Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set...Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.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
In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating...In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.展开更多
文摘Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.
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.
文摘In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.
