,has led to serious economic loss to the global swine industry.In this study,a real-time fluorescence reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay was developed to detect PEDV RNA.The real-time fluorescence RT-LAMP assay was performed at62℃for 60 min,using a simple and portable device,the ESE-Quant Tube Scanner.The detection limit of RNA was 2.9×10^(6) copies/μl,10 times as sensitive as RT-PCR,and the detection was specific only to PEDV.Application of this method to clinical samples yielded a positivity rate of 93%,which was higher than that of RT-PCR.This technique saves time and is efficient,and is thus expected to be useful for the diagnosis of PEDV infection in the field.展开更多
According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 (NC001718) available in GenBank (NC_001718), a pair of specific primer was designed, and the target fragment of 431 bp was obtained by P...According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 (NC001718) available in GenBank (NC_001718), a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.展开更多
Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to des...Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>展开更多
Technological improvements are crucial in the evolution of surgery.Real-time fluorescence-guided surgery(FGS)has spread worldwide,mainly because of its usefulness during the intraoperative decision-making processes.Th...Technological improvements are crucial in the evolution of surgery.Real-time fluorescence-guided surgery(FGS)has spread worldwide,mainly because of its usefulness during the intraoperative decision-making processes.The success of any gastrointestinal oncologic resection is based on the anatomical identification of the primary tumor and its regional lymph nodes.FGS allows also to evaluate the blood perfusion at the gastrointestinal stumps after colorectal or esophageal resections.Therefore,a reduction on the anastomotic leak rates has been postulated as one of the foreseeable benefits provided by the use of FGS in these procedures.Although the use of fluorescence in lymph node detection was initially described in breast cancer surgery,the technique is currently applied in gastric or splenic flexure cancers,as they both present complex and variable lymphatic drainages.FGS allows also to perform intraoperative lymphograms or sentinel lymph node biopsies.New applications of FGS are being developed to assist in the detection of peritoneal metastases or in the evaluation of the tumor resection margins.The present review aims to provide a general overview of the current status of real-time FGS in gastrointestinal oncologic surgery.We put a special focus on the different applications of FGS,discussing the main findings and limitations found in the contemporary literature and also the promising near future applications.展开更多
[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes vir...[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes virus (KHV) pol-ymerase gene (Sph) to establish a rapid and effective fluorescence quantitative PCR method for Koi herpes virus detection. The cell cultures were detected by using the established fluorescence quantitative PCR assay, and the results were com- pared with that of conventional PCR. [ Result] The sensitivity of fluorescence quantitative PCR was higher than that of conventional PCR. The minimum copy num- ber that could be detected was 1.6 - 102 copies/p.1. The established method was adopted for sample detection, and a reliable diagnostic result could be obtained within 4 h. [Conclusion] The established method is rapid, sensitive, specific and repeatable, which is conducive to the rapid detection of Koi herpes virus. Key words Koi herpes virus; Fluorescence quantitative PCR; Detection展开更多
The emerging viruses within the genus Henipavirus in the family Paramyxoviridae pose a great threat to public biosafety.To develop a quadruple real-time fluorescence-based quantitative reverse transcription polymerase...The emerging viruses within the genus Henipavirus in the family Paramyxoviridae pose a great threat to public biosafety.To develop a quadruple real-time fluorescence-based quantitative reverse transcription polymerase chain reaction(qRT-PCR)assay is pivotal for the early warning of the potential of zoonotic infectious diseases.Specific primers and probes were designed for the relatively conserved regions based on whole genome sequences of Langya virus(LayV),Mojiang virus(MojV),Nipah virus(NiV),and Cedar virus(CedV),followed by the establishment of a quadruple real-time fluorescence-based qRT-PCR detection method.No cross-reactivity was observed with other viral nucleic acids.The optimal linear detection range for LayV,MojV,NiV,and CedV was 10^(1)-10^(8)copies/μL,and the lower limit of detection was 10 copies/μL.Three different DNA concentrations of LayV,MojV,NiV,and CedV(10^(4),10^(5),and 10^(6)copies/μL)were tested 14 times,achieving good repeatability.The standard deviation of the cycle threshold values for each concentration was<0.5 and the coefficient of variation was<3%.Furthermore,the amplification efficiency of quadruple real-time fluorescence-based qRT-PCR was>90%,and the correlation coefficient was>0.99.The established quadru-ple real-time fluorescence-based qRT-PCR assay for the detection of LayV,MojV,NiV,and CedV exhibits good sensitivity,specificity,and repeatability.Therefore,it can be used to detect Henipavirus and other related clinical specimens.展开更多
