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Real-time PCR assay for detection of Dickeya fangzhongdai causing bleeding canker of pear disease in China

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Journal of Integrative Agriculture 2020年第4期19卷 898-905页

作者： TIAN Yan-li ZHAO Yu-qiang CHEN Bao-hui CHEN Shuo ZENG Rong HU Bai-shi LI Xiang College of Plant Protection and Key Laboratory of Integrated Management of Crop Diseases and Pests Ministry of EducationNanjing Agricultural UniversityNanjing 210095P.R.China Institute of Botany Jiangsu Province and Chinese Academy of Sciences(Nanjing Botanical Garden Mem.Sun Yat-Sen)/Jiangsu Key Laboratory for the Research and Utilization of Plant ResourcesNanjing 210014P.R.China Charlottetown Laboratory Canadian Food Inspection AgencyCharlottetownPE C1A5T1Canada

Bleeding canker, caused by Dickeya fangzhongdai, is a devastating disease of pear in China. The bacterium causes cankers, branch die-back, and eventually kills pear trees. The typical sign of bleeding canker infection is a rusty-brown bacterial ooze that exudes down from cankers onto branches or trunks. However, early symptoms and signs are inconspicuous, which makes effective disease management difficult. Detection and identification of D. fangzhongdai are time-consuming and difficult because no rapid method exists to date. In this study, a Taq Man real-time PCR assay was developed for D. fangzhongdai based on an elongation factor G(fus A) gene. The real-time PCR assay detected 0.2 pg μL^-1 DNA and 1×10^3 Cfu m L^-1 of D. fangzhongdai. Based on this assay, bleeding canker on asymptomatic pear trees can be diagnosed as early as 5 days after infection. The real-time PCR assay can facilitate disease management by providing early and accurate diagnosis of the bleeding canker disease of pear.

关键词： molecular assay pear bleeding canker detection

A one-step specific assay for continuous detection of sirtuin 2 activity

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Acta Pharmaceutica Sinica B 2019年第6期9卷 1183-1192页

作者： Qi Dai Zhihua Zheng Fan Xia Peiqing Liu Min Li School of Pharmaceutical Sciences Sun Yat-Sen UniversityNational and Local United Engineering Lab of Druggability and New Drugs EvaluationGuangdong Provincial Key Laboratory of New Drug Design and EvaluationGuangzhou 510006China

Sirtuins(SIRTs)are nicotinamide adenine dinucleotide(NAD^+)-dependent histone deacetylases with diverse physiological functions.A variety of small molecules have been developed to interrogate the physiological function of ***,it is desirable to establish efficient and convenient assays to screen SIRTs *** this study,we designed a series of fluorescent nonapeptide probes derived from substrates of *** increment of these probes is based on SIRT-mediated removal of the acyl side chain with fluorophore,which makes this system free of lysine-recognizing *** the reaction of these fluorescent nonapeptide substrates with SIRT1-SIRT3 and SIRT6,it was confirmed that this assessment system was the most suitable for SIRT2activity ***,SIRT2 was used to modify substrates by truncating the amino acids or lysine side chain of ***,two specific and efficient fluorescent probes for SIRT2,ne-D9 and ne-K4a,were *** of the results revealed that ne-K4a based assay was more suitable for modulators screening in vitro,while the other specific substrate ne-D9 was stable in cell lysate and could detect the activity of SIRT2 in the *** summary,this study presents a novel strategy for detecting SIRT2 activity in vitro and in cell lysate.

关键词： Deacetylate Deacylate Fluorescent probe One-step assay Sirtuins SIRT2

Development of a competitive enzyme-linked immunosorbent assay for therapeutic drug monitoring of afatinib

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Journal of Pharmaceutical Analysis 2019年第1期9卷 49-54页

作者： Rintaro Sogawa Tetsuya Saita Yuta Yamamoto Sakiko Kimura Yutaka Narisawa Shinya Kimura Masashi Shin Department of Pharmacy Saga University Hospital Applied Life Science Department Faculty of Biotechnology and Life Science Sojo University Division of Hematology Respiratory Medicine and Oncology Department of Internal Medicine Faculty of Medicine Saga University

