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Quantitative comparison of the expression of myogenic regulatory factors in flounder(Paralichthys olivaceus) embryos and adult tissues 被引量:1
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作者 张玉青 谭训刚 +3 位作者 徐芃 孙威 徐永立 张培军 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2010年第2期248-253,共6页
MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal ... MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, MyJ5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions ofMyf5, MyoD, and myogenin at the early stages have a common characteristic: expression gradually increased to a peak level, and then gradually decreased to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages, the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage. 展开更多
关键词 Paralichthys olivaceus myogenic regulatory factors MYOGENESIS MUSCLE quantitative rt-pcr
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Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai 被引量:1
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作者 邱礽 孙铂光 +2 位作者 房沙沙 孙黎 刘晓 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2013年第2期421-430,共10页
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without val... Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points. 展开更多
关键词 Haliotis discus hannai housekeeping gene normalization factor quantitative real time rt-pcr reference gene
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Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR 被引量:1
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作者 Shengmiao Fu Junhong Cai +5 位作者 Zhihua Tu Yutian Wang Liqun Deng Zhu Liang Zhenqun Lin Xuanju Gong 《The Chinese-German Journal of Clinical Oncology》 CAS 2008年第9期523-526,共4页
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 N... 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. 展开更多
关键词 nasopharyngeal carcinoma (NPC) real-time fluorescence quantitative rt-pcr gene expression apoptosisinhibitor Survivin
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Synchronous Detection of DNA/RNA of Four Shrimp Viruses by Real-time Fluorescence Quantitative RT-PCR 被引量:1
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作者 Biao SHEN Zhongfa WANG +1 位作者 Xingjuan HU Songye GU 《Agricultural Biotechnology》 CAS 2014年第5期48-50,共3页
[ 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 rea... [ 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. 展开更多
关键词 Real-time fluorescence quantitative rt-pcr Shrimp viruses Synchronous amplification of DNA/RNA
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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 被引量:1
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作者 Yuan Lu Runming Jin +3 位作者 Kun Yang Lirong Sun Yan Xia Xiuying Pang 《Journal of Nanjing Medical University》 2008年第3期153-158,共6页
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... 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. 展开更多
关键词 LEUKEMIA CHILDREN multidrug resistance MDR1 gene minimal residual disease real-time fluorescence quantitative rt-pcr
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TaqMan real-time fluorescent quantitative RT-PCR in detection of macrophage inflammatory protein-2γ mRNA in myocarditis murine
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作者 杨佳荟 沈茜 《Journal of Medical Colleges of PLA(China)》 CAS 2003年第5期301-304,共4页
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 an... 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. 展开更多
关键词 TAQMAN real-time fluorescent quantitative rt-pcr MYOCARDITIS MIP-2γ MRNA
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THE DETECTION OF MDR1 GENE EXPRESSION USING FLUOROGENIC PROBE QUANTITATIVE RT-PCR METHOD
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作者 高劲松 马刚 +3 位作者 仝明 陈佩毅 王传华 何蕴韶 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2001年第2期96-100,共5页
Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cance... Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107 copies/μg RNA and (8.49±0.67)×105 copies/μg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer. 展开更多
关键词 Fluorogenic quantitative rt-pcr/MDR1 Expression/Real time DETECTION
