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
Uncertainties in structure properties can result in different responses in hybrid simulations. Quantification of the effect of these tmcertainties would enable researchers to estimate the variances of structural respo...Uncertainties in structure properties can result in different responses in hybrid simulations. Quantification of the effect of these tmcertainties would enable researchers to estimate the variances of structural responses observed from experiments. This poses challenges for real-time hybrid simulation （RTHS） due to the existence of actuator delay. Polynomial chaos expansion （PCE） projects the model outputs on a basis of orthogonal stochastic polynomials to account for influences of model uncertainties. In this paper, PCE is utilized to evaluate effect of actuator delay on the maximum displacement from real-time hybrid simulation of a single degree of freedom （SDOF） structure when accounting for uncertainties in structural properties. The PCE is first applied for RTHS without delay to determine the order of PCE, the number of sample points as well as the method for coefficients calculation. The PCE is then applied to RTHS with actuator delay. The mean, variance and Sobol indices are compared and discussed to evaluate the effects of actuator delay on uncertainty quantification for RTHS. Results show that the mean and the variance of the maximum displacement increase linearly and exponentially with respect to actuator delay, respectively. Sensitivity analysis through Sobol indices also indicates the influence of the single random variable decreases while the coupling effect increases with the increase of actuator delay.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] 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
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
The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle compone...The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle component and multivariate analyses. Twelve media combinations including 0.1% and 3% glucose as carbon source and no (0 mmol/L), low (10 mmol/L) and high (100 mmol/L) ammonium acetate as nitrogen source combined with or without colloidal chitin at 3 time intervals and 2 replications were applied to current study. The real-time RT-PCR analysis showed that the expression of ech30, ech42 and nag1 was regulated by the interaction of nitrogen, glucose and chitin under different growth conditions. The highest and earliest expressions of ech30 were induced by glucose and nitrogen starvation i.e. 0.1% glucose and 10 mmol/L ammonium acetate in the growth media. This was also the case for ech42 and nag1 but at a relatively low level. In contrast, high (3%) glucose and high (100 mmol/L) ammonium acetate concentrations repressed the expression of all the genes studied. These results were confirmed by principle component and multivariate analyses. The effect of chitin on ech30, ech42 and nag1 expression varied depending on the concentrations of glucose and ammonium acetate.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
The species distinctive PCR primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of s...The species distinctive PCR primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR （RT-PCR）. Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.展开更多
A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR （RTFQ-IPCR） assay using a molecular beacon was developed for the determination of trace fluoranthene （FL） in the environment.Under...A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR （RTFQ-IPCR） assay using a molecular beacon was developed for the determination of trace fluoranthene （FL） in the environment.Under optimized assay conditions,FL can be determined in the concentration range from 1 fg/mL to 100 ng/mL,with y=0.194x ＋ 7.859,and a correlation coefficient of 0.967 was identified,with a detection limit of 0.6 fg/mL.Environmental water samples were successfully analyzed,recovery was between 90% and 116%,with intra-day relative standard deviation （RSD） of 6.7%-12.8% and inter-day RSD of 8.4%-15.2%.The results obtained from RTFQ-IPCR were confirmed by ELISA,showing good accuracy and suitability to analyze FL in field samples.As a highly sensitive method,the molecular beacon-based RTFQ-IPCR is acceptable and promising for providing reliable test results to make environmental decisions.展开更多
Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagn...Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.展开更多
[ Objective] To develop a real-time fluorescent PCR assay for rapid detection of Haempohlius parasuis （HPS）. [ Method] According to the conservative sequences of 16 S rRNA genes of HPS published in GenBank, a pair o...[ Objective] To develop a real-time fluorescent PCR assay for rapid detection of Haempohlius parasuis （HPS）. [ Method] According to the conservative sequences of 16 S rRNA genes of HPS published in GenBank, a pair of specific primers was designed. The real-time fluorescent PCR was developed by optimizing primer concentration and annealing temperature. And its specificity and reproducibility were evaluated. Ten HPS- suspected samples were detected by the developed method. [ Result] The lowest detection limit of the developed real-time fluorescent PCR was 50 copies/μl. This method had good reproducibility, and its coefficient of variation was lower than 2%. Only HPS rather than Streptococcus suis type 2, Staphylococcus aureus, E. coli DH5 alpha, and swine Salmonella typhi could be detected by the developed real-time fluorescent PCR. The HPS-pesitive samples detected by this method were also positive when they were detected by isolation of bacteria or conventional PCR. [ Conclusion] The developed real-time fluorescent PCR is rapid, sensitive, specific and highly reproducible; thus, it can be used for rapid detection of HPS.展开更多
