Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas ...Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.展开更多
Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Met...Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Methods: We detected HSV infection in 121 speci-mens collected from patients with genital herpesusing both multiplex fluorescent PCR and serum type-specific antibody detection. HSV viral isolation wasused as the standard control.Results: When compared with the viral isolation, thesensitivity and specificity for multiplex fluorescentPCR were 100% and 88.89%, respectively afterdiscrepant analysis. The sensitivity and specificity fortype-specific antibody detection was 77.68 % and77.78 %, respectively. However, the type-specificantibody detected HSV in two asymptomatic patientswhile the multiplex fluorescent PCR couldn’t detectany HSV DNA from those specimens.Conclusions: Multiplex fluorescent PCR is a verysensitive and specific method for detection and typingof HSV in the lesion of genital herpes, it failed todetect HSV DNA from the asymptomatic patients.Serum type-specific antibody detection was a lesssensitive and specific test but could detect the specificantibody from some asymptomatic patients. Thecombination of these two techniques would allow rapid,sensitive and accurate detection and typing of HSVand help clinical diagnosis and epidemiologic survey-ing of genital herpes.展开更多
A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as th...A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.展开更多
The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced ...The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced by Isopropyl β-D-thiogalactoside （IPTG）, the DHFR/StxB fusion protein was highly expressed to the level of 41.36% in E. coli MI5 cells. The 35 kDa fusion protein with a 6 His-tag was one-step purified from inclusion bodies using Ni-NTA affinity chromatography column under denaturing conditions, and was refolded by dialyzing with a decreasing urea gradient. Purified DHFR/StxB fusion protein was used to immunize Kunming mice for generating the ascitic polyclonal antibody against recombinant StxB protein by injecting sarcoma 180 cells and the titer ofascitic polyclonal antibody is up to 1： 1× 10^6 detected by the indirect enzyme linked immunosorbent assy （ELISA）. Western immunoblotting analysis revealed that the ascitic polyclonal antibody against StxB had a specific affinity for a 70 kDa shiga toxin protein of Shigella dysenteriae type 1. It is a new simple and quick method to produce a large amount of ascitic polyclonal antibody. The antibody is used to develop immunological method for detecting shiga toxin.展开更多
To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expres...To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.展开更多
Genotyping 42 serum samples taken from the pigs in the oubreaks from 2009 to 2013 with RT-PCR and nested RT-PCR, showed more than 80% of samples were positive with Chinese PRRSV clade, and the others were European and...Genotyping 42 serum samples taken from the pigs in the oubreaks from 2009 to 2013 with RT-PCR and nested RT-PCR, showed more than 80% of samples were positive with Chinese PRRSV clade, and the others were European and North American classical PRRSV genotypes. Ten serum samples from unnapprent PRRS herds were examined for antibodies against PRRSV with ELISA and also for PRRSV with RT-PCR. It was clearly that the titer of antibodies against PRRSV by ELISA test could not be used for interpreting PRRSV infection. In despite of PRRS vaccination or non-vaccination, a risk of PRRSV infection and re-infection exist, utilizing RT-PCR in combination with serology will give the producer and veterinarian PRRSV more exact situation in the herds.展开更多
With the purpose to determine the frequency and type of cardiac lesions in naturally exposed dogs to Trypanosoma cruzi, ninety one stray dogs, capture by the Canine and Feline Control Center （dog pound） from the mun...With the purpose to determine the frequency and type of cardiac lesions in naturally exposed dogs to Trypanosoma cruzi, ninety one stray dogs, capture by the Canine and Feline Control Center （dog pound） from the municipality of Merida, were studied. Before euthanasia, blood samples were taken to detect 72 cruzi antigens by indirect immunofluorescence antibody test and Western Blot and to detect the genome of parasite by Polymerase Chain Reaction. Immediately after euthanasia, hearts were macroscopically evaluated and a sample of the middle right atrial wall of each dog was taken for histopathological analyses. DNA was also obtained from paraffin blocks of seropositives animals with microscopic lesions to detect 72 cruzi genome. Of ninety one dogs, thirteen were seropositive. All seropositive dogs showed an association （P 〈 0.05） with lymphocytoplasmatic myocarditis. The presence of the 72 cruzi genome was also detected by PCR in cardiac septum tissue of seropositive dogs and in all the cases with microscopic lesions indicating the high pathogenicity of the local circulating strain. No association with macroscopic lesions was observed in seropositive dogs. Also, the presence of Dirofilaria immitis （D. immitis） was found in 6% of dogs evaluated. This study demonstrates a high tropism to cardiac tissue and virulence of the strains of 72 cruzi circulating in the studied dog population.展开更多
