[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish emb...[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.展开更多
Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification react...Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.展开更多
[Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another ...[Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another pair of primers was designed based on the conserved gene sequences of Mycoplasma. The competitive template, which carried the same primer binding site with the target fragment, was constructed using enzyme digestion method. [Result] The logarithm of concentration of competitive template was treated as the abscissa（X-axis）, and the logarithm of corrected optical density ratio between amplification products by competitive template and target template was treated as the ordinate（Y-axis）. Thus the standard curve was drawn, and the regression equation was also obtained. When Y was assigned as 0, the concentration of the competitive plate was calculated, and then the concentration of Mhp was deduced. The logarithms of Color change unit（CCU） were treated as the abscissa（X-axis）, and the copy numbers of Mhp were treated as the ordinate（Y-axis）, so the standard curve was generated. It was found that the copy number of Mhp was highly correlated to CCU. [Conclusion] A quantitative competitive PCR assay was successfully established for the rapid detection of Mhp in culture.展开更多
[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PC...[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.展开更多
Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a ma...Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.展开更多
Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these char...Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes （18S rRNA （18S）, ubiquitin-conju-ating enzyme （UBC）, actin （ACT）, β-tubulin （TUB）, elongation factors 2 （EF2）, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold （Ct） method and by two different software packages： geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.展开更多
Quantitative real-time PCR （qRT-PCR） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have b...Quantitative real-time PCR （qRT-PCR） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.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
Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate referenc...Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes （ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S） were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EFIA was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages （from oosphere to 90 days old） in this study. However, when divided the Ct value data of the above mentioned early developmental stages into two separate periods （embryo and post-hatching periods）, TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.展开更多
The brown planthopper Nilaparvata lugens Stal （Homoptera： Delphacidae） can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but sever...The brown planthopper Nilaparvata lugens Stal （Homoptera： Delphacidae） can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens, including two different treatments （resistant or susceptible rice） and three virulent N. lugens populations. Three software programs （BestKeeper, Normfinder and geNorm） were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, E-ACT, E-TUB and a-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and a-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and a-TUB were the most suitable reference genes in N. lugens. These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.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
The detection of viable bacteria in wastewater treatment plants （WWTPs） is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was...The detection of viable bacteria in wastewater treatment plants （WWTPs） is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide （PMA） combined with the quantitative polymerase chain reaction （PMA-qPCR） to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 μmol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.展开更多
MicroRNAs （miRNAs） are small noncoding RNAs （18-25 nucleotides） that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Der...MicroRNAs （miRNAs） are small noncoding RNAs （18-25 nucleotides） that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs i n serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction （PCR）-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps： （1） sample collection and preparation; （2） global miRNAs profiling using quantitative real-time PCR （qRT-PCR）; （3） data normalization and analysis; and （4） selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.展开更多
Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila...Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.展开更多
Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this stu...Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.展开更多
The anaerobic ammonium-oxidizing （ANAMMOX） bacteria were enriched from a sequencing batch biofilm reactor （SBBR）. A quantitative competitive polymerase chain reaction （QC-PCR） system was successfully developed t...The anaerobic ammonium-oxidizing （ANAMMOX） bacteria were enriched from a sequencing batch biofilm reactor （SBBR）. A quantitative competitive polymerase chain reaction （QC-PCR） system was successfully developed to detect and quantify ANAMMOX bacteria in environmental samples. For QC-PCR system, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this system was 4 orders of magnitude, from 10^3 to 10^6 copies per PCR, corresponding to the detection limit of 300 target copies per mL. A 312-bp internal standard was constructed, which showed very similar amplification efficiency with the target amxC fragment （349 bp） over 4 orders of magnitude （10^3-10^6）. The linear regressions were obtained with R^2 of 0.9824 for 10^3 copies, 0.9882 for 10^4 copies, 0.9857 for 10^5 copies and 0.9899 for 10^6 copies, respectively. Using this method, ANAMMOX bacteria were quantified in a shortcut nitrification/denitrification-anammox system which was set for piggery wastewater treatment.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
A quantitative PCR（q PCR） assay was used to quantify Ancylostoma caninum ova in wastewater and sludge samples.We estimated the average gene copy numbers for a single ovum using a mixed population of ova.The average ...A quantitative PCR（q PCR） assay was used to quantify Ancylostoma caninum ova in wastewater and sludge samples.We estimated the average gene copy numbers for a single ovum using a mixed population of ova.The average gene copy numbers derived from the mixed population were used to estimate numbers of hookworm ova in A.caninum seeded and unseeded wastewater and sludge samples.The newly developed qP CR assay estimated an average of3.7 × 10~3 gene copies per ovum,which was then validated by seeding known numbers of hookworm ova into treated wastewater.The qP CR estimated an average of（1.1 ± 0.1）,（8.6 ± 2.9）and（67.3 ± 10.4） ova for treated wastewater that was seeded with（1 ± 0）,（10 ± 2） and（100 ± 21）ova,respectively.The further application of the q PCR assay for the quantification of A.caninum ova was determined by seeding a known numbers of ova into the wastewater matrices.The qP CR results indicated that 50%,90% and 67% of treated wastewater（1 L）,raw wastewater（1 L）and sludge（~4 g） samples had variable numbers of A.caninum gene copies.After conversion of the q PCR estimated gene copy numbers to ova for treated wastewater,raw wastewater,and sludge samples,had an average of 0.02,1.24 and 67 ova,respectively.The result of this study indicated that qP CR can be used for the quantification of hookworm ova from wastewater and sludge samples;however,caution is advised in interpreting qP CR generated data for health risk assessment.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
基金Supported by National Natural Science Foundation of Shandong Province (No. SY2008C179)~~
文摘[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.
