[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] 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
Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for q...Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for quantitative detection of T. rotula. The RFQ - PCR assay data showed that the results obtained with the RFQ - PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ - PCR could be a useful method for red tide alga detection.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
A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conse...A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.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 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
Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and vali...Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 (ACT-2), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), β-tubulin (β-TUB), and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene (Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.展开更多
Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in dise...Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.展开更多
Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the...Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.展开更多
To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent...To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction) method was developed for quantitative detection of H.akashiwo. Primer H.akashiwo and TaqMan probe were designed, and the specificity of primer was checked with PCR. A calibration curve was constructed with cycle threshold value against visual counted cell number. And the value of the curve was tested with other H.akashiwo samples, which were assayed with both the RFQ-PCR method and visual count under microscope.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
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
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
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
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 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.
基金The research was supported by the National Natural Science Foundation of China under contract No.40406028the National High Technology Research and Development Program of China under contract No.2001AA635090.
文摘Gene specific primers and DNA probe were designed based on the sequence of 18S rDNA cloned from the red tide alga Thalassiosira rotula. A real-time fluorescent quantitative PCR (RFQ - PCR) method was developed for quantitative detection of T. rotula. The RFQ - PCR assay data showed that the results obtained with the RFQ - PCR quite good agreement with those with the light microscope (LM) counting method, which suggested that the RFQ - PCR could be a useful method for red tide alga detection.
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 emarked fund for Moden Agro-Industry Technology Research System, China (CARS25)the National Natural Science Foundation of China (31201473)the Key Laboratory of Biology and Genetic Improvement of Horticulture Crops, Ministry of Agriculture, China
文摘A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.
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 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 the National Natural Science Foundation of China(No.41176113)the National Basic Research Program of China(973 Program)(No.2010CB126403)+1 种基金the Changjiang Scholars Program for Innovative Research Team in Universities(No.IRT0941)the Earmarked Fund for Modern Agro-Industry Technology Research System(No.nycytx-47)
文摘Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 (ACT-2), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), β-tubulin (β-TUB), and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene (Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.
基金supported by the the National Key Research and Development Program of China (2017YFD0201602)the National Natural Science Foundation of China (31401704)the Beijing Academy of Agriculture and Forestry Foundation, China (KJCX20180203)
文摘Bacterial fruit blotch caused by Acidovorax citrulli is a serious threat to cucurbit industry worldwide.The pathogen is seedtransmitted,so seed detection to prevent distribution of contaminated seed is crucial in disease management.In this study,we adapted a quantitative real-time PCR(qPCR)assay to droplet digital PCR(ddPCR)format for A.citrulli detection by optimizing reaction conditions.The performance of ddPCR in detecting A.citrulli pure culture,DNA,infested watermelon/melon seed and commercial seed samples were compared with multiplex PCR,qPCR,and dilution plating method.The lowest concentrations detected(LCD)by ddPCR reached up to 2 fg DNA,and 102 CFU mL–1 bacterial cells,which were ten times more sensitive than those of the qPCR.When testing artificially infested watermelon and melon seed,0.1%infestation level was detectable using ddPCR and dilution plating method.The 26 positive samples were identified in 201 commercial seed samples through ddPCR,which was the highest positive number among all the methods.High detection sensitivity achieved by ddPCR demonstrated a promising technique for improving seed-transmitted pathogen detection threshold in the future.
基金Supported by the National Natural Science Foundation of China(Nos.41476140,41306151,41676157,41506172)the Strategic Leading Science and Technology Projects of Chinese Academy of Sciences(No.XDA11020404)+1 种基金the China Postdoctoral Science Foundation(No.2015M582153)the Science and Technology Plan of Jiangsu Province(No.BE2016330)
文摘Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.
文摘To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction) method was developed for quantitative detection of H.akashiwo. Primer H.akashiwo and TaqMan probe were designed, and the specificity of primer was checked with PCR. A calibration curve was constructed with cycle threshold value against visual counted cell number. And the value of the curve was tested with other H.akashiwo samples, which were assayed with both the RFQ-PCR method and visual count under microscope.
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 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 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.