Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages

[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.

Application of Real-time Fluorescent Quantitative PCR in Plant

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.

Determining the Copy Number of Exogenous Gene in Transgenic Plant by SYBR Green Real-time Quantitative PCR

[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.

Detection of Lactobacillus acidophilus in Fermented Material by Real-time Fluorescent Quantitative PCR

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.

Application of Real-time Fluorescent Quantitative PCR in Studies on Plants

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.

Detection of Ratoon Stunting Disease in Virus-free Seedcane via Real-time Fluorescence Quantitative PCR

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.

Application of quantitative PCR method in detection of Lymphocystis disease virus China (LCDV-cn) in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease causes serious economic losses in the fish farming industry. The causative agent of the disease is Lymphocystis disease virus China (LCDV-cn), which has a wide range of hosts. Based on competitive quantitative PCR technology, we established a method to quantify the LCDV-cn in tissue. Results demonstrate that the average amount of LCDV-cn in the peripheral blood of infected flounder with evident tumors is about 106virions/ml while the average amount in those flounder with no evident tumor but cultured with the flounder with evident tumor is about 104virions/ml. No virus was found in the negative samples of flounder.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.

Establishment of Real-Time Quantitative PCR Method for the Determination of Transposon Copy Number in Cronobacter sakazakii

[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.

Development of a Fluorescence Quantitative PCR Method for Detection of Marteilia refringens in Shellfish

[Objective] This paper was to develop a fluorescence quantitative PCR method for detection of M. refringens in shellfish. [Method] A pair of primers and a TaqMan probe were designed and synthesized according to the conserved gene sequences of M. refringens in GenBank, so as to develop a fluorescence quantitative PCR method for detection of M. refringens. The developed fluorescence quantitative PCR method was compared with conventional PCR detection. [Result] The fluorescence quantitative PCR could detect 40 template copies of plasmid DNA, and its sensitivity was 100 times higher than the conventional PCR. The detection results of Perkinsus sp, Haplosporidium sp, Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio parahaemolyticu, Vibrio alginolyticu, Vibrio rluvialis and Vibrio mimicus were negtive. [Conclusion] The fluorescence quantitative PCR method for M. refringens established in this paper is specific, sensitive, rapid and quantitative with good repeatability, which can be used for clinical detection of M. refringens infection.

Development and Preliminary Application of SYBR Green I Real-Time Fluorescence Quantitative PCR Method for Detecting Porcine Parvovirus Virus

According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.

Establishment and Application of a Real-time Fluorescent Quantitative PCR Method for Detection of Porcine Circovirus Type 2

[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction （PCR） method with SYBR Green I for the detection of porcine circovirus type 2 （PCV2）. [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.

Establishment and Application of the TaqMan Real-Time Fluorescence Quantitative PCR Detection Assay for Koi Herpes Virus

[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes virus （KHV） pol-ymerase gene （Sph） to establish a rapid and effective fluorescence quantitative PCR method for Koi herpes virus detection. The cell cultures were detected by using the established fluorescence quantitative PCR assay, and the results were com- pared with that of conventional PCR. [ Result] The sensitivity of fluorescence quantitative PCR was higher than that of conventional PCR. The minimum copy num- ber that could be detected was 1.6 - 102 copies/p.1. The established method was adopted for sample detection, and a reliable diagnostic result could be obtained within 4 h. [Conclusion] The established method is rapid, sensitive, specific and repeatable, which is conducive to the rapid detection of Koi herpes virus. Key words Koi herpes virus; Fluorescence quantitative PCR; Detection

Evaluation of Clinical Application of Chemiluminescence and Real-time,Fluorescence-based Quantitative PCR in Diagnosis of Epstein-Barr Virus lnfection

Objective:To compare the effects of clinical application of chemiluminescence and real-time,fluorescence-based quantitative PCR in the detection Epstein-Barr virus(EBV).Methods:The data of chemiluminescence and real-time fluorescent quantitative PCR.fromipaEsfwo were suspected of being infectea w1tn rito1 roro January 2016 to January 2019 in our hospital were analyzed.The specific stage of EBV infection was analyzed,and the differences in results of the two detection methods were compared.Results:Chemiluminescence method was used to detect EBV infection during the active phase.The sensitivity of the chemiluminescence method was 76.7%(56/73)and the real-time quantitative PCRmethod was 90.4%(66/73).There was a statistical difference between the two detection methods(P<0.05).Conclusion:There was no statistical difference in positive predictive values between the chemiluminescence method and the real-time,fluorescence-based quantitative PCR method in the detection of EBV infection,but the sensitivity of chemiluminescence method is slightly lower than the real-time quantitative PCRmethod.It is noteworthy that chemiluminescence method is convenient and fast while the real-time,fluorescence-based quantitative PCR method is more accurate,which can provide a more accurate reference for clinical treatment.

Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.

Detection of the covalently closed circular DNA of duck hepatitis B virus by Taq-Man fluorescent quantitative PCR assay

To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

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.

Identification of a suitable endogenous control gene in porcine blastocysts for use in quantitative PCR analysis of microRNAs

To obtain reliable results in quantitative PCR （qPCR） reactions, an endogenous control （EC） gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the expression levels of microRNA （rniRNA） genes in in vivo fertilized, in vitro fertilized, parthenogenetic and somatic cell nuclear transfer blastocysts. The aim was to identify suitable EC genes for the qPCR analysis of rniRNAs in porcine blastocysts. The results showed that thirty-six miRNAs were commonly expressed in the four kinds of embryos and the expression levels of eleven miRNAs were similar in the different embryo types （P-value〉0.05）. These 11 miRNAs were selected as candidate EC genes for further analysis and, of these, miR-16 was identified as the most stable EC gene by the GeNorm （a tool based on a pair-wise comparison model that calculates the internal control genes stability measure and determines the most reliable pair of EC genes） and NorrnFinder （an excel plug-in that uses an ANOVA-based model to estimate intra- and inter- group variation to indicate the single most stable EC gene） programs. In addition, a cell number normalization method validated miR-16 as a suitable EC gene for use in future qPCR analysis of miRNAs in porcine blastocysts.

Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR(PMA-qPCR)

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.