Real-Time PCR Technique and Its Application in Quantification of Plant Nucleic Acid Molecules

Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.

Real-time fluorescent quantitative PCR非特异性扩增的研究进展

Real-time fluorescent quantitative PCR(RT-qPCR)因其高效快速、操作简便、高度敏感等优点获得广泛应用,但因其强大的放大功能而容易出现非特异性扩增的问题,尤其是荧光染料法中更加明显。文章对RT-qPCR的原理、非特异性扩增的发生因素、解决方案及该技术的应用前景进行了综述。

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.

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.

Real-time fluorescent quantitative immuno-PCR method for determination of fluoranthene in water samples with a molecular beacon

A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR （RTFQ-IPCR） assay using a molecular beacon was developed for the determination of trace fluoranthene （FL） in the environment.Under optimized assay conditions,FL can be determined in the concentration range from 1 fg/mL to 100 ng/mL,with y=0.194x ＋ 7.859,and a correlation coefficient of 0.967 was identified,with a detection limit of 0.6 fg/mL.Environmental water samples were successfully analyzed,recovery was between 90% and 116%,with intra-day relative standard deviation （RSD） of 6.7%-12.8% and inter-day RSD of 8.4%-15.2%.The results obtained from RTFQ-IPCR were confirmed by ELISA,showing good accuracy and suitability to analyze FL in field samples.As a highly sensitive method,the molecular beacon-based RTFQ-IPCR is acceptable and promising for providing reliable test results to make environmental decisions.

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.

Establishment of Real-time Fluorescent RT-LAMP Detection Method for Grouper Nervous Necrosis Virus

Nervous necrosis virus （NNV） is the causative agent of fulminant infectious diseases in marine fishes such as grouper. Specific primers were designed based on the conserved sequence of capsid protein （CP） gene of red-spotted grouper nervous necrosis virus （NNV）. By optimizing the reaction conditions, a rapid and simple reverse transcription loop-mediated isothermal amplification （RT-LAMP） method was established for NNV detection. After adding SYTO-9 fluorescent dye in the reaction system, the amplification curve was monitored in real time using a fluorescence detector, and the result was obviously easy to assess. Moreover, the specificity and sensitivity of the established method was analyzed. The results showed that the established RT-LAMP method has good specificity with a detection limit of 1.3 pg/μl. The detection sensitivity of the established RT-LAMP method is 100 times that of the conventional RT-PCR method, and the detection duration is only 40 min. The established RT-LAMP method is suitable for quarantine and rapid detection of grouper nervous necrosis virus.

Development of Real-Time Fluorescent PCR for Rapid Detection of Haempohlius parasuis

[ Objective] To develop a real-time fluorescent PCR assay for rapid detection of Haempohlius parasuis （HPS）. [ Method] According to the conservative sequences of 16 S rRNA genes of HPS published in GenBank, a pair of specific primers was designed. The real-time fluorescent PCR was developed by optimizing primer concentration and annealing temperature. And its specificity and reproducibility were evaluated. Ten HPS- suspected samples were detected by the developed method. [ Result] The lowest detection limit of the developed real-time fluorescent PCR was 50 copies/μl. This method had good reproducibility, and its coefficient of variation was lower than 2%. Only HPS rather than Streptococcus suis type 2, Staphylococcus aureus, E. coli DH5 alpha, and swine Salmonella typhi could be detected by the developed real-time fluorescent PCR. The HPS-pesitive samples detected by this method were also positive when they were detected by isolation of bacteria or conventional PCR. [ Conclusion] The developed real-time fluorescent PCR is rapid, sensitive, specific and highly reproducible; thus, it can be used for rapid detection of HPS.

TaqMan real-time fluorescent quantitative RT-PCR in detection of macrophage inflammatory protein-2γ mRNA in myocarditis murine

Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis and experimental autoimmune myocarditis and MIP-2γ mRNA expression in mouse was studied by TaqMan real-time fluorescent quantitative RT-PCR. Results: MIP-2γ mRNA expression rose on 3 to 5 d after CVB3 infection, reached peak on 7 d, and returned to normal level until 14 d, which corresponded well with the disease course. The MIP-2γ mRNA expression level rose significantly on the day 18 d after immunization with porcine cardiac myosin, which was consistent with pathological examination. Conclusion: MIP-2γ may be involved in the pathogenesis of myocarditis.