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Analysis of photo inactivation damaged nucleic acid of VSV by means of quantitative RT-PCR
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《中国输血杂志》 CAS CSCD 2001年第S1期349-,共1页
关键词 Analysis of photo inactivation damaged nucleic acid of VSV by means of quantitative rt-pcr VSV ACID RT
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The quantitative expressions of lymphangiogenic factors and metastasis in human colorectal cancers 被引量:2
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作者 Jianyu He Yuanfang Lv 《The Chinese-German Journal of Clinical Oncology》 CAS 2011年第3期170-173,共4页
Objective: The purpose of this paper was to study the expression levels of newly described lymphatic endothelial markers – LYVE-1, Prox-1, podoplanin and 5’-nucleotidase, and their correlation with metastasis of hu... Objective: The purpose of this paper was to study the expression levels of newly described lymphatic endothelial markers – LYVE-1, Prox-1, podoplanin and 5’-nucleotidase, and their correlation with metastasis of human colorectal cancers. Methods: Tumor and corresponding tumor-side normal tissue samples were obtained from resected specimens immediately after operation. Expression level of each factor was determined by quantitative real-time PCR (RT-PCR) and Western blot technique. Results: Expression levels of lymphatic endothelial markers LYVE-1, Prox-1, podoplanin and 5’-nucleotidase were significantly different in tumor and tumor-side normal groups. Expression levels of Prox-1 and podoplanin were higher in patients with positive lymph node metastasis than those without metastasis. LYVE-1, but not 5’-nucleotidase expression level was higher in both cancer and normal groups. Conclusion: These results indicate that combined quantitative analysis of lymphangiogenic markers LYVE-1, Prox-1 and podoplanin in colorectal cancer specimens may be useful in predicting metastasis of colorectal cancer to regional lymph nodes. However, the role of 5’-nucleotidase in predicting metastasis of colorectal cancer still remains to be further analyzed. 展开更多
关键词 colorectal cancer lymphatic endothelial markers quantitative real-time PCR rt-pcr
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Quantitative analysis of a panel of gene expression in prostate cancer——with emphasis on NPY expression analysis 被引量:1
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作者 LIU Ai-jun FURUSATO Bungo +5 位作者 RAVINDRANATH Lakshmi CHEN Yong-mei SRIKANTAN Vasanta MCLEOD David G. PETROVICS Gyorgy SRIVASTAVA Shiv 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2007年第12期853-859,共7页
Objective: To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods: A set of laser captured microdis... Objective: To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods: A set of laser captured microdissected (LCM) specimens from 300 prostate cancer (PCa) patients undergoing radical prostatectomy (RP) were defined. Ten patients representing "aggressive" PCa, and 10 representing "non-aggressive" PCa were selected based on prostate-specific antigen (PSA) recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR (TaqMan), and correlation was analyzed with clinicopathological features. Results: The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas (P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test). The lower expression of NPY showed association with "aggressive" PCas based on a larger PCa patient cohort analysis (P=0.0037, univariate generalized linear model (GLM) analysis). Conclusion: Despite widely noted heterogeneous nature of PCa, gene expression alterations ofAM,4CR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mechanisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression. 展开更多
关键词 Prostate cancer NPY expression quantitative real-time reverse-transcript polymerase chain reaction rt-pcr
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Sequence Analysis and Quantitative Detection of Norwalk-like Viruses in Cultured Oysters of China
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作者 WANG Jun TANG Qingjuan +3 位作者 YUE Zhiqin LI Zhaojie ZHANG Jin XUE Changhu 《Journal of Ocean University of China》 SCIE CAS 2008年第2期223-227,共5页
