猴逆转录病毒(Simian type D retrovirus,SRV)是引起猴获得性免疫缺陷综合征(Simian acquired immunodeficiency syndrome,SAIDS)的病原之一,其严重危害猴的健康,并威胁与猴接触人员的健康,是无特定病原体(SPF)猴必须排除的病毒之一。...猴逆转录病毒(Simian type D retrovirus,SRV)是引起猴获得性免疫缺陷综合征(Simian acquired immunodeficiency syndrome,SAIDS)的病原之一,其严重危害猴的健康,并威胁与猴接触人员的健康,是无特定病原体(SPF)猴必须排除的病毒之一。为了应对口岸对进出境野生及实验用灵长类动物SRV感染情况的监测和流行病学调查的需要,建立了RT-PCR和real-time RT-PCR检测SRV的方法,并对方法的特异性、敏感性和稳定性进行了验证。展开更多
Erythropoietin-producing hepatocellular receptor and its membrane-bound ligands (Eph-Ephrin) system could regulate some mammalian blastocyst attachment and spreading. In order to investigate the involvement of the E...Erythropoietin-producing hepatocellular receptor and its membrane-bound ligands (Eph-Ephrin) system could regulate some mammalian blastocyst attachment and spreading. In order to investigate the involvement of the Eph-Ephrin system in swine embryo attachment, mRNA expression of Eph-Ephrin molecules in endometrium was examined by real-time RT- PCR during embryo implantation in pigs. The results indicated that mRNA expressions of Eph A5, A7 and Ephrin A5 all continually increased from pregnancy day 13 to 24. Ephrin A3 mRNA expression significantly increased from day 13 to 18 and decreased from day 18 to 24, and the expression was the lowest on pregnancy day l 3 and the highest on day 18. However, Ephrin A4 mRNA expression was the lowest on pregnancy day 18 and the highest on day 24, and the expression decreased from day 13 to 18 and increased from day 18 to 24. Furthermore, mRNA expressions of Eph A5 and A7 were both found in other tissues, such as brain, muscle, intestine, stomach, etc. These findings suggest that the Eph-Ephrin system may play an important role in regulating the contact between blastocysts and endometrium during swine embryo implantation.展开更多
Objective To detect Japanese encephalitis virus(JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction(RT-PCR) detection system was developed.Method...Objective To detect Japanese encephalitis virus(JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction(RT-PCR) detection system was developed.Methods By aligning the full-length sequences of JEV(G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.Results With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/μL. The coefficients of variation of this real-time RT-PCR were all 〈 2.8%. The amplification efficiency of this method was between 90% and 103%.Conclusion A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.展开更多
Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify ...Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.展开更多
A real-time RT-PCR assay using Taq Man-MGB probes was developed to detect and type the bovine viral diarrhea virus(BVDV) in cattle.Universal primers and Taq Man-MGB probes were designed from the 5′-untranslated reg...A real-time RT-PCR assay using Taq Man-MGB probes was developed to detect and type the bovine viral diarrhea virus(BVDV) in cattle.Universal primers and Taq Man-MGB probes were designed from the 5′-untranslated region of known pestiviral sequences.Prior to optimizing the assay, c RNAs were transcribed in vitro from the BVDV 1 and BVDV 2 RTPCR products to make standard curves.The detection limit of the assay was 1.72×102 copies for BVDV 1 and 2.14×102copies for BVDV 2.The specificity of the assay evaluated on several BVDV strains including bovine herpesvirus 1(BHV 1), foot and mouth disease virus(FMDV) and several classical swine fever virus(CSFV) strains showed specific detection of the positive virus over 40 cycles.The assay was highly reproducible with the coefficient of variance ranging from 1.04 to 1.33% for BVDV 1 and from 0.83 to 1.48% for BVDV 2, respectively.Using this method, we tested a total of 2 327 cattle from three dairy farms for the presence of BVDV persistently infected(PI) animals.In this assay, each RT-PCR template contained a mixture of ten samples from different animals.The occurrence rate of PI cattle in three farms ranging from 0.9 to 2.54% could represent partly the PI rates in cattle farm in China.In conclusion, using our real-time PCR assay, we could effectively detect and type BVDV and identify PI cattle in a rapid and cost-effective manner.展开更多