Nervous necrosis virus （NNV） is the causative agent of fulminant infectious diseases in marine fishes such as grouper. Specific primers were designed based on the conserved sequence of capsid protein （CP） gene of ...Nervous necrosis virus （NNV） is the causative agent of fulminant infectious diseases in marine fishes such as grouper. Specific primers were designed based on the conserved sequence of capsid protein （CP） gene of red-spotted grouper nervous necrosis virus （NNV）. By optimizing the reaction conditions, a rapid and simple reverse transcription loop-mediated isothermal amplification （RT-LAMP） method was established for NNV detection. After adding SYTO-9 fluorescent dye in the reaction system, the amplification curve was monitored in real time using a fluorescence detector, and the result was obviously easy to assess. Moreover, the specificity and sensitivity of the established method was analyzed. The results showed that the established RT-LAMP method has good specificity with a detection limit of 1.3 pg/μl. The detection sensitivity of the established RT-LAMP method is 100 times that of the conventional RT-PCR method, and the detection duration is only 40 min. The established RT-LAMP method is suitable for quarantine and rapid detection of grouper nervous necrosis virus.展开更多
Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut an...Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut and Ses i 1 gene of sesame.After the optimization of reaction conditions,a real-time fluorescent PCR method was established for simultaneous detection of allergenic ingredients of peanut and sesame in food.Genomic DNA samples of peanut,sesame,rice,wheat,barley,soybean,celery,maize,potato,tomato,walnut,groundnut in shell,cashew nut,sunflower seed,almond,apple,pear and strawberry,pork,beef,mutton and fish were used as templates for PCR amplification with deionized water as negative control template.Results indicated that the established real-time fluorescent PCR method could specifically identify allergenic ingredients of peanut and sesame simultaneously.Sensitivity test showed that the minimum detection limit of this method was 0.01%.Therefore,the established real-time fluorescent PCR method is a specific,sensitive and effective assay for simultaneously detecting allergenic ingredients of peanut and sesame in food.展开更多
In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent ...In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.展开更多
目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-...目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。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National Science Foundation of China under grant No.51378107Fundamental Research Funds for the Central Universities and Doctoral Research Fund by Southeast University under Grant No.YBJJ-1442
文摘Uncertainties in structure properties can result in different responses in hybrid simulations. Quantification of the effect of these tmcertainties would enable researchers to estimate the variances of structural responses observed from experiments. This poses challenges for real-time hybrid simulation （RTHS） due to the existence of actuator delay. Polynomial chaos expansion （PCE） projects the model outputs on a basis of orthogonal stochastic polynomials to account for influences of model uncertainties. In this paper, PCE is utilized to evaluate effect of actuator delay on the maximum displacement from real-time hybrid simulation of a single degree of freedom （SDOF） structure when accounting for uncertainties in structural properties. The PCE is first applied for RTHS without delay to determine the order of PCE, the number of sample points as well as the method for coefficients calculation. The PCE is then applied to RTHS with actuator delay. The mean, variance and Sobol indices are compared and discussed to evaluate the effects of actuator delay on uncertainty quantification for RTHS. Results show that the mean and the variance of the maximum displacement increase linearly and exponentially with respect to actuator delay, respectively. Sensitivity analysis through Sobol indices also indicates the influence of the single random variable decreases while the coupling effect increases with the increase of actuator delay.
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.
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The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle component and multivariate analyses. Twelve media combinations including 0.1% and 3% glucose as carbon source and no (0 mmol/L), low (10 mmol/L) and high (100 mmol/L) ammonium acetate as nitrogen source combined with or without colloidal chitin at 3 time intervals and 2 replications were applied to current study. The real-time RT-PCR analysis showed that the expression of ech30, ech42 and nag1 was regulated by the interaction of nitrogen, glucose and chitin under different growth conditions. The highest and earliest expressions of ech30 were induced by glucose and nitrogen starvation i.e. 0.1% glucose and 10 mmol/L ammonium acetate in the growth media. This was also the case for ech42 and nag1 but at a relatively low level. In contrast, high (3%) glucose and high (100 mmol/L) ammonium acetate concentrations repressed the expression of all the genes studied. These results were confirmed by principle component and multivariate analyses. The effect of chitin on ech30, ech42 and nag1 expression varied depending on the concentrations of glucose and ammonium acetate.
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The species distinctive PCR primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR （RT-PCR）. Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.
基金support by the Scienceand Technology Commission of Shanghai Municipality in China (Key Project of Fundamental Research) (No.09JC1407600)the Science and Technology Commission of Shanghai Municipality in China (Key Project of theScience and Technology Research) (No. 09231202805)the Shanghai Leading Academic Discipline Project(No. B604)
文摘A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR （RTFQ-IPCR） assay using a molecular beacon was developed for the determination of trace fluoranthene （FL） in the environment.Under optimized assay conditions,FL can be determined in the concentration range from 1 fg/mL to 100 ng/mL,with y=0.194x ＋ 7.859,and a correlation coefficient of 0.967 was identified,with a detection limit of 0.6 fg/mL.Environmental water samples were successfully analyzed,recovery was between 90% and 116%,with intra-day relative standard deviation （RSD） of 6.7%-12.8% and inter-day RSD of 8.4%-15.2%.The results obtained from RTFQ-IPCR were confirmed by ELISA,showing good accuracy and suitability to analyze FL in field samples.As a highly sensitive method,the molecular beacon-based RTFQ-IPCR is acceptable and promising for providing reliable test results to make environmental decisions.