We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) r... We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF 1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The results showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient's fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9× 10^8, 1.25× 10^8 and 4.7× 10^1 respectively. The detecting limit of NLVs was 1 × 10^1 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future. 展开更多
关键词 OYSTERS Norwalk-like viruses (NLVs) reverse transcription polymerase chain reaction rt-pcr sequence analysis real time quantitative PCR
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Establishment of a Real-time Fluorescent Quantitative RTPCR Method for Detecting NP Gene of Class Ⅰ Newcastle Disease Virus(NDV)
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作者 Junping CAO Xiaoquan WANG +2 位作者 Han CHENG Xiaowen LIU Xiufan LIU 《Agricultural Biotechnology》 CAS 2018年第6期16-19,24,共5页
Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( cla... Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry. 展开更多
关键词 CLASS Newcastle disease virus NUCLEOCAPSID protein gene FLUORESCENT quantitative rt-pcr
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Establishment of Fluorescence Quantitative RT-PCR Assay for Detection of Equine Arteritis Virus
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作者 Wang Keke Liang Xinxin +7 位作者 Jiang Gangqiang Long Zhixin Hudusi Aierken Liu Zhiling Wang Yan Wu Xiaowei Xiao Yuanyuan Bai Meihua 《Animal Husbandry and Feed Science》 CAS 2024年第1期21-25,共5页
[Objective]The paper was to establish a fluorescence quantitative RT-PCR method for detection of equine arteritis virus(EAV).[Method]Primers and probes were developed for the EAV ORF7 gene sequence,and the reaction sy... [Objective]The paper was to establish a fluorescence quantitative RT-PCR method for detection of equine arteritis virus(EAV).[Method]Primers and probes were developed for the EAV ORF7 gene sequence,and the reaction system was optimized.Standard curves were established,leading to the initial development of the EAV fluorescence quantitative RT-PCR assay.The accuracy,specificity,and sensitivity of this method were subsequently evaluated.[Result]The EAV fluorescence quantitative RT-PCR assay demonstrated optimal performance at an annealing temperature of 61 C,with a final concentration of primer and probe set at 0.6μmol/L.The plasmid standard demonstrated a strong linear correlation with Ct values within the range of 1.6×10^(7)-1.6×10^(2)copies/μL.The equation of the standard curve was determined to be y=-2.68x+32.88,with an R^(2) value of 0.9927.Consequently,the EAV fluorescence quantitative RT-PCR assay was successfully established.The methodology employed was effective in detecting EAV,Theileria equi,equine herpesvirus-1(EHV-1),equine herpesvirus-4(EHV-4),and equine influenza virus(EIV).The findings indicated that the method was specifically capable of detecting EAV,while the other pathogens tested yielded negative results.The method demonstrated a high degree of specificity.It was employed to detect the standard plasmid cRNA synthesized through in vitro transcription following a 10-fold dilution.The results indicated that the minimum detection limit of the method was 1.6×10^(2) copies/μL,and it exhibited high sensitivity.The coefficient of variation,both within and between groups,was maintained at 1.8%,indicating good reproducibility.In this study,the fluorescence quantitative RT-PCR assay developed was utilized alongside the EAV fluorescence quantitative RT-PCR assay established by previous researchers to analyze a total of 234 clinical samples.Both methods yielded a positive detection rate of 14.1%,and the coincidence rate between the two techniques was found to be 100%.[Conclusion]The fluorescence quantitative RT-PCR assay developed in this study offers a novel approach and concept for the prevention and control of equine viral arteritis(EVA). 展开更多
关键词 Equine arteritis virus(EAV) ORF7 gene Fluorescence quantitative rt-pcr
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定量RT-PCR及其在植物学研究中的应用 被引量:15