A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequ...A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.展开更多
Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed t...Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.展开更多
In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin(HA) cleavage site, leading to the emergence of a ...In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin(HA) cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.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 expression of CD44 correlation and the ability of metastasis of tumor cells in gastric carcinoma, and to find the correlation of the quantitative of CD44V6mRNA and the histology expression of ...Objective: To study the expression of CD44 correlation and the ability of metastasis of tumor cells in gastric carcinoma, and to find the correlation of the quantitative of CD44V6mRNA and the histology expression of CD44v6 in tumors with the clinic-pathologic features, and to make the quantitative expression of CD44v6mRNA. Methods: Twenty patients with gastric carcinoma, 4 patients with gastritis, and 10 apparently healthy controls were recruited. Blood samples were obtained before surgery. 10 days after surgery, the blood samples were obtained again. Serum CIM4v6mRNA in all cases was measured by real-time quantitative PCR. Results: Serum CIM4V6mRNA was detectable in 20 of 20( 100% ) gastric carcinoma cases, The expression level ranged from 4.9×10^2 copies/μg RNA to 3.2 ×10^5 copies/μg RNA, the average levels of peripheral blood was 3.9 ×10^4 copies/μg RNA, The expression level of pedpheral blood of gastric cancer after curative operation ranged from 5.5×10^0 copies/μg RNA to 7.6 ×10^3 copies/μg RNA. After curative operation the expression level was decreased markedly. Conclusion: Serurn CIM4v6mRNA is expressed in the peripheral blood of gastric carcinoma patients. The expression level of CD44V6mRNA is obviously decreased after curative operation. An elevated level of CD44v6mRNA may serve as an indicator of lymph node metastasis (especially early metastasis) and bad prognosis in patients with gastric carcinoma.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
Ret finger protein(RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger of motif, a B-box, and a coiled-coil domain(they are called RBCC generally). Although RFP was k...Ret finger protein(RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger of motif, a B-box, and a coiled-coil domain(they are called RBCC generally). Although RFP was known to be an oncogene when its RBCC moiety was connected with a tyrosine kinase domain by DNA rearrangement, its biological function was not well defined. In this study, by using real-time RT-PCR, the RFP expressions in human and mouse normal tissues, and in the cervical squamous cell carcinoma, endometrial adenocarcinoma, gastric adenocarcinoma, esophageal squamous cell carcinoma, and brain cancer tissues were analyzed. The result of the study proved that the highest level of mRNA reverse transcription appeared in the normal testical tissue, whereas that in other normal tissues of human and mice were low. The mRNA reverse transcription level of RFP was higher in the endometrial adenocarcinoma tissue than in the cervical squamous cell carcinoma tissue; the mRNA reverse transcription level of RFP in the gastric adenocarcinoma tissue was significantly higher than that in the esophageal squamous cell carcinoma tissue. It was also found that the mRNA reverse transcription level of RFP in the brain cancer tissue was higher than that in the normal brain tissue. These results suggested that RFP could possibly be a useful molecular target for the development of new therapeutics for malignant tumors.展开更多
文摘猴逆转录病毒(Simian type D retrovirus,SRV)是引起猴获得性免疫缺陷综合征(Simian acquired immunodeficiency syndrome,SAIDS)的病原之一,其严重危害猴的健康,并威胁与猴接触人员的健康,是无特定病原体(SPF)猴必须排除的病毒之一。为了应对口岸对进出境野生及实验用灵长类动物SRV感染情况的监测和流行病学调查的需要,建立了RT-PCR和real-time RT-PCR检测SRV的方法,并对方法的特异性、敏感性和稳定性进行了验证。
基金supported by the National Natural Science Foundation of China (30771540)the National High-Technology Research Development Program of China (2007AA10Z166)