In liver tumor surgery,the recognition of tumor margin and radical resection of microcancer focis have always been the crucial points to reduce postoperative recurrence of tumor.However,naked-eye inspection and palpat...In liver tumor surgery,the recognition of tumor margin and radical resection of microcancer focis have always been the crucial points to reduce postoperative recurrence of tumor.However,naked-eye inspection and palpation have limited effectiveness in identifying tumor boundaries,and traditional imaging techniques cannot consistently locate tumors in real time.As an intraoperative real-time navigation imaging method,NIRfluorescence imaging has been extensively studied for its simplicity,reliable safety,and superior sensitivity,and is expected to improve the accuracy of liver tumor surgery.In recent years,the research focus of NIRfluorescence has gradually shifted from the-rst near-infrared window(NIR-I,700–900 nm)to the second near-infrared window(NIR-II,1000–1700 nm).Fluorescence imaging in NIR-II reduces the scattering effect of deep tissue,providing a preferable detection depth and spatial resolution while signi-cantly eliminating liver autofluorescence background to clarify tumor margin.Developingfluorophores combined with tumor antibodies will further improve the precision offluorescence-guided surgical navigation.With the development of a bunch offluorophores with phototherapy ability,NIR-II can integrate tumor detection and treatment to explore a new therapeutic strategy for liver cancer.Here,we review the recent progress of NIR-IIfluorescence technology in liver tumor surgery and discuss its challenges and potential development direction.展开更多
Deep engineering disasters,such as rockbursts and collapses,are more related to the shear slip of rock joints.A novel multifunctional device was developed to study the shear failure mechanism in rocks.Using this devic...Deep engineering disasters,such as rockbursts and collapses,are more related to the shear slip of rock joints.A novel multifunctional device was developed to study the shear failure mechanism in rocks.Using this device,the complete shearedeformation process and long-term shear creep tests could be performed on rocks under constant normal stiffness(CNS)or constant normal loading(CNL)conditions in real-time at high temperature and true-triaxial stress.During the research and development process,five key technologies were successfully broken through:(1)the ability to perform true-triaxial compressioneshear loading tests on rock samples with high stiffness;(2)a shear box with ultra-low friction throughout the entire stress space of the rock sample during loading;(3)a control system capable of maintaining high stress for a long time and responding rapidly to the brittle fracture of a rock sample as well;(4)a refined ability to measure the volumetric deformation of rock samples subjected to true triaxial shearing;and(5)a heating system capable of maintaining uniform heating of the rock sample over a long time.By developing these technologies,loading under high true triaxial stress conditions was realized.The apparatus has a maximum normal stiffness of 1000 GPa/m and a maximum operating temperature of 300C.The differences in the surface temperature of the sample are constant to within5C.Five types of true triaxial shear tests were conducted on homogeneous sandstone to verify that the apparatus has good performance and reliability.The results show that temperature,lateral stress,normal stress and time influence the shear deformation,failure mode and strength of the sandstone.The novel apparatus can be reliably used to conduct true-triaxial shear tests on rocks subjected to high temperatures and stress.展开更多
Chymosin is one of the critical enzymes in cheese making.Herein,we proposed a novel fluorometric assay for chymosin determination.Firstly,covalent organic frameworks(COF)were synthesized and exfoliated to 2-dimensiona...Chymosin is one of the critical enzymes in cheese making.Herein,we proposed a novel fluorometric assay for chymosin determination.Firstly,covalent organic frameworks(COF)were synthesized and exfoliated to 2-dimensional COF nanosheets(COF NS)by ultrasound treatment.Gold nanoparticles(Au NPs)were loaded with COF NS to prepare AuNPs/COF NS(Au@COF NS).Secondly,rhodamine B(RhB)modified substrate peptide(Pep)for chymosin was linked with Au@COF NS to construct a Pep-Au@COF NS nanocomposite.For the sensing principle,fluorescence of RhB was quenched by Au@COF NS and the fluorescence intensity was weak due to the fluorescence resonance energy transfer between COF NS and RhB of Pep.However,in the presence of chymosin,the RhB was released by specific cleavage of the substrate peptide by chymosin and resulted in the recovery of fluorescence.The increased fluorescence intensity was proportional to the increase of chymosin concentration and thus a“turn on”fluorescent sensor for chymosin was constructed.The sensor showed a linear range in the concentration of 0.05-60.00μg/mL for the detection of chymosin with a detection limit of 20 ng/mL.The sensor was used to quantify chymosin in rennet product with good selectivity,which has the potential applications in cheese manufacturing.展开更多
