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.

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 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.

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.

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.

Quantification of hookworm ova from wastewater matrices using quantitative PCR

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.

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time PCR

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.

A Comparison Between Northern Blotting and Quantitative Real-Time PCR as a Means of Detecting the Nutritional Regulation of Genes Expressed in Roots of Arabidopsis thaliana

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.

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole(Cynoglossus semilaevis)

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.

Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

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.

Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

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.

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

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.