Afatinib is an oral tyrosine kinase inhibitor(TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug monitoring(TDM) in clinical settings. This study sought to develop a simple and sensitive competitive enzyme-linked immunosorbent assay(ELISA) to quantify afatinib in plasma for routine pharmacokinetic applications. An anti-afatinib antibody was obtained using(S)-N-4-(3-chloro-4-fluorophenyl)-7-(tetrahydrofuran-3-yloxy)-quinazoline-4,6-diamine(CTQD), which has the same substructure as afatinib, as a hapten. Enzyme labeling of afatinib with horseradish peroxidase was similarly performed using CTQD. A simple competitive ELISA for afatinib was developed based on the principle of direct competition between afatinib and the enzyme marker for the anti-afatinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Plasma afatinib concentrations below the limit of quantification of 30 pg/mL were reproducibly measurable. Also, the values of plasma afatinib levels measured from 20 patients were comparable with those measured by high-performance liquid chromatography, and there was a strong correlation between the values determined by both methods(Y=0.976 X – 0.207, r=0.975). As indicated by its specificity and sensitivity, this newly developed ELISA for afatinib is an important tool for TDM and studies of the pharmacokinetics of afatinib.

关键词： Afatinib Enzyme-linked immunosorbent assay Therapeutic drug monitoring Tyrosine-kinase inhibitor

Rapid one-step enzyme immunoassay and lateral flow immunochromatographic assay for colistin in animal feed and food

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Journal of Animal Science and Biotechnology 2020年第1期11卷 280-289页

作者： Jiayi Wang Jinyu Zhou Yiqiang Chen Xinpei Zhang Yongpeng Jin Xiaojing Cui Dongting He Wenqing Lai Lidong He Beijing Advanced Innovation Center for Food Nutrition and Human Health and State Key Laboratory of Animal NutritionCollege of Animal Science and TechnologyChina Agricultural UniversityBeijingChina Department of Chemistry and Biochemistry Florida State UniversityTallahasseeFLUSA.

Background:Colistin(polymyxin E)is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention,treatment,and growth ***,the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings,and its residue in animal-origin food may also pose serious health hazards to ***,it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin ***:A one-step enzyme-linked immunosorbent assay(ELISA)and a lateral flow immunochromatographic assay(LFIA)for colistin were developed based on a newly developed monoclonal *** ELISA showed a 50%inhibition value(IC50)of 9.7 ng/m L with assay time less than 60 min,while the LFIA had a strip reader-based detection limit of 0.87 ng/m L in phosphate buffer with assay time less than 15 *** reducing the non-specific adsorption of colistin onto sample vial,the components of sample extraction solution were optimized and proved to greatly improve the assay *** spiked recovery experiment showed that the recoveries of colistin from feed,milk and meat samples were in the range of 77.83%to 113.38%with coefficient of variations less than 13%by ELISA analysis and less than 18%by LFIA analysis,***,actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental ***:The developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.

关键词： Colistin ELISA Feed Food Gold nanoparticle Lateral flow immunochromatographic assay Monoclonal antibody

Stability Indicating RP-UPLC Method for Quantification of Glycopyrrolate, Methylparaben and Propylparaben assay in Liquid Oral Formulation

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American Journal of Analytical Chemistry 2022年第12期13卷 538-552页

作者： Sreenivas Pippalla Arjuna Rao Nekkalapudi Suresh Babu Jillellamudi Sikkim Professional University Gangtok India Asphar Research Labs Pvt. Ltd. Hyderabad India

Stability indicating RP-UPLC technique was developed for the simultaneous quantification of glycopyrrolate, methylparaben, and propylparaben in glycopyrrolate oral solution. The method was established by using gradient UPLC and a Waters Acquity UPLC BEH C18, 100 mm 2.1 mm, i.d 1.7 μm particle size column with a gradient program of mobile phase A and mobile phase B with a flow rate of 0.25 mL/minute, UV wavelength detection at 222 nm, column temperature of 40°C, injection volume of 2 μL, mobile phase A contains 0.05% trifluoro acetic acid in water and Acetonitrile (90:10) v/v and mobile phase-B contains 0.05% trifluoro acetic acid in water and Acetonitrile (10:90) v/v. The current research describes a single UPLC method for developing an assay method for Glycopyrrolate Oral solution that includes Glycopyrrolate (Active), Methylparaben (Preservative), and Propylparaben (Preservative). The assay method was validated in accordance with ICH guidelines. The retention times of glycopyrrolate, methyl paraben and propylparaben were 6.051 min, 3.458 min and 8.095 min, respectively. Linearity range of glycopyrrolate, methyl paraben and propylparaben were in the range of 4 - 32 μg per mL, 35 - 290 μg per mL and 4 - 32 μg per mL, respectively. Recovery of glycopyrrolate, methylparaben and propylparaben ranged from 100.1% to 98.9%, 100.2% - 100.8%, and 100.2% - 100.8%. Validation of analytical method demonstrated that the method is suitable, specific, linear, accurate, precise, rugged and stability indicating for estimating three components in the pharmaceutical dosage form.