基金Supported by National Natural Science Foundation of China(31260406)Natural Science Fund Project of Inner Mongolia(2012MS0502)~~
文摘Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.
基金Supported by Jiangsu Agricultural Science and Technology Innovation Fund[CX(133066]~~
文摘[Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another pair of primers was designed based on the conserved gene sequences of Mycoplasma. The competitive template, which carried the same primer binding site with the target fragment, was constructed using enzyme digestion method. [Result] The logarithm of concentration of competitive template was treated as the abscissa（X-axis）, and the logarithm of corrected optical density ratio between amplification products by competitive template and target template was treated as the ordinate（Y-axis）. Thus the standard curve was drawn, and the regression equation was also obtained. When Y was assigned as 0, the concentration of the competitive plate was calculated, and then the concentration of Mhp was deduced. The logarithms of Color change unit（CCU） were treated as the abscissa（X-axis）, and the copy numbers of Mhp were treated as the ordinate（Y-axis）, so the standard curve was generated. It was found that the copy number of Mhp was highly correlated to CCU. [Conclusion] A quantitative competitive PCR assay was successfully established for the rapid detection of Mhp in culture.
基金Supported by National Natural Science Foundation Project(30270086)~~
文摘[Objective] To explore the feasibility of using SYBR Green real-time quantitative PCR technique to estimate the copy numbers of exogenous gene in a transgenic plant.[Methods] Using SYBR Green real-time quantitative PCR technique,we have determined the copy numbers of the exogenous CYCD3;1 in transgenic Arabidopsis by comparing an endogenous single copy reference gene with CYCD3;1 copy numbers in transgenic plant,meanwhile comparing CYCD3;1 copy numbers between wild plant and transgenic plant.[Results]The exogenous CYCD3;1 copy numbers calculated by this method is identical with results of traditional Southern blot analysis which is highly accurate.[Conclusion]This method is simple,effective and safe for estimating transgene copy numbers.
文摘Objective Shellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. Methods Oyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction（RT-ddPCR）. Results A total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. Conclusion This study provides a systematic analysis of norovirus contamination ‘from the farm to the fork＇ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.
基金The National High Technology Research&Development Program of China under contract No.2012AA10A411the National Natural Science Foundation of China under contract Nos 41176151 and 41276177
文摘Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes （18S rRNA （18S）, ubiquitin-conju-ating enzyme （UBC）, actin （ACT）, β-tubulin （TUB）, elongation factors 2 （EF2）, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold （Ct） method and by two different software packages： geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.
基金supported by the Fundamental Research Funds for the Central Universities of China(2009QNA6023)the International Scientific and Technological Cooperation Project of Ministry of Science and Technology of China (2010DFA34430)
文摘Quantitative real-time PCR （qRT-PCR） has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate, phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12, AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.
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The National Natural Science Foundation of China under contract No.31201981China Postdoctoral Science Foundation under contract No.2013M531658the Special Scientific Research Funds for Central Non-profit Institutes,Yellow Sea Fisheries Research Institutes under contract No.20603022012032
文摘Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes （ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S） were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EFIA was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages （from oosphere to 90 days old） in this study. However, when divided the Ct value data of the above mentioned early developmental stages into two separate periods （embryo and post-hatching periods）, TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.