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作者 胡丹丹 顾金刚 +1 位作者 姜瑞波 董金皋 《植物营养与肥料学报》 CAS CSCD 北大核心 2007年第3期520-525,共6页
定量RT-PCR(Quantitative reverse transcriptase-PCR)是在反转录和定量PCR的基础上发展起来的一种特异性检测基因表达的技术。主要包括相对定量RT-PCR(Relative quantitative RT-PCR)、竞争性定量RT-PCR(Competitive quan-titative RT-... 定量RT-PCR(Quantitative reverse transcriptase-PCR)是在反转录和定量PCR的基础上发展起来的一种特异性检测基因表达的技术。主要包括相对定量RT-PCR(Relative quantitative RT-PCR)、竞争性定量RT-PCR(Competitive quan-titative RT-PCR)、比较定量RT-PCR(Comparative quantitative RT-PCR)和实时定量RT-PCR(Real time quantitative RT-PCR)四种。目前定量RT-PCR在植物学研究中的应用越来越广泛,如植物营养学研究、植物发育学研究、植物抗逆机理研究、转基因植物的检测、病原菌的检测、植物与微生物互作机理研究、植物抗病性检测等方面。本文综述了定量RT-PCR的原理及在植物学中的应用。 展开更多
关键词 相对定量RT—PCR 竞争性定量rt-pcr 比较定量rt-pcr 实时定量rt-pcr 植物学
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Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation 被引量:4
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作者 赵业 陈慕雁 +3 位作者 王天明 孙丽娜 徐冬雪 杨红生 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2014年第6期1248-1256,共9页
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal ... Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPSI 8), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation. 展开更多
关键词 Apostichopus japonicus sea cucumber AESTIVATION quantitative real-time rt-pcr reference gene normalization factor
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Establishment and Application of Digital RT-PCR Assay for Detection of Avian Influenza Virus H9 Subtype 被引量:1
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作者 Bin Wu Lin Zhang +2 位作者 Liming Su Huijun Zhao Xiaoping Cai 《Advances in Microbiology》 2017年第11期760-768,共9页
A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of... A digital RT-PCR method for rapid detection of H9 subtype influenza was established by comparing the two methods of digital RT-PCR and real-time quantitative RT-PCR. The sensitivity, specificity and reproducibility of the two methods for H9 were determined by gradient dilution using the same pair of primers and probes. Both methods were able to detect 104 times diluted H9 pathogens, while digital RT-PCR could detect H9 in single droplets, and its sensitivity was higher than real-time quantitative RT-PCR. At the same time, the specificities of both methods were very strong, with no amplification reactions for H3N2, H4N2, H6N2. The reproducibility of the two methods were also good. Digital RT-PCR has a higher sensitivity than real-time quantitative RT-PCR and could play an important role in the rapid detection of H9 subtype influenza virus. 展开更多
关键词 AVIAN Influenza Virus H9 SUBTYPE (H9) DIGITAL rt-pcr Real-Time quantitative rt-pcr Sensitivity Specificity
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QUANTIFICATION OF P4HA2 mRNA OF FIBROBLASTS WITH SYBR GREEN BASED RT-PCR FOR CORRECTING CMV INACTIVATION EFFICIENCY IN DONOR BLOOD
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作者 方风琴 张玥 +2 位作者 陆萍 章莉 季育华 《Journal of Shanghai Second Medical University(Foreign Language Edition)》 2009年第2期81-86,共6页
Objective To quantify proline 4-hydroxylase, alpha polypeptide ii ( P4HA2 ) mRNA of human embryo lung fibroblast (HELF) with SYBR green based reversed transcript PCR (RT-PCR) for correcting cytomegalovirus (CMV... Objective To quantify proline 4-hydroxylase, alpha polypeptide ii ( P4HA2 ) mRNA of human embryo lung fibroblast (HELF) with SYBR green based reversed transcript PCR (RT-PCR) for correcting cytomegalovirus (CMV) inactivation or clearance efficiency in donor blood. Methods A pair of specific primers of exon 12a of P4HA2 was designed, and the related PCR-reaction system and condition were optimized. Then the recombinant plasmid containing the target fragment was constructed for making standard curve with SYBR green based real-time RT-PCR. Finally, the sensitivity, reproducibility, and specificity of this method were fully estimated. Results The sensitivity of the method was 1.5E + 04 copies/mL of P4HA2 mRNA, corresponding to 10^3 fibroblasts. In addition, existence of 8. 67E + 06 leukocytes could not interfere with the accurate quantification of HELF in the large dynamic range. The intra-assay variability and inter-assay variability both varied in different concentrations, being higher in low concentrations and lower in high concentrations. But all of them were below 13. 76% in variation, which showed acceptable stability of this method. Conclusion SYBR green and specific primer based real-time RT-PCR show up a good quality for quantifying HELF P4HA2 mRNA with good specificity, stability, and high sensitivity. Approximate 10 copies of P4HA2 mRNA per cell in average can be detected by the method. Therefore, this method can be used to deduct fibroblast-associated CMV for correcting CMV inactivation efficiency in leukocytes. 展开更多
