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Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2010年第1期11卷 52-60页

作者： Jian-xiang WU Shao-en ZHANG xue-ping zhou Institute of Biotechnology Zhejiang University Hangzhou 310029 China Nankai Biotechnology Limited Company Hangzhou 310051 China

A protein conjugate of streptomycin （streptomycin-bovine serum albumin （BSA） conjugate） was prepared and used as immunogen to produce monoclonal antibodies （MAb）. One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentra- tion （IC50） value of 4.65 ng/ml and the IC20value of 0.21 ng/ml in phosphate buffered saline （PBS）. At optimum con- ditions, an indirect competitive enzyme-linked immunosorbent assay （ELISA） and a colloidal gold-based immuno- chromatographic assay （CGIA） were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/rnl were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum de- tection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.

关键词： Streptomycin Monoclonal antibody Antibiotic residue Enzyme-linked immunosorbent assay (ELISA) Colloidal gold-based immunochromatographic assay (CGIA)

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Interaction between a Nanovirus-like Component and the Tobacco Curly Shoot Virus/Satellite Complex

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Acta Biochimica et Biophysica Sinica 2005年第1期37卷 25-31页

作者： Pei-Jun WU xue-ping zhou~* Institute of Biotechnology Zhejiang University,Hangzhou 310029,China Institute of Biotechnology Zhejiang UniversityHangzhou 310029 China

The biological role of DNA1,a nanovirus-like component shown to be associated with thebegomovirus/satellite complex,has not yet been ***,we demonstrated that DNAl of Tobaccocurly shoot virus isolate Y35(TbCSV-Y35)attenuated leaf-curling symptoms induced by TbCSV-Y35 orTbCSV-Y35 plus Y35 DNAβ in the early stage of symptom development and induced leaf cluster at a laterstage of symptom development in Nicotiana benthamiana *** leaf disc assay demonstrated thatTbCSV-Y35 DNA 1 replicated *** blot analysis revealed that TbCSV-Y35 DNA 1 re-duced viral DNA *** DNA accumulation was not reduced when plants were co-inoculatedwith TbCSV-Y35 DNAβ,but the TbCSV-Y35 DNAβ level was dramatically reduced in the presence ofTbCSV-Y35 DNA *** determine whether the interaction between TbCSV/satellite complex and DNA 1 hadisolate specificity,DNAI of TbCSV isolate Y132 was cloned and *** was found to have 75%nucleotide sequence identity with TbCSV-Y35 DNA *** tests showed that TbCSV-Y 132 DNA 1 hadno effect on the symptoms induced by TbCSV-Y35 or TbCSV-Y35 and Y35 DNAβ in ***,although Y 132 DNA1 could replicate in these plants.

关键词： Tobacco curly shoot virus DNA1 DNAβ symptom replication interaction

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Molecular characterization and infectivity of Papaya leaf curl China virus infecting tomato in China

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2010年第2期11卷 109-114页

作者： Hui ZHANG Xin-ying MA Ya-juan QIAN xue-ping zhou Key Laboratory of Molecular Biology of Crop Pathogens and Insects of Ministry of Agriculture Institute of Biotechnology Zhejiang University Hangzhou 310029 China Horticultural Research Institute Shanghai Academy of Agriculture Sciences Shanghai 201106 China

Papaya leaf curl China virus (PaLCuCNV) was previously reported as a distinct begomovirus infecting papaya in southern China. Based on molecular diagnostic survey, 13 PaLCuCNV isolates were obtained from tomato plants showing leaf curl symptoms in Henan and Guangxi Provinces of China. Complete nucleotide sequences of 5 representative isolates (AJ558116, AJ558117, AJ704604, FN256260, and FN297834) were determined to be 2738–2751 nucleotides, which share 91.7%–97.9% sequence identities with PaLCuCNV isolate G2 (AJ558123). DNA-β was not found to be associated with PaLCuCNV isolates. To investigate the infectivity of PaLCuCNV, an in-fectious clone of PaLCuCNV-[CN:HeNZM1] was constructed and agro-inoculated into Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants, which induced severe leaf curling and crinkling symptoms in these plants. Southern blot analysis and polymerase chain reaction (PCR) indicated a systemic infection of test plants by the agro-infectious clone.