Objective： The aim of our study was to investigate whether Jinshuixian has the abilities of inhibiting tumor growth and radio-sensitivity effect. Methods： Cultured lung A549 ceils were randomly divided into 6 groups...Objective： The aim of our study was to investigate whether Jinshuixian has the abilities of inhibiting tumor growth and radio-sensitivity effect. Methods： Cultured lung A549 ceils were randomly divided into 6 groups： normal control group （NC）, the Jinshuixian group （JSX）, radiotherapy （RT）, JSX for the first day and the next day followed by RT group （JSX~ RT）, RT for the first day and the next day followed by JSX group （RT→JSX） and RT ＋ JSX concomitantly group （JSX ＋ RT）. MTT was applied to measure the cell viability, RT-PCR and ELISA were used to test the expression of mRNA and protein of VEGF. Results： The proliferation of A549 cells were inhibited in JSX and combined groups and the inhibiting effects were time dependent. The expression of VEGF in RT group was increased, however, VEGF in JSX and combination groups were largely decreased over time when compared to NC group. Results in JSX→RT, RT→JSX and JSX ＋ RT groups did not achieve significantly differences. Conclusion： JSX has the ability of anti-tumor growth in vitro accompanied down-regulating of VEGF, especially when combined with radiotherapy, and its effect is time-dependent. However, more studies in vivo and in vitro are needed to further supporting these effects.展开更多
Objective： To investigate the molecular pathway of substance P （SP） to induce osteoblastic differentiation. Methods： Mesenchymal stem cells were isolated and cultured. The cultures were divided into four groups w...Objective： To investigate the molecular pathway of substance P （SP） to induce osteoblastic differentiation. Methods： Mesenchymal stem cells were isolated and cultured. The cultures were divided into four groups with Group A （control group） cultured without any factors, Group B cultured with SP, Group C cultured with SP and SP receptor neurokinin-1 （NK1） antagonist, and Group D cultured with SP NK1 antagonist respectively to induce osteoblastic cells differentiation. Osterix gene expression was detected by reverse transcription-polymerase chain reaction （RT-PCR） for three times after 1-2 weeks of cultivation and the results were analyzed by one-way analysis of variance （ANOVA）. Results： The log phase of bone marrow stromal cells appeared at 4-6 days. ALP staining revealed that the majority of cells, more than 95%, were positive and small bluepurple granules were found in the cytoplasm. And Group B, treated with SP, showed a higher level of ALP activity than the other three groups. Meanwhile, RT-PCR found that Osterix expression in Group B was obviously up-regulated, compared with other groups. But Osterix expression in Group D had no remarkable differences, compared with the controls. Conclusions： SP can up-regulate Osterix gene expression to stimulate differentiation of mesenchymal stem cells into osteoblastic cells at the final stage. The regulatory effect of SP on Osterix expression was dependant on SP NK1 receptors.展开更多
基金Supported by Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province[CX(12)1003]Science and Technology Support Program of Jiangsu Province(BE2013301)Special Fund for the Construction of Modern Agriculture Industry System of China(CARS-01-47)~~
文摘Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.
文摘Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Methods: We detected HSV infection in 121 speci-mens collected from patients with genital herpesusing both multiplex fluorescent PCR and serum type-specific antibody detection. HSV viral isolation wasused as the standard control.Results: When compared with the viral isolation, thesensitivity and specificity for multiplex fluorescentPCR were 100% and 88.89%, respectively afterdiscrepant analysis. The sensitivity and specificity fortype-specific antibody detection was 77.68 % and77.78 %, respectively. However, the type-specificantibody detected HSV in two asymptomatic patientswhile the multiplex fluorescent PCR couldn’t detectany HSV DNA from those specimens.Conclusions: Multiplex fluorescent PCR is a verysensitive and specific method for detection and typingof HSV in the lesion of genital herpes, it failed todetect HSV DNA from the asymptomatic patients.Serum type-specific antibody detection was a lesssensitive and specific test but could detect the specificantibody from some asymptomatic patients. Thecombination of these two techniques would allow rapid,sensitive and accurate detection and typing of HSVand help clinical diagnosis and epidemiologic survey-ing of genital herpes.
文摘A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.
文摘The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced by Isopropyl β-D-thiogalactoside （IPTG）, the DHFR/StxB fusion protein was highly expressed to the level of 41.36% in E. coli MI5 cells. The 35 kDa fusion protein with a 6 His-tag was one-step purified from inclusion bodies using Ni-NTA affinity chromatography column under denaturing conditions, and was refolded by dialyzing with a decreasing urea gradient. Purified DHFR/StxB fusion protein was used to immunize Kunming mice for generating the ascitic polyclonal antibody against recombinant StxB protein by injecting sarcoma 180 cells and the titer ofascitic polyclonal antibody is up to 1： 1× 10^6 detected by the indirect enzyme linked immunosorbent assy （ELISA）. Western immunoblotting analysis revealed that the ascitic polyclonal antibody against StxB had a specific affinity for a 70 kDa shiga toxin protein of Shigella dysenteriae type 1. It is a new simple and quick method to produce a large amount of ascitic polyclonal antibody. The antibody is used to develop immunological method for detecting shiga toxin.
