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
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
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
[ 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
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
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
Dear Editor,Influenza A viruses(IAVs)are single-stranded,negative sense RNA viruses.IAV subtype is determined on the basis of the viral surface glycoproteins,hemagglutinin(HA),and neuraminidase(NA).To date,18 HA and 1...Dear Editor,Influenza A viruses(IAVs)are single-stranded,negative sense RNA viruses.IAV subtype is determined on the basis of the viral surface glycoproteins,hemagglutinin(HA),and neuraminidase(NA).To date,18 HA and 11NA subtypes have been reported(Tong et al.,2012).展开更多
[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
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
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
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
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
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
基金a grant from the National New Technology Program (No. 1998-345).
文摘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.
基金Supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No.KSCX2-EW-G-12B)the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KZCX2-EW-Q213)the National High Technology Research and Development Program of China (863 Program)(No.2012AA10A412)
文摘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.
基金the National Natural Science Foundation of China (No. 30460145).
文摘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.
文摘[ 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.
基金This work was supported by Science Project from Science and Tech- nology Department of HuBei province(2006AA301B56-3)
文摘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.
文摘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.
基金partially supported by the National Institutes of Health(grant no.P20GM103646)the United States Department of Agriculture Animal and Plant Health Inspection Service(agreement 14-7428-1041-CA)
文摘Dear Editor,Influenza A viruses(IAVs)are single-stranded,negative sense RNA viruses.IAV subtype is determined on the basis of the viral surface glycoproteins,hemagglutinin(HA),and neuraminidase(NA).To date,18 HA and 11NA subtypes have been reported(Tong et al.,2012).
基金Supported by Research Project of General Administration of Customs(2022HK126)Youth Science Foundation(2022D01B08).
文摘[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).
基金Supported by the Key Laboratory of Experimental Marine Biology,Academia Sinica,Qingdao,Chinathe National High Technology Research and Development Program of China(863 Program)(No.2006AA10AA402)+1 种基金the National Basic Research Program of China(973Program)(Nos.2004CB117402,2010CB126304)the National Natural Science Foundation of China(No.30871929)
文摘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.
文摘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.
基金Project supported by the Center for Prostate Disease Researchthe Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, MD, USA
文摘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.
文摘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.
基金Supported by National Natural Science Foundation of China(30630048)National Science and Technology Support Program(2006BAD06A03)
文摘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.