Fluorescent Voltage Imaging Technique for the Measurement of Molluscan Neural Activities

The electrophysiological methods using microelectrodes are not appropriate for the simultaneous measurement of neural activities of many neurons. To overcome the difficulty, the fluorescent imaging technique using voltage sensitive dyes can be a powerful technique. The voltage sensitive dyes, however, generally exhibit a relatively small change in their fluorescence intensities, resulting in a low S/N ratio. Additionally, they often exhibit photobleaching and phototoxity. We have therefore improved the fluorescent voltage imaging technique by using a LED as the light source and an electron multiplying (EM)-CCD camera as the fluorescence detector. In this study, we applied our imaging system for the measurement of two kind of molluscan neural activities;one of which is involved in the olfactory processing of the land slug Limax valentianus and the other is involved in the feeding rhythm of the pond snail Lymnaea stagnalis. The system enabled us to measure the neural activities for a long time with a high speed and a high S/N ratio, and the obtained results showed some new physiological findings.

Synchronously Detecting Allergenic Ingredients of Peanut and Sesame in Food by Real-time Fluorescent PCR

Peanut,sesame and other raw materials of food are allergens for special populations.In this study,specific primers and TaqMan probes labeled by different fluorescences were designed targeting Ara h 2 gene of peanut and Ses i 1 gene of sesame.After the optimization of reaction conditions,a real-time fluorescent PCR method was established for simultaneous detection of allergenic ingredients of peanut and sesame in food.Genomic DNA samples of peanut,sesame,rice,wheat,barley,soybean,celery,maize,potato,tomato,walnut,groundnut in shell,cashew nut,sunflower seed,almond,apple,pear and strawberry,pork,beef,mutton and fish were used as templates for PCR amplification with deionized water as negative control template.Results indicated that the established real-time fluorescent PCR method could specifically identify allergenic ingredients of peanut and sesame simultaneously.Sensitivity test showed that the minimum detection limit of this method was 0.01%.Therefore,the established real-time fluorescent PCR method is a specific,sensitive and effective assay for simultaneously detecting allergenic ingredients of peanut and sesame in food.

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.

The Double Projecting Neuron of the Cat Spinal Dorsal Horn——A Study with Double Retrograde Fluorescent Tracing Technique

The new double projecting neurons were found in the cat spinal dorsal horn by the double retrograde fluorescent tracing technique.Fast Blue(FB)was injected into unilateral dorsal column nucleus(DCN)of adult cats anesthetized with pentobarbital.Nuclear Yellow(NY)was injected ipsilaterally into the lateral cervical nucleus(LCN)8-9 days later.After an additional 18-30 hrs.

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.

Effects of Different Temperature and Time Durations of Virus Inactivation on Results of Real-time Fluorescence PCR Testing of COVID-19 Viruses

Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.

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.

Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR

Objective： To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma （NPC） tissues. Methods： The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients＇ nasopharynx tissues treated as control group. Results： The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients＇ nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients＇ nasopharynx tissues, the difference was significant （P 〈 0.01）. The expression of Survivin mRNA could be detected both in stage Ⅰ ＋ Ⅱ and stage Ⅲ ＋ Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups （P 〉 0.05）. There was no relationship between Survivin mRNA expression and age and sex of NPC patients （P 〉 0.05）. Conclusion： Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.

The Preliminary Report on Rumen Protozoa Grazing Rate on Bacteria with a Fluorescence-Labeled Technique

Studies on the bacterial predation rate by rumen protozoa were carried out under laboratory conditions using a technique of fluorescence-labeled bacteria （FLB）. Four Xuhuai goats were used in this experiment to obtain rumen protozoa and bacteria. Two groups were designed as follows： One group was the whole bacteria which were labeled using fluorescence through removing free bacteria from rumen fluid （WFLB）; the other group was the bacteria which were labeled using fluorescence without removing free bacteria from rumen fluid （FLB）. The result indicated that the bacterial predation rates of rumen protozoa was 398.4 cells/（cell h） for the group WFLB, 230.4 cells/（cell h） for the group FLB, when the corresponding values expressed as bacteria-N, they were 2.15 pg N/（cell h） for the group WFLB, and 1.24 pg N/（cell h） for the group FLB, respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat, there were 103.2 mg N/（d capita） for the group WFLB, and 59.5 mg N/（d capita） for the group FLB, respectively. It was estimated that protein losses due to microbial recycling were 0.645 g pro/（d capita） for the group WFLB and 0.372 g pro/（d capita） for the group FLB, respectively. In addition, the fluorescence-labeled technique would be a potential assay for the determination of bacterial predation rate by rumen protozoa.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective： Multidrug resistance（MDR） is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia（ALL） which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction（FQ-RT-PCR）, and combine minimal residual desease（MRD） detection by flow cytometry（FCM） and to study their relationship with treatment response and prognosis of ALL. Methods：The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission（CR）, 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results：Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group（P 〉 0.05）, but significantly higher in the recurrence group than that in newly diagnosed disease group and control group（0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05）. 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration（0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01）. For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group（P〉 0.05）. In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group（0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05）. Conclusion：The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration（within 3 years）. Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

Synchronous Detection of DNA/RNA of Four Shrimp Viruses by Real-time Fluorescence Quantitative RT-PCR

[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.