A real-time data processing system is designed for the carbon dioxide dispersion interferometer(CO_(2)-DI)on EAST.The system utilizes the parallel and pipelining capabilities of an fieldprogrammable gate array(FPGA)to...A real-time data processing system is designed for the carbon dioxide dispersion interferometer(CO_(2)-DI)on EAST.The system utilizes the parallel and pipelining capabilities of an fieldprogrammable gate array(FPGA)to digitize and process the intensity of signals from the detector.Finally,the real-time electron density signals are exported through a digital-to-analog converter(DAC)module in the form of analog signals.The system has been successfully applied in the CO_(2)-DI system to provide low-latency electron density input to the plasma control system on EAST.Experimental results of the latest campaign with long-pulse discharges on EAST(2022–2023)demonstrate that the system can respond effectively in the case of rapid density changes,proving its reliability and accuracy for future electron density calculation.展开更多
The co-frequency vibration fault is one of the common faults in the operation of rotating equipment,and realizing the real-time diagnosis of the co-frequency vibration fault is of great significance for monitoring the...The co-frequency vibration fault is one of the common faults in the operation of rotating equipment,and realizing the real-time diagnosis of the co-frequency vibration fault is of great significance for monitoring the health state and carrying out vibration suppression of the equipment.In engineering scenarios,co-frequency vibration faults are highlighted by rotational frequency and are difficult to identify,and existing intelligent methods require more hardware conditions and are exclusively time-consuming.Therefore,Lightweight-convolutional neural networks(LW-CNN)algorithm is proposed in this paper to achieve real-time fault diagnosis.The critical parameters are discussed and verified by simulated and experimental signals for the sliding window data augmentation method.Based on LW-CNN and data augmentation,the real-time intelligent diagnosis of co-frequency is realized.Moreover,a real-time detection method of fault diagnosis algorithm is proposed for data acquisition to fault diagnosis.It is verified by experiments that the LW-CNN and sliding window methods are used with high accuracy and real-time performance.展开更多
To address the impact of wind-power fluctuations on the stability of power systems,we propose a comprehensive approach that integrates multiple strategies and methods to enhance the efficiency and reliability of a sys...To address the impact of wind-power fluctuations on the stability of power systems,we propose a comprehensive approach that integrates multiple strategies and methods to enhance the efficiency and reliability of a system.First,we employ a strategy that restricts long-and short-term power output deviations to smoothen wind power fluctuations in real time.Second,we adopt the sliding window instantaneous complete ensemble empirical mode decomposition with adaptive noise(SW-ICEEMDAN)strategy to achieve real-time decomposition of the energy storage power,facilitating internal power distribution within the hybrid energy storage system.Finally,we introduce a rule-based multi-fuzzy control strategy for the secondary adjustment of the initial power allocation commands for different energy storage components.Through simulation validation,we demonstrate that the proposed comprehensive control strategy can smoothen wind power fluctuations in real time and decompose energy storage power.Compared with traditional empirical mode decomposition(EMD),ensemble empirical mode decomposition(EEMD),and complete ensemble empirical mode decomposition with adaptive noise(CEEMDAN)decomposition strategies,the configuration of the energy storage system under the SW-ICEEMDAN control strategy is more optimal.Additionally,the state-of-charge of energy storage components fluctuates within a reasonable range,enhancing the stability of the power system and ensuring the secure operation of the energy storage system.展开更多
基金supported by National Natural Science Foundation of China(Grant No.30671397 and No.30871655)the Public Beneficial Research Project of Agricultural Ministry,China(Grant No.nyhyzx07-056)
文摘Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.