关键词： Papaya leaf curl China virus (PaLCuCNV) Tomato Infectivity

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Genetic variability and evolution of rice stripe virus

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2013年第10期14卷 875-885页

作者： Ling-zhe HUANG Li-xia RAO xue-ping zhou Jian-xiang WU State Key Laboratory of Rice Biology Institute of Biotechnology Zhejiang University

Rice stripe virus(RSV) is the type member of the genus *** is known to have four segmented,single-stranded RNA molecules and causes rice stripe disease in the rice fields of China,Japan,and *** on the complete genomic sequences of the determined 6 RSV isolates(from Yunnan,Jiangsu,Zhejiang,and Liaoning Provinces,China) and 27 other RSV isolates(from Yunnan,Jiangsu,Anhui,Henan,and Shandong Provinces of China,also Japan and Korea) downloaded from GenBank,we provided a genotyping profile of RSV field isolates and described the population structure of *** RSV isolates,except isolate CX,could be divided into two subtypes,one including 6 isolates from Yunnan Province,and the other including 26 isolates from different parts of China,Japan,and Korea,which were referred to as subtype II and subtype I,*** amino acid distances between subtypes range from 0.053 to *** isolates in Yunnan Province were genetically differentiated from other parts of China,Japan,and Korea and showed infrequent gene *** RSV populations collected from other parts of China,Japan,and Korea were only composed of subtype I and showed very low genetic *** speculated that isolate CX may be the result of recombination of isolates from two *** potential recombination events were detected in RNA4 of isolate CX.

关键词： Rice stripe virus Genetic variability Genetic evolution

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Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

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Journal of Integrative Plant Biology 2007年第12期49卷 1726-1733页

作者： You-ping Xu Lu-ping Zheng Qiu-Fang Xu Chang-Chun Wang xue-ping zhou Zu-Jian Wu Xin-Zhong Cai Institute of Biotechnology Zhejiang University Hangzhou 310029 China Centre of Analysis and Measurement Zhejiang University Hangzhou 310029 China Institute of Plant Virology Fujian Agriculture and Forestry University Fuzhou 350002 China

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.

关键词： DNAβ Nicotiana tobacco rattle virus tomato yellow leaf curl China virus virus-induced gene silencing.

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Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2011年第2期12卷 83-92页

作者： Chang-jun HUANG Tong ZHANG Fang-fang LI Xin-yue ZHANG xue-ping zhou State Key Laboratory of Rice Biology Institute of Biotechnology Zhejiang University Hangzhou 310029 China

Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18-32°C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.

关键词： Virus induced gene silencing Geminivirus Alphasatellite Nicotiana tabacum

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Monoclonal antibody-based serological methods for maize chlorotic mottle virus detection in China

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2013年第7期14卷 555-562页

作者： Jian-xiang WU Qiang WANG Huan LIU Ya-juan QIAN Yan XIE xue-ping zhou State Key Laboratory of Rice Biology Institute of Biotechnology Zhejiang University

Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.

关键词： Maize chlorotic mottle virus (MCMV) Immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) Triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) Monoclonal antibody (MAb) Dot-immunobinding assay (DIBA)

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Viral infection of tobacco plants improves performance of Bemisia tabaci but more so for an invasive than for an indigenous biotype of the whitefly

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2010年第1期11卷 30-40页

作者： Jian LIU Meng LI Jun-min LI Chang-jun HUANG xue-ping zhou Fang-cheng XU Shu-sheng LIU Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects Institute of Insect Sciences Zhejiang University Hangzhou 310029 China) Institute of Biotechnology Zhejiang University Hangzhou 310029 China Wenzhou Academy of Agricultural Sciences Wenzhou 325006 China

The ecological effects of plant-virus-vector interactions on invasion of alien plant viral vectors have been rarely investigated. We examined the transmission of Tomato yellow leaf cur/China virus （TYLCCNV） by the invasive Q biotype and the indigenous ZHJ2 biotype of the whitefly Bemisia tabaci, a plant viral vector, as well as the influence of TYLCCNV-infection of plants on the performance of the two whitefly biotypes. Both whitefly biotypes were able to acquire viruses from infected plants and retained them in their bodies, but were unable to transmit them to either tobacco or tomato plants. However, when the Q biotype fed on tobacco plants infected with TYLCCNV, its fecundity and longevity were increased by 7- and 1-fold, respectively, compared to those of the Q biotype fed on uninfected tobacco plants. When the ZHJ2 biotype fed on virus-infected plants, its fecundity and longevity were increased by only 2- and 0.5-fold, respectively. These data show that the Q biotype acquired higher beneficial effects from TYLCCNV-infection of tobacco plants than the ZHJ2 biotype. Thus, the Q biotype whitefly may have advantages in its invasion and displacement of the indigenous ZHJ2 biotype.

