Pub Date : 2009-04-01DOI: 10.1017/S1479236209002599
Shi Lijun, Lü MaoMin, L. Gang, Li Cheng-yao, Zhang Jingang
A rapid Real-time polymerase chain reaction (RT-PCR) for West nile virus(WNV) detection was established. Primers were designed according to Capsid protein gene by Primer Premier5.0. In response, a cheap assay with the intercalating dye SYBR green Ⅰ was developed and validated. Amplifying curve showed that this method could successfully amplify 102 copies/μL WNV gene, meanwhile reference Japanese encephalitis virus(JEV) and blank control were all negative. Ten-fold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay showed high reproducibility with CV2%, indicating that the developed RT-PCR assay, a rapid, sensitive and specific test, has been built for detecting WNV.
{"title":"Establishment and evaluation of real-time PCR for West Nile virus detection.","authors":"Shi Lijun, Lü MaoMin, L. Gang, Li Cheng-yao, Zhang Jingang","doi":"10.1017/S1479236209002599","DOIUrl":"https://doi.org/10.1017/S1479236209002599","url":null,"abstract":"A rapid Real-time polymerase chain reaction (RT-PCR) for West nile virus(WNV) detection was established. Primers were designed according to Capsid protein gene by Primer Premier5.0. In response, a cheap assay with the intercalating dye SYBR green Ⅰ was developed and validated. Amplifying curve showed that this method could successfully amplify 102 copies/μL WNV gene, meanwhile reference Japanese encephalitis virus(JEV) and blank control were all negative. Ten-fold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay showed high reproducibility with CV2%, indicating that the developed RT-PCR assay, a rapid, sensitive and specific test, has been built for detecting WNV.","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"62 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115023617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-04-01DOI: 10.1017/S1479236209002605
Wu Cheng-long, Shi Cheng-yin, H. Jie, Kong Xiaoyu
A rapid and sensitive Real-time PCR assay coupled with SYBR Green Ⅰchemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from TRBIV major capsid protein (MCP) gene was involved in the Real-time PCR assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescence dye, SYBR Green Ⅰ, which binding to dsDNA exhibited fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid pUCm-T/TRBIV MCP containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (r2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has been spread widely in turbot farms locating at Shandong peninsula. The virus number was varied from 1.27×102 to 2.33×106 of viral genome copies/mg tissue in spleen of infected turbot. These results suggest that the Real-time PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.
{"title":"Real-time PCR assay for sensitive organ detection and epidemic investigation of Turbot reddish body iridovirus","authors":"Wu Cheng-long, Shi Cheng-yin, H. Jie, Kong Xiaoyu","doi":"10.1017/S1479236209002605","DOIUrl":"https://doi.org/10.1017/S1479236209002605","url":null,"abstract":"A rapid and sensitive Real-time PCR assay coupled with SYBR Green Ⅰchemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from TRBIV major capsid protein (MCP) gene was involved in the Real-time PCR assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescence dye, SYBR Green Ⅰ, which binding to dsDNA exhibited fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid pUCm-T/TRBIV MCP containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (r2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has been spread widely in turbot farms locating at Shandong peninsula. The virus number was varied from 1.27×102 to 2.33×106 of viral genome copies/mg tissue in spleen of infected turbot. These results suggest that the Real-time PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"21 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"129382699","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-04-01DOI: 10.1017/S1479236209002678
Z. Li-hui, Xuan Hai-feng, Wang Hui-Quang, W. Shaohua, Zhuang Yi-xuan
{"title":"Construction and utilization of plant expression vectors with PMI gene as selection marker to recover transgenic plants of Citrus sinensis L. Osbeck","authors":"Z. Li-hui, Xuan Hai-feng, Wang Hui-Quang, W. Shaohua, Zhuang Yi-xuan","doi":"10.1017/S1479236209002678","DOIUrl":"https://doi.org/10.1017/S1479236209002678","url":null,"abstract":"","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"44 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134263514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-04-01DOI: 10.1017/S1479236209002630
W. Han, Dou Zhong-ying, Wang Hua-yan
Amniotic fluid stem (AFS) cells derived from human amniotic fluid were cultured in vitro and the factors of affecting the primary culture of human AFS cells were investigated. The isolated AFS cells, which express embryonic stem cell markers, such as Oct-4, and adult stem cell markers, such as CD29, grew faster and had high proliferation ability. Moreover, AFS cells could differentiate into neurons and cardiomyocytes which expressed Nestin and α-actin, respectively. There were several factors, such as the date of gestation, the level of blood contamination and the volume of amniotic fluid, could significantly affect the attachment time and numbers of AFS cells in the primary culture. The results showed that cell attachment time of the 2nd trimester of gestation (4.7±0.6) d was significantly different from that of the 3rd trimester of gestation (6±0.5) d (P 0.05), suggesting that cells collected from the fluid of 3rd trimester of gestation needed longer attachment time. Blood contamination could significantly affect the cell attachment time. The attachment time of brown-color fluid group (10.8±0.3) d was significantly different with the blood-cell-free fluid group (6±0.5) d and blood-cell group (6.3±0.6) d (P 0.05). The volume of amniotic fluid influenced on cell attachment numbers and time to some extent. With the increase of amniotic fluid volume, cell attachment numbers significantly increased (P 0.05), and cell attachment time extended but no significant difference(P 0.05). The present studies systematically examined the factors, effection on primary culture of human AFS cells and provided some useful data for AFS cell research. Additionally, the isolated AFS cells maintain the capabilities of differentiation into other cell types and are able to become seed cells for the clinical application.