基金This work was supported by grants from the Special Science and Technology Cooperation Project of Ningxia Hui Autonomous Region Key R&D Program(No.2018BFG02008)the National Science and Technology Key Projects on"Major Infectious Diseases such as HIV/AIDS,Viral Hepatitis Prevention and Treatment"(No.2017ZX10103005).
文摘Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.
基金the National Natural Science Foundation of China (No. 30460145).
文摘Objective: To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma (NPC) tissues. Methods: The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients' nasopharynx tissues treated as control group. Results: The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients' nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients' nasopharynx tissues, the difference was significant (P 〈 0.01). The expression of Survivin mRNA could be detected both in stage Ⅰ + Ⅱ and stage Ⅲ + Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups (P 〉 0.05). There was no relationship between Survivin mRNA expression and age and sex of NPC patients (P 〉 0.05). Conclusion: Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.
基金Supported by Special Funds for Basic Scientific Research of Guangxi Sugarcane Research Institute(G2009006,G2010006,G2009015)Sci-tech Research and Development Program of Guangxi Academy of Agricultural Sciences(200805)
文摘This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease (RSD) in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.
基金This work was supported by Science Project from Science and Tech- nology Department of HuBei province(2006AA301B56-3)
文摘Objective: Multidrug resistance(MDR) is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia(ALL) which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction(FQ-RT-PCR), and combine minimal residual desease(MRD) detection by flow cytometry(FCM) and to study their relationship with treatment response and prognosis of ALL. Methods:The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission(CR), 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results:Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group(P 〉 0.05), but significantly higher in the recurrence group than that in newly diagnosed disease group and control group(0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05). 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration(0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01). For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group(P〉 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group(0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05). Conclusion:The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration(within 3 years). Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.
文摘[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses (WSSV, IHHNV, TSV and YHV ). [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies (E = 1.06, 1.07, 0.92 and 0.92, respectively), good hnear relationship ( r = 1 ), uniform repeatability ( standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ) and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR (average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ). The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.
基金Supported by Science and Technology Research Project of Universities in Hebei Province,China(QN2014220)
文摘Porcine epidemic diarrhea,a highly contagious enteric infectious disease caused by the porcine epidemic diarrhea virus(PEDV)with symptoms of vomit,diarrhea,loss of appetite of suckling pig,has led to serious economic loss to the global swine industry.In this study,a real-time fluorescence reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay was developed to detect PEDV RNA.The real-time fluorescence RT-LAMP assay was performed at62℃for 60 min,using a simple and portable device,the ESE-Quant Tube Scanner.The detection limit of RNA was 2.9×10^(6) copies/μl,10 times as sensitive as RT-PCR,and the detection was specific only to PEDV.Application of this method to clinical samples yielded a positivity rate of 93%,which was higher than that of RT-PCR.This technique saves time and is efficient,and is thus expected to be useful for the diagnosis of PEDV infection in the field.
文摘According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 (NC001718) available in GenBank (NC_001718), a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.