关键词： Bemisia tabaci Biotypes Begomovirus Vector-virus interaction Biological invasion

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Micrometastasis in surrounding liver and the minimal length of resection margin of primary liver cancer

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World Journal of Gastroenterology 2007年第33期13卷 4498-4503页

作者： xue-ping zhou Zhi-Wei Quan Wen-Ming Cong Ning Yang Hai-Bin Zhang Shu-Hui Zhang Guang-Shun Yang Department of General Surgery Xinhua Hospital Shanghai Jiao Tong University Shanghai200092 China Department of Pathology Eastern Hepatobiliary Surgery Hospital Second Military Medical University Shanghai 200438 China Department of Hepatobiliary Surgery Eastern Hepatobiliary Surgery Hospital Second Military Medical University Shanghai 200438 China

AIM： To describe the distribution of micrometastases in the surrounding liver of patients with primary liver cancer （PLC）, and to describe the minimal length of resection margin （RM） for hepatectomy. METHODS： From November 2001 to March 2003, 120 histologically verfied PLC patients without macroscopic tumor thrombi or macrosatellites or extrahepatic metastases underwent curative hepatectomy. Six hundreds and twenty-nine routine pathological sections from these patients were re-examined retrospectively by light microscopy. In the prospective study, curative hepatectomy was performed from November 2001 to March 2003 for 76 histologically verfied PLC patients without definite macroscopic tumor thrombi or macrosatellites or extrahepatic metastases in preoperative imaging. Six hundreds and forty-five pathological sections from these patients were examined by light microscopy. The resected liver specimens were minutely examined to measure the resection margin and to detect the number of daughter tumor nodules, dominant lesions, and macroscopic tumor thrombi inside the lumens of the major venous system. The paraffin sections were microscopically examined to detect the microsatellites, microscopic tumor thrombi, fibrosis tumor capsules, as well as capsule invasion and the distance of histological spread of the micrometastases. RESULTS： In the retrospective study, 70 micrometastases were found in surrounding liver in 26 of the 120 cases （21.7%）. The farthest distance of histological micrometastasis was 3.5 mm, 5.3 mm and 6.0 mm in 95%, 99% and 100% cases, respectively. Macroscopic tumor thrornbi or rnacrosatellites were observed in 18 of 76 cases, and 149 rnicrometastases were found in the surrounding live in 25 （43.1%） of 58 cases with no macroscopic tumor thrombi. The farthest distance of histological micrometastasis was 4.5 mm, 5.5 mm and 6.0 mm in 95%, 99% and 100% cases, respectively. Two hundred and sixty-seven rnicrometastases were found in surrounding liver in 14 （

关键词： Primary liver cancer Micrometastases Resection margin Hepatectomy

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Short-term microbiological effects of scaling and root planing and essential-oils mouthwash in Chinese adults

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2013年第5期14卷 416-425页

作者： Jia-yan HE Gang-gang QI Wu-jing HUANG Xu-dong SUN Yu TONG Chun-mei PENG xue-ping zhou Hui CHEN Institute of Biotechnology Zhejiang University Department of Conservative Dentistry and Periodontics Affiliated Hospital of StomatologySchool of MedicineZhejiang University Department of Stomatology Hangzhou Aeromedicine Certification and Training Center of the PLA Air Force

Objective: To assess the short-term effect of scaling and root planing (SRP) and essential-oils mouthwash on the levels of specific bacteria in Chinese adults. Methods: Fifty Chinese adults with chronic periodontitis were randomly assigned to full-mouth SRP or a 7-d essential-oils mouthwash regimen. In addition, 22 periodontally healthy adults used essential-oils mouthwash for 7 d. Clinical examination and plaque/saliva sampling were performed at baseline and on Day 7. Quantitative real-time polymerase chain reaction (PCR) was used to measure Aggregatibacter actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and total bacterial loads in saliva, supra- and sub-gingival plaque samples. Results: The detection frequencies of four tested species remained unchanged after either treatment. However, the bacterial loads of Fn, Pg, and Pi were significantly reduced by SRP; the mean reduction of bacterial counts in saliva ranged from 52.2% to 62.5% (p<0.01), in supragingival plaque from 68.2% to 81.0% (p<0.05), and in subgingival plaque from 67.9% to 93.0% (p<0.01). Total bacterial loads were reduced after SRP in supra- and sub-gingival plaque (p<0.05). Essential-oils mouthwash reduced Fn levels in supragingival plaque by a mean of 53.2%, and reduced total bacterial loads in supra- and sub-gingival plaque (p<0.01). In subgingival plaque from periodontal patients, Pg and Pi reductions were high after SRP compared to essential-oils mouthwash (93.0% vs. 37.7% and 87.0% vs. 21.0%, p<0.05). No significant bacterial reduction was observed in periodontally healthy subjects using essential-oils mouthwash. Conclusions: SRP and essential-oils mouthwash both have an impact on saliva and gingival plaque flora in Chinese periodontitis patients in 7 d, with greater microbiological improvement by SRP.

关键词： Oral microbiota Chronic periodontitis Scaling and root planing Essential-oils Quantitative detection Real-time PCR

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