基金supported by the National Basic Research Program of China (973 Program) (Grant No. 2010CB126206)Central Public-Interest Scientific Institution Basal Research Program (Grant No. 2009RG004-3)+1 种基金National Natural Science Foundation of China (Grant No. 3120512)Natural Science Foundation of Zhejiang Province, China (Grant No. Y3110461)
文摘The brown planthopper Nilaparvata lugens Stal （Homoptera： Delphacidae） can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but several N. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels in N. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes in N. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated from N. lugens, including two different treatments （resistant or susceptible rice） and three virulent N. lugens populations. Three software programs （BestKeeper, Normfinder and geNorm） were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes 18S, E-ACT, E-TUB and a-TUB as the most stable reference genes. BestKeeper identified ETIF1 as the optimal reference gene with the least overall variation, whereas 18S and a-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes 18S and a-TUB were the most suitable reference genes in N. lugens. These results will facilitate future transcript profiling studies on N. lugens populations that show variation in virulence levels on different rice varieties.
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supported by the National Natural Science Foundation of China (No. 51178242)the Tsinghua University Initiative Scientific Reserch Program (No. 20121087922)the Program of Changjiang Scholars and Innovation Research Team in University
文摘The detection of viable bacteria in wastewater treatment plants （WWTPs） is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide （PMA） combined with the quantitative polymerase chain reaction （PMA-qPCR） to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 μmol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.
基金supported by National Natural Science Fundation of China(grant No. 81170047)Science Industry Trade and Information Technology Commission of Shenzhen Municipality, China (grant No.JC201006010725A)
文摘MicroRNAs （miRNAs） are small noncoding RNAs （18-25 nucleotides） that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs i n serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction （PCR）-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps： （1） sample collection and preparation; （2） global miRNAs profiling using quantitative real-time PCR （qRT-PCR）; （3） data normalization and analysis; and （4） selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.
基金supported by research grants from the Science and Technology Innovation Program of the Laoshan Laboratory(No.LSKJ202203803)the National Natural Science Foundation of China(No.32273107)+2 种基金supported by the Central Public-Interest Scientific Institution Basal Research Fund,Yellow Sea Fisheries Research Institute,CAFS(No.20603022022001)the project of Putian Science and Technology Department(No.2021NJJ002)the Shinan District Science and Technology Plan Project(No.2022-2-026-ZH).
文摘Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.
基金supported by the Western Light Project of Chinese Academy of Sciencesthe National Natural Science Foundation of China(31060057)the National Natural Science Foundation of Inner Mongolia,China(2015MS0305)
文摘Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.
基金supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No.KZCX3-SW-442, KSCX2-YW-G-008)
文摘The anaerobic ammonium-oxidizing （ANAMMOX） bacteria were enriched from a sequencing batch biofilm reactor （SBBR）. A quantitative competitive polymerase chain reaction （QC-PCR） system was successfully developed to detect and quantify ANAMMOX bacteria in environmental samples. For QC-PCR system, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this system was 4 orders of magnitude, from 10^3 to 10^6 copies per PCR, corresponding to the detection limit of 300 target copies per mL. A 312-bp internal standard was constructed, which showed very similar amplification efficiency with the target amxC fragment （349 bp） over 4 orders of magnitude （10^3-10^6）. The linear regressions were obtained with R^2 of 0.9824 for 10^3 copies, 0.9882 for 10^4 copies, 0.9857 for 10^5 copies and 0.9899 for 10^6 copies, respectively. Using this method, ANAMMOX bacteria were quantified in a shortcut nitrification/denitrification-anammox system which was set for piggery wastewater treatment.
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financial support from the Water Corporation(No.XX-09-13910)WA and the Commonwealth Scientific and Industry Research Organisation(CSIRO)(Grant No:EOP 77576),Land and Water Flagship
文摘A quantitative PCR（q PCR） assay was used to quantify Ancylostoma caninum ova in wastewater and sludge samples.We estimated the average gene copy numbers for a single ovum using a mixed population of ova.The average gene copy numbers derived from the mixed population were used to estimate numbers of hookworm ova in A.caninum seeded and unseeded wastewater and sludge samples.The newly developed qP CR assay estimated an average of3.7 × 10~3 gene copies per ovum,which was then validated by seeding known numbers of hookworm ova into treated wastewater.The qP CR estimated an average of（1.1 ± 0.1）,（8.6 ± 2.9）and（67.3 ± 10.4） ova for treated wastewater that was seeded with（1 ± 0）,（10 ± 2） and（100 ± 21）ova,respectively.The further application of the q PCR assay for the quantification of A.caninum ova was determined by seeding a known numbers of ova into the wastewater matrices.The qP CR results indicated that 50%,90% and 67% of treated wastewater（1 L）,raw wastewater（1 L）and sludge（~4 g） samples had variable numbers of A.caninum gene copies.After conversion of the q PCR estimated gene copy numbers to ova for treated wastewater,raw wastewater,and sludge samples,had an average of 0.02,1.24 and 67 ova,respectively.The result of this study indicated that qP CR can be used for the quantification of hookworm ova from wastewater and sludge samples;however,caution is advised in interpreting qP CR generated data for health risk assessment.
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.