文摘Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false<span style="font-family:;" "=""> </span><span style="font-family:Verdana;">negatives, and benefitting early diagnosis.</span>
文摘Technological improvements are crucial in the evolution of surgery.Real-time fluorescence-guided surgery(FGS)has spread worldwide,mainly because of its usefulness during the intraoperative decision-making processes.The success of any gastrointestinal oncologic resection is based on the anatomical identification of the primary tumor and its regional lymph nodes.FGS allows also to evaluate the blood perfusion at the gastrointestinal stumps after colorectal or esophageal resections.Therefore,a reduction on the anastomotic leak rates has been postulated as one of the foreseeable benefits provided by the use of FGS in these procedures.Although the use of fluorescence in lymph node detection was initially described in breast cancer surgery,the technique is currently applied in gastric or splenic flexure cancers,as they both present complex and variable lymphatic drainages.FGS allows also to perform intraoperative lymphograms or sentinel lymph node biopsies.New applications of FGS are being developed to assist in the detection of peritoneal metastases or in the evaluation of the tumor resection margins.The present review aims to provide a general overview of the current status of real-time FGS in gastrointestinal oncologic surgery.We put a special focus on the different applications of FGS,discussing the main findings and limitations found in the contemporary literature and also the promising near future applications.
基金Supported by Project of Jilin Province Science and Technology Commission(20080218)
文摘[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes virus (KHV) pol-ymerase gene (Sph) to establish a rapid and effective fluorescence quantitative PCR method for Koi herpes virus detection. The cell cultures were detected by using the established fluorescence quantitative PCR assay, and the results were com- pared with that of conventional PCR. [ Result] The sensitivity of fluorescence quantitative PCR was higher than that of conventional PCR. The minimum copy num- ber that could be detected was 1.6 - 102 copies/p.1. The established method was adopted for sample detection, and a reliable diagnostic result could be obtained within 4 h. [Conclusion] The established method is rapid, sensitive, specific and repeatable, which is conducive to the rapid detection of Koi herpes virus. Key words Koi herpes virus; Fluorescence quantitative PCR; Detection
基金supported by the National Key R&D Program of China(Grant No.2022YFC2601200).
文摘The emerging viruses within the genus Henipavirus in the family Paramyxoviridae pose a great threat to public biosafety.To develop a quadruple real-time fluorescence-based quantitative reverse transcription polymerase chain reaction(qRT-PCR)assay is pivotal for the early warning of the potential of zoonotic infectious diseases.Specific primers and probes were designed for the relatively conserved regions based on whole genome sequences of Langya virus(LayV),Mojiang virus(MojV),Nipah virus(NiV),and Cedar virus(CedV),followed by the establishment of a quadruple real-time fluorescence-based qRT-PCR detection method.No cross-reactivity was observed with other viral nucleic acids.The optimal linear detection range for LayV,MojV,NiV,and CedV was 10^(1)-10^(8)copies/μL,and the lower limit of detection was 10 copies/μL.Three different DNA concentrations of LayV,MojV,NiV,and CedV(10^(4),10^(5),and 10^(6)copies/μL)were tested 14 times,achieving good repeatability.The standard deviation of the cycle threshold values for each concentration was<0.5 and the coefficient of variation was<3%.Furthermore,the amplification efficiency of quadruple real-time fluorescence-based qRT-PCR was>90%,and the correlation coefficient was>0.99.The established quadru-ple real-time fluorescence-based qRT-PCR assay for the detection of LayV,MojV,NiV,and CedV exhibits good sensitivity,specificity,and repeatability.Therefore,it can be used to detect Henipavirus and other related clinical specimens.
基金supported by the National Key R&D Program of China(No.2020YFA0710700)the National Natural Science Foundation of China(Nos.51873201 and 82172071)+2 种基金Key Research and Development Program of Anhui Province(No.202104b11020025)the Fundamental Research Funds for the Central Universities(No.YD2060002015)the CAS Youth Interdisciplinary Team(No.JCTD-2021-08).
