1. 研究目的与意义

Content: OverviewSteps in molecular cloningResultsDiscussionConnotation:collate and summarize learning has been completed promote the transformation from knowledge to abilityImprove the capacity of Article WritingPrepare for future work and study

2. 文献综述

Basis and application of real-time quantitative fluorescence polymerase chain reactionCheng Zihang, School of Pharmacy, Nanjing University of Chinese Medicine (210046)Abstract: The polymerase chain reaction (PCR) is a technique of DNA amplification in vitro, which main applied in specific gene detection and biological identification. Since the invention of PCR technology in 1983, biological technology developed rapidly. Early ration of PCR method is based on the determination of the end concentration of product. The invention of the real-time PCR in 90s of the 20th century promoted the development and application of PCR technology. Real-time quantitative PCR technology is a method that put fluorophores into PCR reaction system, and then monitor the PCR process according to the accumulation of fluorescent signal at real-time, finally analyze unknown template quantitatively according to the standard curve. Real time quantitative PCR technique is a new kind of nucleic acid quantitative analysis technology. Compared to common qualitative PCR, this technique has the advantages of wide range, high sensitivity, high accuracy and good reproducibility. Its application field is more and more now. Studies have shown that real-time quantitative PCR can fast, accurately and specifically detect and identify Streptococcus suis type 2, Bacillus anthracis, Campylobacter jejuni, porcine reproductive and respiratory syndrome virus, toxoplasma gondii bacterial, viral pathogens and food micro biomass. It can also detect food borne microorganisms, food allergens, genetically modified foods, dairy and so on. The RQ-PCR technology can also be used in early diagnosis of tumor, typing, staging and prognosis. Operation principle, fluorescent type, and application in the field of environmental monitoring, food detection and disease diagnosis of real-time quantitative PCR are reviewed in this paper.Key words: Real-time fluorescent quantitative PCR; Environment monitoring, Disease Diagnosis, Food detectionReal-time fluorescent quantitative PCR（FQ-PCR) is a technique of adding PCR reaction system fluorophore fluorescence，using signal accumulated real-time monitoring of the entire PCR process, and finally quantify unknown template through a standard curve[1]. Launched in 1996 by the American company Applied Biosystems.FQ-PCR was developed on the basis of general qualitative PCR technique on nucleic acid quantification. In Fluorescent quantitative PCR technology development process, the two important discoveries play a key role [2]: First, Taq DNA polymerase 5 exonuclease activity is found, it can degrade specific fluorescent-labeled probes, such indirect detection of PCR products were possible; the other is a pair of fluorescent labeled probes are used in a closed reaction tube capable of monitoring the entire process of reaction in real time. Since the PCR technology to achieve a leap from qualitative to quantitative, with more specificity, high sensitivity, good reproducibility and accurate, high degree of automation, closed reaction, etc., it has become an important research tool in molecular biology. Real-time PCR has a very wide range of applications, including the study of mRNA expression, DNA copy number detection, single nucleotide polymorphisms measured and so on[3]. A more accurate quantitative detection of Mycobacterium tuberculosis, hepatitis B, hepatitis C, AIDS virus, Neisseria gonorrhoeae, Chlamydia trachomatis and other pathogens[4]. Its quantitative range is extremely wide, no need for dilution, more specifically, to overcome the false positive.1 Introduction 1.1 Basis theoryReal-timefluorescentquantitativePCR(FQ-PCR) are made through the system to the base with fluorescent light signal changes in real time PCR detection escalations respond in each of the loop escalations output volume changes [5]. Through the loop threshold (CT) and standard curve analysis on the starter templates for quantitative analysis. Ct value is the PCR augmentation over the expansion of product of the fluorescent signal reaches the threshold[6] (the fluorescent value calculated according to the average baseline fluorescent signal and average standard deviation based on 99. 