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
This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcin...This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quanti-tative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.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
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： 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
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Project (No. 021103004) supported by the Science and TechnologyDevelopment Program of Zhejiang Province, China
文摘This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quanti-tative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.
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 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.
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.