文摘In liver tumor surgery,the recognition of tumor margin and radical resection of microcancer focis have always been the crucial points to reduce postoperative recurrence of tumor.However,naked-eye inspection and palpation have limited effectiveness in identifying tumor boundaries,and traditional imaging techniques cannot consistently locate tumors in real time.As an intraoperative real-time navigation imaging method,NIRfluorescence imaging has been extensively studied for its simplicity,reliable safety,and superior sensitivity,and is expected to improve the accuracy of liver tumor surgery.In recent years,the research focus of NIRfluorescence has gradually shifted from the-rst near-infrared window(NIR-I,700–900 nm)to the second near-infrared window(NIR-II,1000–1700 nm).Fluorescence imaging in NIR-II reduces the scattering effect of deep tissue,providing a preferable detection depth and spatial resolution while signi-cantly eliminating liver autofluorescence background to clarify tumor margin.Developingfluorophores combined with tumor antibodies will further improve the precision offluorescence-guided surgical navigation.With the development of a bunch offluorophores with phototherapy ability,NIR-II can integrate tumor detection and treatment to explore a new therapeutic strategy for liver cancer.Here,we review the recent progress of NIR-IIfluorescence technology in liver tumor surgery and discuss its challenges and potential development direction.
基金financial support from the National Natural Science Foundation of China(Grant Nos.52209125 and 51839003).
文摘Deep engineering disasters,such as rockbursts and collapses,are more related to the shear slip of rock joints.A novel multifunctional device was developed to study the shear failure mechanism in rocks.Using this device,the complete shearedeformation process and long-term shear creep tests could be performed on rocks under constant normal stiffness(CNS)or constant normal loading(CNL)conditions in real-time at high temperature and true-triaxial stress.During the research and development process,five key technologies were successfully broken through:(1)the ability to perform true-triaxial compressioneshear loading tests on rock samples with high stiffness;(2)a shear box with ultra-low friction throughout the entire stress space of the rock sample during loading;(3)a control system capable of maintaining high stress for a long time and responding rapidly to the brittle fracture of a rock sample as well;(4)a refined ability to measure the volumetric deformation of rock samples subjected to true triaxial shearing;and(5)a heating system capable of maintaining uniform heating of the rock sample over a long time.By developing these technologies,loading under high true triaxial stress conditions was realized.The apparatus has a maximum normal stiffness of 1000 GPa/m and a maximum operating temperature of 300C.The differences in the surface temperature of the sample are constant to within5C.Five types of true triaxial shear tests were conducted on homogeneous sandstone to verify that the apparatus has good performance and reliability.The results show that temperature,lateral stress,normal stress and time influence the shear deformation,failure mode and strength of the sandstone.The novel apparatus can be reliably used to conduct true-triaxial shear tests on rocks subjected to high temperatures and stress.
基金supported by Major Science and Technology Project of Yunnan Province(202302AE090022)Key Research and Development Program of Yunnan(202203AC100010)+4 种基金the National Natural Science Foundation of China(32160597,32160236,32371463)National Key Research and Development Program of China(2022YFC2601604)Cardiovascular Ultrasound Innovation Team of Yunnan Province(202305AS350021)Spring City Plan:the High-Level Talent Promotion and Training Project of Kunming(2022SCP001)Graduate Tutor Team of Yunnan Province,and the Second Phase of"Double-First Class"Program Construction of Yunnan University.
文摘Chymosin is one of the critical enzymes in cheese making.Herein,we proposed a novel fluorometric assay for chymosin determination.Firstly,covalent organic frameworks(COF)were synthesized and exfoliated to 2-dimensional COF nanosheets(COF NS)by ultrasound treatment.Gold nanoparticles(Au NPs)were loaded with COF NS to prepare AuNPs/COF NS(Au@COF NS).Secondly,rhodamine B(RhB)modified substrate peptide(Pep)for chymosin was linked with Au@COF NS to construct a Pep-Au@COF NS nanocomposite.For the sensing principle,fluorescence of RhB was quenched by Au@COF NS and the fluorescence intensity was weak due to the fluorescence resonance energy transfer between COF NS and RhB of Pep.However,in the presence of chymosin,the RhB was released by specific cleavage of the substrate peptide by chymosin and resulted in the recovery of fluorescence.The increased fluorescence intensity was proportional to the increase of chymosin concentration and thus a“turn on”fluorescent sensor for chymosin was constructed.The sensor showed a linear range in the concentration of 0.05-60.00μg/mL for the detection of chymosin with a detection limit of 20 ng/mL.The sensor was used to quantify chymosin in rennet product with good selectivity,which has the potential applications in cheese manufacturing.