7% confidence level greater than average fluorescence values ) set by the time of the expansion of the number of occasions on ring, it has a linear relationship with the template with the number of initial copies to the logarithm, the greater the amount of DNA template, the smaller the CT values. Starting copy number of a known standard can draw the standard curve, so given the Ct values of unknown samples, it is available calculates the number of copies of the start of the sample from the standard curve.1.2Types1.2.1SYBR Green ISYBR Green I is a kind of dye which can emit green excitation wavelength just when binding with minor groove of double stranded DNA [7]. It is most common DNA binding dye that used in fluorescence quantitative polymerase chain reaction (PCR). When combining with DNA, it can emit fluorescence. SYBR Green I has the advantages of low cost, good versatility. Also, its detective method is simple. People do not have to design a probe and can identify specificity of PCR reaction by analyzing melt curve. But the disadvantage is that SYBR Green I can combine with all double stranded DNA, and therefore cannot be used in multiplex PCR reaction [8].1.2.2TaqMan probe TaqMan fluorescent quantitative PCR technique is based on TaqMan fluorescent probe, using DNA poly-enzyme 5 '→ 3' exonuclease activity of the probes to hybridize to the sequence of hydrolysis[9]. TaqMan probe technology has the advantage of specificity, low false positive, good linearity, high accuracy, but the need to design specific probes, so the higher the cost. In addition, using Taq DNA polymerase 5 '→ 3' exonuclease activity, the process of quantitation is often affected by the performance of the enzyme[10].1.2.3 Molecular BeaconMolecular beacon is a stem-loop hairpin dual-labeled oligonucleotide probes were, is a derivative form of TaqMan probe [11]. Both ends of the nucleic acid sequence complementary pairing, thus marking the end of the fluorophore and labeled at the other end of the quencher group tightly outskirt. The method of molecular beacons has advantages of high sensitivity and specificity, low background fluorescence. The disadvantage is that the design requirements, cannot be completely hybridization probes and template binding, relatively low stability, more complex when the probe synthesis marked [12].2Applications 2.1 Food detectionSo far, amplify nucleic acid through PCR is the most common method used in food detection [13]. Real-time quantitative PCR technology with its unique advantages, it has been widely used in food and agricultural and livestock research in various fields, including mRNA expression analysis, food-borne pathogens, genetically modified foods, and a variety of strains isolated in foods of animal origin detection [14].2.1.1 Food-borne PathogenDetectionSalmonella is one of the most important food-borne pathogens in food [15]. Rodriguezs[16] team using real-time quantitative PCR to detect and compare salmonella within raw pork and poultry meat, lettuce salads and dairy sheep, the results show that this method is very practical, fast and efficient with a wide detection range, correspond to ISO standards.Du Xiongweis[17] team designed primers for inva gene, using fluorescent dyes Act establishes the salmonella RTQ-PCR detection method for the detection of Salmonella offline reach 101 CFU / mL, with high sensitivity, fast, accurate detection of Salmonella in meat, and simple operation, time-saving.2.1.2 DetectionofGeneticallyModifiedFoodCao Jijuans[18] team, using genetically exogenous recombinant gene fragment and the chromosome of wheat border sequences, primers were designed to establish real-time PCR reaction system to detect genetically modified wheat B73-6-1, B72-8-11b and B102-1-2 strain, and with GM corn, soybeans, rice and wheat as non-transgenic controls. Specificity of identification of this method is rather good, the sensitivity can reach 0. 01% (m / m), provides a more efficient method for the detection of different wheat lines containing the same exogenous gene transgenic, which means it performs great value.2.2 Environment monitoringThe technology could illustrate environmental pollution situation (i.e. biological monitoring) through detecting biological species status of the environment, to supplement the shortage of physical and chemical analysis methods [19].2.2.1 Detection of pathogenic bacteria in surface water and drinking waterVancomycin resistant enterococci (VRE) is a new surface water contaminants [20]. Pushpa[21] researched a method of molecular probe PCR technology for rapid detection of surface water and VanA gene of macrophytes. The detection limits was 1 CFU / mL (correlation is 0.943, validity is 99.7%) under the experimental conditions that 2-fold dilution and cultured under 2. 