{"title":"Growth and identification of human amniotic fluid stem cells and analysis of their influencing factors","authors":"W. Han, Dou Zhong-ying, Wang Hua-yan","doi":"10.1017/S1479236209002630","DOIUrl":"https://doi.org/10.1017/S1479236209002630","url":null,"abstract":"Amniotic fluid stem (AFS) cells derived from human amniotic fluid were cultured in vitro and the factors of affecting the primary culture of human AFS cells were investigated. The isolated AFS cells, which express embryonic stem cell markers, such as Oct-4, and adult stem cell markers, such as CD29, grew faster and had high proliferation ability. Moreover, AFS cells could differentiate into neurons and cardiomyocytes which expressed Nestin and α-actin, respectively. There were several factors, such as the date of gestation, the level of blood contamination and the volume of amniotic fluid, could significantly affect the attachment time and numbers of AFS cells in the primary culture. The results showed that cell attachment time of the 2nd trimester of gestation (4.7±0.6) d was significantly different from that of the 3rd trimester of gestation (6±0.5) d (P 0.05), suggesting that cells collected from the fluid of 3rd trimester of gestation needed longer attachment time. Blood contamination could significantly affect the cell attachment time. The attachment time of brown-color fluid group (10.8±0.3) d was significantly different with the blood-cell-free fluid group (6±0.5) d and blood-cell group (6.3±0.6) d (P 0.05). The volume of amniotic fluid influenced on cell attachment numbers and time to some extent. With the increase of amniotic fluid volume, cell attachment numbers significantly increased (P 0.05), and cell attachment time extended but no significant difference(P 0.05). The present studies systematically examined the factors, effection on primary culture of human AFS cells and provided some useful data for AFS cell research. Additionally, the isolated AFS cells maintain the capabilities of differentiation into other cell types and are able to become seed cells for the clinical application.","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130796325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"In vitro effect of lentinan on the activation of immunological cells in Cyprinus carpio","authors":"Zhang Liu, Cao Liping, Ding Weidong, Galina Jeney, X. Pao, Xiong Chuan-xi, Yin Guojun","doi":"10.1017/S1479236209002496","DOIUrl":"https://doi.org/10.1017/S1479236209002496","url":null,"abstract":"","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121935437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-04-01DOI: 10.1017/S1479236209002526
Li Wenzheng, Song Li-min, Li Yongping, L. Xiuping, Lu Hongmei, Dai Chengen, Fang Yongqi, Dong Hai-tao, L. Debao
{"title":"Expressed sequence tag analysis and cDNA array establishment of Nicotiana tabacum : application to salinity stress","authors":"Li Wenzheng, Song Li-min, Li Yongping, L. Xiuping, Lu Hongmei, Dai Chengen, Fang Yongqi, Dong Hai-tao, L. Debao","doi":"10.1017/S1479236209002526","DOIUrl":"https://doi.org/10.1017/S1479236209002526","url":null,"abstract":"","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"89 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114561984","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-04-01DOI: 10.1017/S1479236208002362
Chen Mei-ling, N. Dong, Ruan Hui, Pan Bing-qing, Wan Jin-ling, Wu De, Zhang Jia-jia, Chen Qihe, He Guoqing
Epidermal growth factor receptor (EGFR) is becoming a perfect target for killing carcinoma cells, especially because of its overexpression on the surface of these cells. Cationic antimicrobial peptides (CAP) have their own special mechanism of membrane-lytic cytotoxicity. In this study, a membrane-lytic immunotoxin (IT), chimeric protein MEGFMEL, was constructed to kill carcinoma cells with EGFR overexpression. This protein is composed of mouse ( Mus musculus ) epidermal growth factor (MEGF), as the target part, and melittin (MEL), as the cytotoxic part. Using Escherichia coli BL21 and pET30a as expression strain and vector, respectively, 63.45 μg/ml of MEGFMEL (68% purity) was obtained through low-temperature induction of expression and a thawing-freezing purification procedure (without cytolysis). In vitro activity measurement showed that this MEGFMEL significantly induced a lethal effect on A 431 carcinoma cells overexpressing EGFR on the surface, with an LD 50 value 52.6 μg/ml. The results suggest that the use of CAP as the toxin in the construction of unique membrane-lytic ITs aimed at EGFR is feasible.