基金funded and supported by the Comprehensive Research Facility for Fusion Technology Program of China(No.2018-000052-73-01-001228)the HFIPS Director’s Fund(No.YZJJKX202301)+1 种基金the Anhui Provincial Major Science and Technology Project(No.2023z020004)Task JB22001 from the Anhui Provincial Department of Economic and Information Technology。
文摘A real-time data processing system is designed for the carbon dioxide dispersion interferometer(CO_(2)-DI)on EAST.The system utilizes the parallel and pipelining capabilities of an fieldprogrammable gate array(FPGA)to digitize and process the intensity of signals from the detector.Finally,the real-time electron density signals are exported through a digital-to-analog converter(DAC)module in the form of analog signals.The system has been successfully applied in the CO_(2)-DI system to provide low-latency electron density input to the plasma control system on EAST.Experimental results of the latest campaign with long-pulse discharges on EAST(2022–2023)demonstrate that the system can respond effectively in the case of rapid density changes,proving its reliability and accuracy for future electron density calculation.
基金Supported by National Natural Science Foundation of China(Grant Nos.51875031,52242507)Beijing Municipal Natural Science Foundation of China(Grant No.3212010)Beijing Municipal Youth Backbone Personal Project of China(Grant No.2017000020124 G018).
文摘The co-frequency vibration fault is one of the common faults in the operation of rotating equipment,and realizing the real-time diagnosis of the co-frequency vibration fault is of great significance for monitoring the health state and carrying out vibration suppression of the equipment.In engineering scenarios,co-frequency vibration faults are highlighted by rotational frequency and are difficult to identify,and existing intelligent methods require more hardware conditions and are exclusively time-consuming.Therefore,Lightweight-convolutional neural networks(LW-CNN)algorithm is proposed in this paper to achieve real-time fault diagnosis.The critical parameters are discussed and verified by simulated and experimental signals for the sliding window data augmentation method.Based on LW-CNN and data augmentation,the real-time intelligent diagnosis of co-frequency is realized.Moreover,a real-time detection method of fault diagnosis algorithm is proposed for data acquisition to fault diagnosis.It is verified by experiments that the LW-CNN and sliding window methods are used with high accuracy and real-time performance.
基金supported by the National Natural Science Foundation of China(Grant No.51677058)。
文摘To address the impact of wind-power fluctuations on the stability of power systems,we propose a comprehensive approach that integrates multiple strategies and methods to enhance the efficiency and reliability of a system.First,we employ a strategy that restricts long-and short-term power output deviations to smoothen wind power fluctuations in real time.Second,we adopt the sliding window instantaneous complete ensemble empirical mode decomposition with adaptive noise(SW-ICEEMDAN)strategy to achieve real-time decomposition of the energy storage power,facilitating internal power distribution within the hybrid energy storage system.Finally,we introduce a rule-based multi-fuzzy control strategy for the secondary adjustment of the initial power allocation commands for different energy storage components.Through simulation validation,we demonstrate that the proposed comprehensive control strategy can smoothen wind power fluctuations in real time and decompose energy storage power.Compared with traditional empirical mode decomposition(EMD),ensemble empirical mode decomposition(EEMD),and complete ensemble empirical mode decomposition with adaptive noise(CEEMDAN)decomposition strategies,the configuration of the energy storage system under the SW-ICEEMDAN control strategy is more optimal.Additionally,the state-of-charge of energy storage components fluctuates within a reasonable range,enhancing the stability of the power system and ensuring the secure operation of the energy storage system.