关键词 human cytomegalovirus human embryo lung fibroblast leukocyteproline 4-hydroxylase alpha polypeptide II quantitative rt-pcr SYBR green
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The application of sequence specific primer and RT-PCR to LRRK2 gene polymorphism typing
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作者 Biao G Gaisheng T +1 位作者 Qinxue L Fengrui L 《Discussion of Clinical Cases》 2019年第2期17-19,共3页
Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA... Objective:To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease(PD)related gene LRRK2.Methods:Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR(RT-PCR).100 cases of PD samples with unknown genotypes were tested,and verified by use of polymerase chain reaction linked restriction fragment length polymorphism(PCR-RLFP).Results:The genotyping results of DNA markers proved to be correct,and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions:Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2. 展开更多
关键词 Parkinson’s disease LRRK2 gene Sequence specific primer quantitative fluorescence rt-pcr GENOTYPE
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基于定量CT身体组分、^(18)F-FDG PET/CT显像预测手术联合新辅助化疗治疗早期乳腺癌预后的价值
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作者 白丽 苏雪娟 陈体 《海南医学》 CAS 2024年第5期713-718,共6页
目的 分析定量CT (QCT)身体组分、^(18)F-氟脱氧葡萄糖正电子发射型计算机断层显像(^(18)F-FDG PET-CT)对手术联合新辅助化疗(NAC)治疗早期乳腺癌患者预后的预测效能,为临床治疗提供参考。方法 选取2020年3月至2022年10月于南阳市第二... 目的 分析定量CT (QCT)身体组分、^(18)F-氟脱氧葡萄糖正电子发射型计算机断层显像(^(18)F-FDG PET-CT)对手术联合新辅助化疗(NAC)治疗早期乳腺癌患者预后的预测效能,为临床治疗提供参考。方法 选取2020年3月至2022年10月于南阳市第二人民医院接受手术联合NAC治疗的82例早期乳腺癌患者纳入研究,在NAC前和化疗1、3个周期后检测记录QCT参数[L_(1)、L_(2)水平的皮下脂肪面积(SFA)、内脏脂肪面积(VFA)、骨密度(BMD)、L_(3)水平的椎旁肌肉面积(TMA)]、^(18)F-FDG PET/CT显像的最大标准摄取值(SUV_(max))、肿瘤代谢体积(MTV)。NAC结束后进行手术,随访12个月(失访2例),依据有无复发转移分为预后良好组43例和预后不良组37例,比较不同预后患者的QCT参数、^(18)F-FDG PET/CT显像指标,采用受试者工作特征(ROC)曲线获取曲线下面积(AUC)分析其对早期乳腺癌患者预后的预测效能。结果化疗1个周期后,预后良好组患者的SFA、BMD、VFA、MA水平分别为(45.23±4.07) cm^(2)、(128.97±26.53) mg/m^(2)、(78.07±6.69) cm^(2)、(37.36±5.74) cm^(2),明显高于预后不良组的(42.52±3.32) cm^(2)、(112.54±25.82) mg/m^(2)、(73.73±7.25) cm^(2)、(32.94±5.31) cm^(2),差异均有统计学意义(P<0.05);化疗3个周期后,预后良好组患者的SFA、BMD、VFA、MA水平分别为(40.95±3.92) cm^(2)、(113.55±15.87) mg/m^(2)、(73.59±6.17) cm^(2)、(32.67±4.98) cm^(2),明显高于预后不良组的(37.51±3.56) cm^(2)、(95.18±17.45) mg/m^(2)、(70.30±5.14) cm^(2)、(28.52±4.42) cm^(2),差异均有统计学意义(P<0.05);化疗1个周期后,预后良好组患者的SUV_(max)、MTV分别为5.43±1.25、(3.86±0.87)×10^(4)mm,明显低于预后不良组的6.04±1.07、(4.27±0.85)×10^(4)mm,差异均有统计学意义(P<0.05);化疗3个周期后,预后良好组患者的SUV_(max)、MTV分别为3.94±1.06、(2.61±0.70)×10^(4)mm,明显低于预后不良组的4.73±1.21、(3.05±0.93)×10^(4)mm,差异均有统计学意义(P<0.05);经ROC曲线分析结果显示,SFA、BMD、VFA、MA、SUV_(max)、MTV联合预测手术联合NAC治疗早期乳腺癌患者预后的AUC分别为0.898 (95%CI:0.809~0.954)、0.919 (95%CI:0.836~0.968)。结论 NAC过程中检测QCT参数、^(18)F-FDG PET/CT显像指标可预测手术联合NAC治疗早期乳腺癌患者的预后,联合预测的价值较高。 展开更多
关键词 乳腺癌 ^(18)F-氟脱氧葡萄糖正电子发射型计算机断层显像 定量CT 新辅助化疗 骨密度 肿瘤代谢体积
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量化积分绩效分配模式在消毒供应中心工勤人员中的应用研究
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作者 杨晓丽 刘玲 卢凯旋 《现代临床护理》 2024年第4期67-72,共6页
目的探讨量化积分在消毒供应中心工勤人员绩效分配中的应用效果,为消毒供应中心提供一种有效的绩效分配方法。方法采用前-后对照研究方法,选取2020年1月至12月本院消毒供应中心绩效分配模式为对照组,选取2021年1月至12月本院消毒供应中... 目的探讨量化积分在消毒供应中心工勤人员绩效分配中的应用效果,为消毒供应中心提供一种有效的绩效分配方法。方法采用前-后对照研究方法,选取2020年1月至12月本院消毒供应中心绩效分配模式为对照组,选取2021年1月至12月本院消毒供应中心绩效分配模式为试验组。对照组采用按工勤人员工龄绩效分配模式,试验组采用量化积分绩效分配模式,即从技术层级、工作质量、工作效率、工作量方面实施量化绩效分配模式。比较改进前后两组工勤人员工作效率、工作质量缺陷发生率(包装器械缺失、器械种类错误、包装标识错误、收送物品错误、信息系统操作错误)、临床科室满意度情况。结果实施量化积分绩效分配模式后,试验组工勤人员工作效率和临床科室满意度均高于对照组,工作缺陷发生率低于对照组,两组比较,差异具有统计学意义(均P<0.05)。结论量化积分绩效分配模式在消毒供应中心工勤人员中的应用,能够提高工勤人员的工作能动性,提高工作效率和工作质量。 展开更多
关键词 消毒供应中心 工勤人员 量化积分 绩效分配 前-后对照研究方法
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