{"title":"Construction of a membrane-lytic immunotoxin using melittin and epidermal growth factor","authors":"Chen Mei-ling, N. Dong, Ruan Hui, Pan Bing-qing, Wan Jin-ling, Wu De, Zhang Jia-jia, Chen Qihe, He Guoqing","doi":"10.1017/S1479236208002362","DOIUrl":"https://doi.org/10.1017/S1479236208002362","url":null,"abstract":"Epidermal growth factor receptor (EGFR) is becoming a perfect target for killing carcinoma cells, especially because of its overexpression on the surface of these cells. Cationic antimicrobial peptides (CAP) have their own special mechanism of membrane-lytic cytotoxicity. In this study, a membrane-lytic immunotoxin (IT), chimeric protein MEGFMEL, was constructed to kill carcinoma cells with EGFR overexpression. This protein is composed of mouse ( Mus musculus ) epidermal growth factor (MEGF), as the target part, and melittin (MEL), as the cytotoxic part. Using Escherichia coli BL21 and pET30a as expression strain and vector, respectively, 63.45 μg/ml of MEGFMEL (68% purity) was obtained through low-temperature induction of expression and a thawing-freezing purification procedure (without cytolysis). In vitro activity measurement showed that this MEGFMEL significantly induced a lethal effect on A 431 carcinoma cells overexpressing EGFR on the surface, with an LD 50 value 52.6 μg/ml. The results suggest that the use of CAP as the toxin in the construction of unique membrane-lytic ITs aimed at EGFR is feasible.","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"25 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"133649584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-04-01DOI: 10.1017/S1479236209002575
Xu Wentao, Bai Wei-bin, Luo Yunbo, Yuan Yanfang, Huang Kunlun
{"title":"Research progress in techniques for detecting genetically modified organisms","authors":"Xu Wentao, Bai Wei-bin, Luo Yunbo, Yuan Yanfang, Huang Kunlun","doi":"10.1017/S1479236209002575","DOIUrl":"https://doi.org/10.1017/S1479236209002575","url":null,"abstract":"","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"15 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132239277","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2009-04-01DOI: 10.1017/S1479236209002502
Yang Jin-guang, Wang Wen-ting, Ding Xin-Lun, Guo Li-juan, Fang Zhen-Xing, Xie Liyan, Lin Qi-ying, Wu Zujian, Xie Lian-hui
The expression of YUCAA1 gene and the amount of endogenous IAA in rice(Oryza sativa subsp,japonica)plants and rice suspension cells infected by Rice stripe virus(RSV)were investigated by Real-time RT-PCR and high performance liquid chromatography,respectively.And the results showed that the expression of YUCAA1 gene and the amount of endogenous IAA increased at various times(16,32,48 and 64 h)after post-infection by RSV in rice suspension cells.In rice plants infected by RSV,the expression of YUCAA1 gene and the amount of endogenous IAA increased at 4～8 d after post-infection as comparison with that of healthy rice plants,and decreased at 12 and 16 d.These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally,the expression of RSV CP increased 2.9 times in rice plants after it was treated with KPSC buffer to deplete the endogenous auxins,and decreased 45% after 30μmol/L IAA treatment.All of these results suggest that the auxin may play a role among RSV replication in rice plant.
{"title":"Auxin regulation in the interaction between Rice stripe virus and rice.","authors":"Yang Jin-guang, Wang Wen-ting, Ding Xin-Lun, Guo Li-juan, Fang Zhen-Xing, Xie Liyan, Lin Qi-ying, Wu Zujian, Xie Lian-hui","doi":"10.1017/S1479236209002502","DOIUrl":"https://doi.org/10.1017/S1479236209002502","url":null,"abstract":"The expression of YUCAA1 gene and the amount of endogenous IAA in rice(Oryza sativa subsp,japonica)plants and rice suspension cells infected by Rice stripe virus(RSV)were investigated by Real-time RT-PCR and high performance liquid chromatography,respectively.And the results showed that the expression of YUCAA1 gene and the amount of endogenous IAA increased at various times(16,32,48 and 64 h)after post-infection by RSV in rice suspension cells.In rice plants infected by RSV,the expression of YUCAA1 gene and the amount of endogenous IAA increased at 4～8 d after post-infection as comparison with that of healthy rice plants,and decreased at 12 and 16 d.These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally,the expression of RSV CP increased 2.9 times in rice plants after it was treated with KPSC buffer to deplete the endogenous auxins,and decreased 45% after 30μmol/L IAA treatment.All of these results suggest that the auxin may play a role among RSV replication in rice plant.","PeriodicalId":236932,"journal":{"name":"Chinese Journal of Agricultural Biotechnology","volume":"7 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2009-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125483233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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