文摘Erythropoietin-producing hepatocellular receptor and its membrane-bound ligands (Eph-Ephrin) system could regulate some mammalian blastocyst attachment and spreading. In order to investigate the involvement of the Eph-Ephrin system in swine embryo attachment, mRNA expression of Eph-Ephrin molecules in endometrium was examined by real-time RT- PCR during embryo implantation in pigs. The results indicated that mRNA expressions of Eph A5, A7 and Ephrin A5 all continually increased from pregnancy day 13 to 24. Ephrin A3 mRNA expression significantly increased from day 13 to 18 and decreased from day 18 to 24, and the expression was the lowest on pregnancy day l 3 and the highest on day 18. However, Ephrin A4 mRNA expression was the lowest on pregnancy day 18 and the highest on day 24, and the expression decreased from day 13 to 18 and increased from day 18 to 24. Furthermore, mRNA expressions of Eph A5 and A7 were both found in other tissues, such as brain, muscle, intestine, stomach, etc. These findings suggest that the Eph-Ephrin system may play an important role in regulating the contact between blastocysts and endometrium during swine embryo implantation.
基金supported by grants from the National Key Research and Development Program[2016YFD0500401]Development Grant of State Key Laboratory of Infectious Disease Prevention and Control[2015SKLID505,2014SKLID03]
文摘Objective To detect Japanese encephalitis virus(JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction(RT-PCR) detection system was developed.Methods By aligning the full-length sequences of JEV(G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.Results With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/μL. The coefficients of variation of this real-time RT-PCR were all 〈 2.8%. The amplification efficiency of this method was between 90% and 103%.Conclusion A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.
基金supported by the Center of Excellence in Clinical Virology.Chulalongkorn University,CU Centenary Academic Development ProjectKing Chulalongkorn Memorial Hospital,the National Research University Project of CHEthe Ratchadaphiseksonphot Endowment Fund(HR1155A)
文摘Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.
基金supported by the China Agriculture Research System(CARS-37)
文摘A real-time RT-PCR assay using Taq Man-MGB probes was developed to detect and type the bovine viral diarrhea virus(BVDV) in cattle.Universal primers and Taq Man-MGB probes were designed from the 5′-untranslated region of known pestiviral sequences.Prior to optimizing the assay, c RNAs were transcribed in vitro from the BVDV 1 and BVDV 2 RTPCR products to make standard curves.The detection limit of the assay was 1.72×102 copies for BVDV 1 and 2.14×102copies for BVDV 2.The specificity of the assay evaluated on several BVDV strains including bovine herpesvirus 1(BHV 1), foot and mouth disease virus(FMDV) and several classical swine fever virus(CSFV) strains showed specific detection of the positive virus over 40 cycles.The assay was highly reproducible with the coefficient of variance ranging from 1.04 to 1.33% for BVDV 1 and from 0.83 to 1.48% for BVDV 2, respectively.Using this method, we tested a total of 2 327 cattle from three dairy farms for the presence of BVDV persistently infected(PI) animals.In this assay, each RT-PCR template contained a mixture of ten samples from different animals.The occurrence rate of PI cattle in three farms ranging from 0.9 to 2.54% could represent partly the PI rates in cattle farm in China.In conclusion, using our real-time PCR assay, we could effectively detect and type BVDV and identify PI cattle in a rapid and cost-effective manner.
基金supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China(No.2013ZX10004-101)
文摘A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.
基金supported by grants from the Development Grant of State Key Laboratory of Infectious Disease Prevention and Control(2012SKLID204,2015SKLID505)the Ministry of Science and Technology of People’s Republic of China(No.2013ZX10004101)
文摘Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.
