Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis an...Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis and experimental autoimmune myocarditis and MIP-2γ mRNA expression in mouse was studied by TaqMan real-time fluorescent quantitative RT-PCR. Results: MIP-2γ mRNA expression rose on 3 to 5 d after CVB3 infection, reached peak on 7 d, and returned to normal level until 14 d, which corresponded well with the disease course. The MIP-2γ mRNA expression level rose significantly on the day 18 d after immunization with porcine cardiac myosin, which was consistent with pathological examination. Conclusion: MIP-2γ may be involved in the pathogenesis of myocarditis.展开更多
BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in di...BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.展开更多
AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA seque...AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis.展开更多
The species distinctive PCR primer of Lactobacillus acidophilus( L. acidophilus) was designed according to 16 S rRNA gene sequences of common Lactobacillus species in fermented material. Bacterial genome DNA of separa...The species distinctive PCR primer of Lactobacillus acidophilus( L. acidophilus) was designed according to 16 S rRNA gene sequences of common Lactobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template,and L. acidophilus in fermented material was conducted the quantitative determination by real-time quantitative PCR( RT-PCR). Analysis on RT-PCR results shown that contents of L. acidophilus 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 applied 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 opt...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-time 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-time 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 disadvantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the application and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.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 real-tim...[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 generated typical amplification curves with high amplification efficiencies(E = 1. 06,1. 07,0. 92 and 0. 92,respectively),good linear 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 real-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
Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( cla...Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry.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 quant...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 replications.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 4;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 organisms and products in China.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) whi...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<50×109 group(P > 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD>103 group than that in MRD<103 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 marrow 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]To establish a real-time fluorescent quantitative polymerase chain reaction(PCR) method with SYBR Green I for the detection of porcine circovirus type 2(PCV2).[Methods]Specific primers were designed to ampl...[Objective]To establish a real-time fluorescent quantitative polymerase chain reaction(PCR) method with SYBR Green I for the detection of porcine circovirus type 2(PCV2).[Methods]Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR.The amplified gene was cloned into the vector of pMD~18-T and transformed into DH5α to screen positive clones.After being extracted and purified,the recombinant plasmids pMD~ 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2,and the PCR reaction conditions were optimized.[Results]Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21×10~0-4.16×10~8 copies /μL,the correlation coefficient was 0.998 8 and the slope was-3.286.The method did not show any cross-reactions with the genomes of PRRSV,PCV1,CSFV,PRV,PPV and Escherichia coli.Sensitivity of this method was proved to be 3.21×10° copies/μL,which was 1 000 times higher as conventional PCR method.Variation coefficients of the repeated trials among same batch or different batches were both less than 3.00%.Positive rate of clinical samples detected by the established PCR method was 58.94%,which was significantly higher than the detection rate by conventional PCR.[Conclusions]A real-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established,which was better for conducting the quantitative analysis and the early diagnosis of PCV2 infection.展开更多
An accurate and reasonable technique combining direct absorption spectroscopy and laser-induced fluorescence(LIF)methods is developed to quantitatively measure the concentrations of hydroxyl in CH_4/air flat laminar f...An accurate and reasonable technique combining direct absorption spectroscopy and laser-induced fluorescence(LIF)methods is developed to quantitatively measure the concentrations of hydroxyl in CH_4/air flat laminar flame. In our approach, particular attention is paid to the linear laser-induced fluorescence and absorption processes, and experimental details as well. Through measuring the temperature, LIF signal distribution and integrated absorption, spatially absolute OH concentrations profiles are successfully resolved. These experimental results are then compared with the numerical simulation. It is proved that the good quality of the results implies that this method is suitable for calibrating the OH-PLIF measurement in a practical combustor.展开更多
The aim of the present work is to quantitatively measure the hydroxyl radical concentration by using LIF(laserinduced fluorescence) in flame.The detailed physical models of spectral absorption lineshape broadening,col...The aim of the present work is to quantitatively measure the hydroxyl radical concentration by using LIF(laserinduced fluorescence) in flame.The detailed physical models of spectral absorption lineshape broadening,collisional transition and quenching at elevated pressure are built.The fine energy level structure of the OH molecule is illustrated to understand the process with laser-induced fluorescence emission and others in the case without radiation,which include collisional quenching,rotational energy transfer(RET),and vibrational energy transfer(VET).Based on these,some numerical results are achieved by simulations in order to evaluate the fluorescence yield at elevated pressure.These results are useful for understanding the real physical processes in OH-LIF technique and finding a way to calibrate the signal for quantitative measurement of OH concentration in a practical combustor.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 six...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 samples. 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