文摘To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.
文摘Genotyping 42 serum samples taken from the pigs in the oubreaks from 2009 to 2013 with RT-PCR and nested RT-PCR, showed more than 80% of samples were positive with Chinese PRRSV clade, and the others were European and North American classical PRRSV genotypes. Ten serum samples from unnapprent PRRS herds were examined for antibodies against PRRSV with ELISA and also for PRRSV with RT-PCR. It was clearly that the titer of antibodies against PRRSV by ELISA test could not be used for interpreting PRRSV infection. In despite of PRRS vaccination or non-vaccination, a risk of PRRSV infection and re-infection exist, utilizing RT-PCR in combination with serology will give the producer and veterinarian PRRSV more exact situation in the herds.
文摘With the purpose to determine the frequency and type of cardiac lesions in naturally exposed dogs to Trypanosoma cruzi, ninety one stray dogs, capture by the Canine and Feline Control Center （dog pound） from the municipality of Merida, were studied. Before euthanasia, blood samples were taken to detect 72 cruzi antigens by indirect immunofluorescence antibody test and Western Blot and to detect the genome of parasite by Polymerase Chain Reaction. Immediately after euthanasia, hearts were macroscopically evaluated and a sample of the middle right atrial wall of each dog was taken for histopathological analyses. DNA was also obtained from paraffin blocks of seropositives animals with microscopic lesions to detect 72 cruzi genome. Of ninety one dogs, thirteen were seropositive. All seropositive dogs showed an association （P 〈 0.05） with lymphocytoplasmatic myocarditis. The presence of the 72 cruzi genome was also detected by PCR in cardiac septum tissue of seropositive dogs and in all the cases with microscopic lesions indicating the high pathogenicity of the local circulating strain. No association with macroscopic lesions was observed in seropositive dogs. Also, the presence of Dirofilaria immitis （D. immitis） was found in 6% of dogs evaluated. This study demonstrates a high tropism to cardiac tissue and virulence of the strains of 72 cruzi circulating in the studied dog population.
文摘Objective： The aim of our study was to investigate whether Jinshuixian has the abilities of inhibiting tumor growth and radio-sensitivity effect. Methods： Cultured lung A549 ceils were randomly divided into 6 groups： normal control group （NC）, the Jinshuixian group （JSX）, radiotherapy （RT）, JSX for the first day and the next day followed by RT group （JSX~ RT）, RT for the first day and the next day followed by JSX group （RT→JSX） and RT ＋ JSX concomitantly group （JSX ＋ RT）. MTT was applied to measure the cell viability, RT-PCR and ELISA were used to test the expression of mRNA and protein of VEGF. Results： The proliferation of A549 cells were inhibited in JSX and combined groups and the inhibiting effects were time dependent. The expression of VEGF in RT group was increased, however, VEGF in JSX and combination groups were largely decreased over time when compared to NC group. Results in JSX→RT, RT→JSX and JSX ＋ RT groups did not achieve significantly differences. Conclusion： JSX has the ability of anti-tumor growth in vitro accompanied down-regulating of VEGF, especially when combined with radiotherapy, and its effect is time-dependent. However, more studies in vivo and in vitro are needed to further supporting these effects.
文摘Objective： To investigate the molecular pathway of substance P （SP） to induce osteoblastic differentiation. Methods： Mesenchymal stem cells were isolated and cultured. The cultures were divided into four groups with Group A （control group） cultured without any factors, Group B cultured with SP, Group C cultured with SP and SP receptor neurokinin-1 （NK1） antagonist, and Group D cultured with SP NK1 antagonist respectively to induce osteoblastic cells differentiation. Osterix gene expression was detected by reverse transcription-polymerase chain reaction （RT-PCR） for three times after 1-2 weeks of cultivation and the results were analyzed by one-way analysis of variance （ANOVA）. Results： The log phase of bone marrow stromal cells appeared at 4-6 days. ALP staining revealed that the majority of cells, more than 95%, were positive and small bluepurple granules were found in the cytoplasm. And Group B, treated with SP, showed a higher level of ALP activity than the other three groups. Meanwhile, RT-PCR found that Osterix expression in Group B was obviously up-regulated, compared with other groups. But Osterix expression in Group D had no remarkable differences, compared with the controls. Conclusions： SP can up-regulate Osterix gene expression to stimulate differentiation of mesenchymal stem cells into osteoblastic cells at the final stage. The regulatory effect of SP on Osterix expression was dependant on SP NK1 receptors.