基金supported by the National Key R&D Program of China(2016YFD0500800)the International Science&Technology Cooperation Program of China(2014DFR31260)
文摘In 2013, a human influenza outbreak caused by a novel H7N9 virus occurred in China. Recently, the H7N9 virus acquired multiple basic amino acids at its hemagglutinin(HA) cleavage site, leading to the emergence of a highly pathogenic virus. The development of an effective diagnostic method is imperative for the prevention and control of highly pathogenic H7N9 influenza. Here, we designed and synthesized three pairs of primers based on the nucleotide sequence at the HA cleavage site of the newly emerged highly pathogenic H7N9 influenza virus. One of the primer pairs and the corresponding probe displayed a high level of amplification efficiency on which a real-time RT-PCR method was established. Amplification using this method resulted in a fluorescent signal for only the highly pathogenic H7N9 virus, and not for any of the H1–H15 subtype reference strains, thus demonstrating high specificity. The method detected as low as 39.1 copies of HA-positive plasmid and exhibited similar sensitivity to the virus isolation method using embryonated chicken eggs. Importantly, the real-time RT-PCR method exhibited 100% consistency with the virus isolation method in the diagnosis of field samples. Collectively, our data demonstrate that this real-time RT-PCR assay is a rapid, sensitive and specific method, and the application will greatly aid the surveillance, prevention, and control of highly pathogenic H7N9 influenza viruses.
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 expression of CD44 correlation and the ability of metastasis of tumor cells in gastric carcinoma, and to find the correlation of the quantitative of CD44V6mRNA and the histology expression of CD44v6 in tumors with the clinic-pathologic features, and to make the quantitative expression of CD44v6mRNA. Methods: Twenty patients with gastric carcinoma, 4 patients with gastritis, and 10 apparently healthy controls were recruited. Blood samples were obtained before surgery. 10 days after surgery, the blood samples were obtained again. Serum CIM4v6mRNA in all cases was measured by real-time quantitative PCR. Results: Serum CIM4V6mRNA was detectable in 20 of 20( 100% ) gastric carcinoma cases, The expression level ranged from 4.9×10^2 copies/μg RNA to 3.2 ×10^5 copies/μg RNA, the average levels of peripheral blood was 3.9 ×10^4 copies/μg RNA, The expression level of pedpheral blood of gastric cancer after curative operation ranged from 5.5×10^0 copies/μg RNA to 7.6 ×10^3 copies/μg RNA. After curative operation the expression level was decreased markedly. Conclusion: Serurn CIM4v6mRNA is expressed in the peripheral blood of gastric carcinoma patients. The expression level of CD44V6mRNA is obviously decreased after curative operation. An elevated level of CD44v6mRNA may serve as an indicator of lymph node metastasis (especially early metastasis) and bad prognosis in patients with gastric carcinoma.
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Supported by the National Natural Science Foundation of China(No. 30371757)
文摘Ret finger protein(RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger of motif, a B-box, and a coiled-coil domain(they are called RBCC generally). Although RFP was known to be an oncogene when its RBCC moiety was connected with a tyrosine kinase domain by DNA rearrangement, its biological function was not well defined. In this study, by using real-time RT-PCR, the RFP expressions in human and mouse normal tissues, and in the cervical squamous cell carcinoma, endometrial adenocarcinoma, gastric adenocarcinoma, esophageal squamous cell carcinoma, and brain cancer tissues were analyzed. The result of the study proved that the highest level of mRNA reverse transcription appeared in the normal testical tissue, whereas that in other normal tissues of human and mice were low. The mRNA reverse transcription level of RFP was higher in the endometrial adenocarcinoma tissue than in the cervical squamous cell carcinoma tissue; the mRNA reverse transcription level of RFP in the gastric adenocarcinoma tissue was significantly higher than that in the esophageal squamous cell carcinoma tissue. It was also found that the mRNA reverse transcription level of RFP in the brain cancer tissue was higher than that in the normal brain tissue. These results suggested that RFP could possibly be a useful molecular target for the development of new therapeutics for malignant tumors.