The introduction of multi-methods of Geo-logging at wellsite has become the major measurement of oil & gas exploration. From the early stage of manually geo-logging to the modern mudlogging with new techonlogies o...The introduction of multi-methods of Geo-logging at wellsite has become the major measurement of oil & gas exploration. From the early stage of manually geo-logging to the modern mudlogging with new techonlogies of MWD, LWD and QFT etc. The new technologies have played very important roles in the exploring of oil & gas. Being one of the newest technology of mudlogging, QFT has been widely used in oilfield for about 3 years. When it is put in operation in some oilfields of China in 1997, its advantages in oil & gas detection at wellsite have been continuously recognized,especially in the detection of shows of light oil and condensed oil. A set of powerful classification standard of resource rock oil bearing grades and the interpretation standards have been summarized by the application of the quantitative fluorescencelogging techniques (QFT) in Basins of China, together with gas-logging data, and other information got from the Geo-logging procedures at wellsite.展开更多
Objective: In the prenatal screening, several different methods were used to detect the presence of group B streptococcus (GBS) infection, in this assay, the diagnostic value and clinical significance of the applicati...Objective: In the prenatal screening, several different methods were used to detect the presence of group B streptococcus (GBS) infection, in this assay, the diagnostic value and clinical significance of the application of realtime fluorescent PCR were explored. Methods: A total of 86 women with 35-37 weeks pregnancy were enrolled, vaginal secretion samples were collected. Fluorescence PCR, bacterial culture and gene sequencing were used to detect whether there was GBS infection, and the results obtained were compared and analyzed. Results: 10 subjects were detected to be positive for GBS by fluorescence PCR (the positive rate was 11.6%), however, only 4 cases were positive for GBS by bacterial culture method (the positive rate was 4.7%). There was a statistically significant difference in the positive rate between the two methods (P<0.01). Compared with the results of gene sequencing, the detection of GBS infection by fluorescence PCR has an accuracy of 95.2%, and the sensitivity was 90.9% with 100% specificity. Conclusion: The application of realtime fluorescence quantitative PCR for the detection of GBS infection is significantly better than the use of bacterial culture method. Compared with the gold standard method (gene sequencing method), its detection efficiency, accuracy, sensitivity and specificity are relatively high. In summary, PCR for prenatal screening of GBS is worthy of promotion in clinical practice.展开更多
文摘Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis and experimental autoimmune myocarditis and MIP-2γ mRNA expression in mouse was studied by TaqMan real-time fluorescent quantitative RT-PCR. Results: MIP-2γ mRNA expression rose on 3 to 5 d after CVB3 infection, reached peak on 7 d, and returned to normal level until 14 d, which corresponded well with the disease course. The MIP-2γ mRNA expression level rose significantly on the day 18 d after immunization with porcine cardiac myosin, which was consistent with pathological examination. Conclusion: MIP-2γ may be involved in the pathogenesis of myocarditis.
文摘BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.
基金Supported by The National Key Technology R&D Program of China, No. 2004B A901A03Program for Chang Jiang Scholars and Innovative Research Team in University, No. IRTO753+2 种基金Program for New Century Excellent Talents in University, No. NCET-04-0906Sichuan Province Basic Research Program, No. 04JY0290061Program for Key Disciplines Construction of Sichuan Province, No. SZD0418
文摘AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis.
文摘The species distinctive PCR primer of Lactobacillus acidophilus( L. acidophilus) was designed according to 16 S rRNA gene sequences of common Lactobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template,and L. acidophilus in fermented material was conducted the quantitative determination by real-time quantitative PCR( RT-PCR). Analysis on RT-PCR results shown that contents of L. acidophilus 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 applied 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-time 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 disadvantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the application and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.
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 generated typical amplification curves with high amplification efficiencies(E = 1. 06,1. 07,0. 92 and 0. 92,respectively),good linear 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 real-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 National Natural Science Foundation of China(30630048)National Science and Technology Support Program(2006BAD06A03)
文摘Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry.
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 replications.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 4;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 organisms and products in China.
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<50×109 group(P > 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD>103 group than that in MRD<103 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 marrow 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.
