[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-s...[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.展开更多
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
The objective of this paper was to analyze the E2 genetic characterization of HB-DCZ strain of Bovine viral diaxrhca Virus （BVDV） which wcrc amplified by RT-PCR and isolated from China. The product of PCK was cloned...The objective of this paper was to analyze the E2 genetic characterization of HB-DCZ strain of Bovine viral diaxrhca Virus （BVDV） which wcrc amplified by RT-PCR and isolated from China. The product of PCK was cloned into pMD18-T vector, and then transfected Escherichia Coli JMI00. The recombinant plasmids were amplified by PCR and were sequenced. From the nucleotide sequence of the amplified products, phylogenetie analyses were performed and genotypes or subgenotypes were identified. The results indicated that the E2 gene fragment of HB-DCZ strain contained 1277bp nucleotides, and had 89.4%, 70.7%, 97.6%, 68.9%, 67.2% sequence similarity with Osloss, OregonC24V, Changchun184, ZM195, NADL, respectively. In conclusion, HB-DCZ strain is closely related to BVDV Osloss, Changchun184, and belongs to subgenotype lb.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
Objective: To investigate the prevalence of bovine coronavirus(BCo V), bovine rotavirus, and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and Octob...Objective: To investigate the prevalence of bovine coronavirus(BCo V), bovine rotavirus, and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and October of 2016 in the Republic of Korea. Methods: Samples were obtained from 50 normal and 93 diarrheic(56 semi-formed, 28 loose, and 9 watery feces) calves in six different regions of northern and southern Korea. These fecal samples were tested for BCo V, bovine rotavirus, and bovine viral diarrhea virus by RT-PCR. Results: Among the three pathogens examined, infection with BCo V was especially prominent in relation to diarrhea among calves aged 1-21 days [odds ratio(OR)=9.3, 95% confidence interval(CI): 1.1-78.9; P=0.02). Infection with BCo V alone(OR=2.9; 95% CI: 1.1-7.6; P=0.03) or coinfection of BCo V with bovine viral diarrhea virus(OR=3.6; 95% CI: 1.0-12.4; P=0.04) was significantly associated with the development of loose feces. Grazing and colostrum intake strongly reduced the occurrence of diarrhea as compared to housed calves(OR=0.2; 95% CI: 0.1-0.4; P=0.00) and calves that had not been fed colostrum(OR=0.2; 95% CI: 0.1-0.7; P=0.02), respectively. Conclusions: The present study suggests that BCo V is involved in calf diarrhea in the Republic of Korea. Therefore, grazing and colostrum intake is recommended for preventing and controlling calf diarrhea caused by BCoV.展开更多
基金Supported by Science and Technology Development Plan Fund Project of Jilin Province(20210202101NC)YDZJ202203C G Z H 050.
文摘[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.
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.
文摘The objective of this paper was to analyze the E2 genetic characterization of HB-DCZ strain of Bovine viral diaxrhca Virus （BVDV） which wcrc amplified by RT-PCR and isolated from China. The product of PCK was cloned into pMD18-T vector, and then transfected Escherichia Coli JMI00. The recombinant plasmids were amplified by PCR and were sequenced. From the nucleotide sequence of the amplified products, phylogenetie analyses were performed and genotypes or subgenotypes were identified. The results indicated that the E2 gene fragment of HB-DCZ strain contained 1277bp nucleotides, and had 89.4%, 70.7%, 97.6%, 68.9%, 67.2% sequence similarity with Osloss, OregonC24V, Changchun184, ZM195, NADL, respectively. In conclusion, HB-DCZ strain is closely related to BVDV Osloss, Changchun184, and belongs to subgenotype lb.
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.
基金supported by the National Research Foundation of Korea(NRF)funded by the Korea government(No.2015R1C1A2A01053080)the "Cooperative Research Program for Agriculture Science&Technology Development(Project No.PJ01194503)" from the Rural Development Administration,the Republic of Korea
文摘Objective: To investigate the prevalence of bovine coronavirus(BCo V), bovine rotavirus, and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and October of 2016 in the Republic of Korea. Methods: Samples were obtained from 50 normal and 93 diarrheic(56 semi-formed, 28 loose, and 9 watery feces) calves in six different regions of northern and southern Korea. These fecal samples were tested for BCo V, bovine rotavirus, and bovine viral diarrhea virus by RT-PCR. Results: Among the three pathogens examined, infection with BCo V was especially prominent in relation to diarrhea among calves aged 1-21 days [odds ratio(OR)=9.3, 95% confidence interval(CI): 1.1-78.9; P=0.02). Infection with BCo V alone(OR=2.9; 95% CI: 1.1-7.6; P=0.03) or coinfection of BCo V with bovine viral diarrhea virus(OR=3.6; 95% CI: 1.0-12.4; P=0.04) was significantly associated with the development of loose feces. Grazing and colostrum intake strongly reduced the occurrence of diarrhea as compared to housed calves(OR=0.2; 95% CI: 0.1-0.4; P=0.00) and calves that had not been fed colostrum(OR=0.2; 95% CI: 0.1-0.7; P=0.02), respectively. Conclusions: The present study suggests that BCo V is involved in calf diarrhea in the Republic of Korea. Therefore, grazing and colostrum intake is recommended for preventing and controlling calf diarrhea caused by BCoV.