基金Supported by National Natural Science Foundation of China ( 30800885,30871726)
文摘Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.
基金funded by the Key Technologies R&D Program of Guangxi of China (0993009-1)
文摘[ Objective] To develop a real-time fluorescent PCR assay for rapid detection of Haempohlius parasuis （HPS）. [ Method] According to the conservative sequences of 16 S rRNA genes of HPS published in GenBank, a pair of specific primers was designed. The real-time fluorescent PCR was developed by optimizing primer concentration and annealing temperature. And its specificity and reproducibility were evaluated. Ten HPS- suspected samples were detected by the developed method. [ Result] The lowest detection limit of the developed real-time fluorescent PCR was 50 copies/μl. This method had good reproducibility, and its coefficient of variation was lower than 2%. Only HPS rather than Streptococcus suis type 2, Staphylococcus aureus, E. coli DH5 alpha, and swine Salmonella typhi could be detected by the developed real-time fluorescent PCR. The HPS-pesitive samples detected by this method were also positive when they were detected by isolation of bacteria or conventional PCR. [ Conclusion] The developed real-time fluorescent PCR is rapid, sensitive, specific and highly reproducible; thus, it can be used for rapid detection of HPS.
基金Supported by Application Technology R&D and Demonstration Project of Hainan Province(ZDXM2015025)
文摘Nervous necrosis virus （NNV） is the causative agent of fulminant infectious diseases in marine fishes such as grouper. Specific primers were designed based on the conserved sequence of capsid protein （CP） gene of red-spotted grouper nervous necrosis virus （NNV）. By optimizing the reaction conditions, a rapid and simple reverse transcription loop-mediated isothermal amplification （RT-LAMP） method was established for NNV detection. After adding SYTO-9 fluorescent dye in the reaction system, the amplification curve was monitored in real time using a fluorescence detector, and the result was obviously easy to assess. Moreover, the specificity and sensitivity of the established method was analyzed. The results showed that the established RT-LAMP method has good specificity with a detection limit of 1.3 pg/μl. The detection sensitivity of the established RT-LAMP method is 100 times that of the conventional RT-PCR method, and the detection duration is only 40 min. The established RT-LAMP method is suitable for quarantine and rapid detection of grouper nervous necrosis virus.
基金Supported by Scientific Research Project of Anhui Bureau of Quality and Technical Supervision(13zj370033)
文摘Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut and Ses i 1 gene of sesame.After the optimization of reaction conditions,a real-time fluorescent PCR method was established for simultaneous detection of allergenic ingredients of peanut and sesame in food.Genomic DNA samples of peanut,sesame,rice,wheat,barley,soybean,celery,maize,potato,tomato,walnut,groundnut in shell,cashew nut,sunflower seed,almond,apple,pear and strawberry,pork,beef,mutton and fish were used as templates for PCR amplification with deionized water as negative control template.Results indicated that the established real-time fluorescent PCR method could specifically identify allergenic ingredients of peanut and sesame simultaneously.Sensitivity test showed that the minimum detection limit of this method was 0.01%.Therefore,the established real-time fluorescent PCR method is a specific,sensitive and effective assay for simultaneously detecting allergenic ingredients of peanut and sesame in food.
基金Supported by Project of Standardization Technical System from the Administration of Quality and Technology Supervision of Sichuan Province(ZYBZ2013-39)
文摘In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.
文摘目的建立快速、特异性好、灵敏度高的Real-Time PCR方法定量检测沙门菌。方法根据编码沙门菌肠毒素基因stn的核苷酸序列,设计荧光探针和一对引物,通过对荧光定量PCR反应体系和反应条件的摸索,建立定量检测沙门菌的方法。结果建立的Real-Ti me PCR方法有很好的特异性与敏感性,所检测沙门菌结果均为阳性,而非沙门菌均为阴性;标准曲线相关系数为R2=0.993,其敏感性为5CFU。运用该方法对108份鸡粪便、50份鸡肉以及58份水样进行检测,阳性率分别为3.7%(6/108)、4%(2/50)和3.4%(2/58),与传统细菌分离检测结果相符。结论结果表明该方法具有简便、快速、特异性强、敏感性高等特点,此研究为环境及疾病诊断中沙门菌快速检测提供了新方法。