基金Supported by Shandong Province Natural Science Fund Project
文摘[Objective]To establish a real-time fluorescent quantitative polymerase chain reaction(PCR) method with SYBR Green I for the detection of porcine circovirus type 2(PCV2).[Methods]Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR.The amplified gene was cloned into the vector of pMD~18-T and transformed into DH5α to screen positive clones.After being extracted and purified,the recombinant plasmids pMD~ 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2,and the PCR reaction conditions were optimized.[Results]Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21×10~0-4.16×10~8 copies /μL,the correlation coefficient was 0.998 8 and the slope was-3.286.The method did not show any cross-reactions with the genomes of PRRSV,PCV1,CSFV,PRV,PPV and Escherichia coli.Sensitivity of this method was proved to be 3.21×10° copies/μL,which was 1 000 times higher as conventional PCR method.Variation coefficients of the repeated trials among same batch or different batches were both less than 3.00%.Positive rate of clinical samples detected by the established PCR method was 58.94%,which was significantly higher than the detection rate by conventional PCR.[Conclusions]A real-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established,which was better for conducting the quantitative analysis and the early diagnosis of PCV2 infection.
基金supported by the National Natural Science Foundation of China(Grant No.11272338)the Science and Technology on Scramjet Key Laboratory Funding,China(Grant No.STSKFKT 2013004)the China Scholarship Council
文摘An accurate and reasonable technique combining direct absorption spectroscopy and laser-induced fluorescence(LIF)methods is developed to quantitatively measure the concentrations of hydroxyl in CH_4/air flat laminar flame. In our approach, particular attention is paid to the linear laser-induced fluorescence and absorption processes, and experimental details as well. Through measuring the temperature, LIF signal distribution and integrated absorption, spatially absolute OH concentrations profiles are successfully resolved. These experimental results are then compared with the numerical simulation. It is proved that the good quality of the results implies that this method is suitable for calibrating the OH-PLIF measurement in a practical combustor.
基金Project supported by the National Natural Science Foundation of China(Grant No.11272338)the Fund from the Science and Technology on Scramjet Key Laboratory,China(Grant No.STSKFKT2013004)
文摘The aim of the present work is to quantitatively measure the hydroxyl radical concentration by using LIF(laserinduced fluorescence) in flame.The detailed physical models of spectral absorption lineshape broadening,collisional transition and quenching at elevated pressure are built.The fine energy level structure of the OH molecule is illustrated to understand the process with laser-induced fluorescence emission and others in the case without radiation,which include collisional quenching,rotational energy transfer(RET),and vibrational energy transfer(VET).Based on these,some numerical results are achieved by simulations in order to evaluate the fluorescence yield at elevated pressure.These results are useful for understanding the real physical processes in OH-LIF technique and finding a way to calibrate the signal for quantitative measurement of OH concentration in a practical combustor.
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 samples. 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The introduction of multi-methods of Geo-logging at wellsite has become the major measurement of oil & gas exploration. From the early stage of manually geo-logging to the modern mudlogging with new techonlogies of MWD, LWD and QFT etc. The new technologies have played very important roles in the exploring of oil & gas. Being one of the newest technology of mudlogging, QFT has been widely used in oilfield for about 3 years. When it is put in operation in some oilfields of China in 1997, its advantages in oil & gas detection at wellsite have been continuously recognized,especially in the detection of shows of light oil and condensed oil. A set of powerful classification standard of resource rock oil bearing grades and the interpretation standards have been summarized by the application of the quantitative fluorescencelogging techniques (QFT) in Basins of China, together with gas-logging data, and other information got from the Geo-logging procedures at wellsite.
文摘Objective: In the prenatal screening, several different methods were used to detect the presence of group B streptococcus (GBS) infection, in this assay, the diagnostic value and clinical significance of the application of realtime fluorescent PCR were explored. Methods: A total of 86 women with 35-37 weeks pregnancy were enrolled, vaginal secretion samples were collected. Fluorescence PCR, bacterial culture and gene sequencing were used to detect whether there was GBS infection, and the results obtained were compared and analyzed. Results: 10 subjects were detected to be positive for GBS by fluorescence PCR (the positive rate was 11.6%), however, only 4 cases were positive for GBS by bacterial culture method (the positive rate was 4.7%). There was a statistically significant difference in the positive rate between the two methods (P<0.01). Compared with the results of gene sequencing, the detection of GBS infection by fluorescence PCR has an accuracy of 95.2%, and the sensitivity was 90.9% with 100% specificity. Conclusion: The application of realtime fluorescence quantitative PCR for the detection of GBS infection is significantly better than the use of bacterial culture method. Compared with the gold standard method (gene sequencing method), its detection efficiency, accuracy, sensitivity and specificity are relatively high. In summary, PCR for prenatal screening of GBS is worthy of promotion in clinical practice.