5 h, and prove that the specificity detection of enterococci in certain environment.VRE can be usually detected in contaminated river flows downstream and regeneration water, nutrition excessive surface water with wild growth of water hyacinth could be detected VRE [22]. Molecular beacon fluorescence quantitative PCR technology can rapidly detect surface water Vancomycin resistant enterococci (VRE), provides a quick and simple method for the detection of fecal contamination of surface water, and timely notice of such situation and take measures.2.2.2 Detection of environmental hormoneEnvironment hormones refers to a class of endocrine exogenous chemical substances that interfere with the body's normal biological function, which appears increasingly serious harm to human [23].Pang Xiaoqian[24] completed the environmental hormones pentachlorophenol (PCP) fluorescence quantitative PCR study. Research were carried out in conventional PCR experiments and SYBR Green I fluorescence quantitative PCR experiments. Analyzed results, it shows a significant linear relationship (R2 = 0. 9975) when the PCP concentration is in the range of 10 - 13 ~ 10 - 9mol / L. When reduce the concentration of PCP, test results were negative. Therefore, it can be determined that the minimum detection limit of PCP of fluorescence quantitative PCR is 10 - 13mol / L; repeated experimental results show that fluorescence quantitative PCR the character of good repeatability. Also compared quantitative PCR and HPLC, when the PCP concentration of 10-9mol / L, HPLC cannot detect PCP, but FQ-PCR could, further study confirm that fluorescence quantitative PCR is able to detect lower limit of pentachlorophenol.2.3 Disease DiagnosisThe appearance of Fluorescent quantitative PCR overcome the defects of traditional PCR techniques, which contains false positive and not quantitatively widespread problems [25]. This PCR technology is more widely used in clinical and drugs, now in cancer research, diagnosis of hepatitis B virus detection, detection and defense of animal diseases, drug efficacy evaluation and assessment aspects [26], provides an objective material basis for theoretical clinical research, drug development and treatment of disease.2.3.1 Tumor studyFluorescence in situ hybridization (FISH) showed 10% to 15% of CML (chronic myelogenous leukemia) patients with bcr / abl loss of heterozygous, thus the prognosis is very poor [27]. FQ-PCR method can identify CML patients with micro-deletions of heterozygous on zygote subunit, tiny deletions cannot be identified by FISH method. Kolomietzs[28] team detected CML 71 patients, using FQ-PCR, results showed that 25 patients with poor prognosis (less than 96 months of remission, survival is less than 86 months) present micro-deletions of bcr/abl gene.2.3.2 Pulmonary tuberculosis study860 sputum samples of the patients with pulmonary tuberculosis were detected by methods of FQ-PCR, staining and culture, respectively. The sensitivity and specificity of FQ-PCR were confirmed by comparing three different results [29]. Results show that the sensitivity of FQ-PCR can reach 10-100 CFU/ml .The results of other pathogens in respiratory system were negative using FQ-PCR .The positive rate of FQ-PCR in testing sputum samples was higher than staining and cultures counterparts , and the difference was statistically significant (P

3. 设计方案和技术路线

Molecular CloningmRNA IsolationcDNA Library ConstructionPolymerase Chain ReactionGel AnalysisPCR Products Purification Ligation TransformationCloning Isolation of RecombinantPlasmid DNA PCRGel AnalysisSelected PCR Products PurificationSequencing ReactionExtension Products PurificationSequencing

4. 工作计划

3.13.10：Read articles related to research project and do summary3.113.31: Conduct experiment4.15.20: Write research paper

5. 难点与创新点

The material of the experiment is directly obtained from the skin secretion of certain species, using electrical stimulation causing a low degree of stress to the animals.