关键词： Glycopyrrolate Oral Solution UPLC assay Methylparaben Propylparaben

Quantitative determination of erlotinib in human serum using competitive enzyme-linked immunosorbent assay

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Journal of Pharmaceutical Analysis 2018年第2期8卷 119-123页

作者： Yuta Yamamoto Tetsuya Saita Yutaro Yamamoto Masashi Shin Applied Life Science Department Faculty of Biotechnology and Life Science Sojo University

A selective and sensitive competitive enzyme-linked immunosorbent assay(ELISA) method was developed and validated for the quantification of erlotinib in 50 mL of samples of human serum. Anti-erlotinib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and 3,4-bis(2-methoxyethoxy)benzoic acid using the N-succinimidyl ester method. Enzyme labeling of erlotinib with horseradish peroxidase was similarly performed using 3,4-bis(2-methoxyethoxy)benzoic acid. A simple competitive ELISA for erlotinib was developed using the principle of direct competition between erlotinib and the enzyme marker for anti-erlotinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Serum erlotinib concentrations lower than 40 ng/mL were reproducibly measurable using the ELISA. This ELISA was specific to erlotinib and showed very slight cross-reactivity(6.7%) with a major metabolite, O-desmethyl erlotinib. Using this assay, drug levels were easily measured in the blood of mice after oral administration of erlotinib at a single dose of 30 mg/kg. ELISA should be used as a valuable tool for therapeutic drug monitoring and in pharmacokinetic studies of erlotinib.

关键词： Erlotinib Enzyme-linked immunosorbent assay O-desmethyl erlotinib Tyrosine-kinase inhibitor

Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

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Science China(Life Sciences) 2010年第2期53卷 241-247页

作者： HE HongQiu,LIU Bin,ZHANG XiaoYi,CHEN WeiZu & WANG CunXin College of Life Science and Bioengineering,Beijing University of Technology,Beijing 100124,China College of Life Science and Bioengineering Beijing University of Technology Beijing China

Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in *** this study,IN and IN-CCD proteins were expressed and purified,and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration *** exhibited a marked preference for Mn2+ over Mg2+ as the divalent cation cofactor in ***,a known IN inhibitor,was found to be an IN-CCD *** assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN,especially targeting IN-CCD.

关键词： HIV-1 integrase IN-CCD disintegration high-throughput assay

Gene analysis of an antiviral protein SP-2 from Chinese wild silkworm, Bombyx mandarina Moore and its bioactivity assay

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Science China(Life Sciences) 2008年第10期51卷 879-884页

作者： YAO HuiPeng, HE FangQing, GUO AiQin, CAO CuiPing, LU XingMeng & WU XiaoFeng College of Animal Science, Zhejiang University, Hangzhou 310029, China College of Animal Science Zhejiang University Hangzhou China

The cDNA encoding an antiviral protein SP-2 against BmNPV was cloned from the midgut of Chinese wild silkworm, Bombyx mandarina Moore (GenBank access AY945210) based on the available informa- tion of the domesticated silkworm. Its cDNA was 855 bp encoding 284 amino acids with predicted mo- lecular weight of 29.6 kDa. Its full length in genomics was 1376 bp, including 5 exons and 4 introns. The expression analysis indicated that it was only expressed in midgut, and its expression level was higher during feeding stage of larval instars while very lower during the moltism and mature stages. The de- duced amino acid sequence of this protein showed eight-amino-acid variation compared with the counterpart of domesticated silkworm. Its antiviral activity was assayed through in vitro test. The re- sults indicated that it showed strong bioactivity against BmNPV, and its activity was 1.6 fold higher that the counterpart of domesticated silkworm.

关键词： Bombyx mandarina Moore antiviral protein bioactivity assay

Performance of QuantiFERON(R)-TB Gold In-Tube assay in children receiving disease modifying anti-rheumatic drugs

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World Journal of Pediatrics 2017年第5期13卷 472-478页

作者： Francesca Gabriele Maria Trachana Maria Simitsopoulou Polixeni Pratsidou-Gertsi Elias Iosifidiso Zoi Dorothea Pana Emmanuel Roilides Infectious Diseases Unit 3rd Department of PediatricsAristotle UniversityThessalonikiGreece Pediatric Immunology and Rheumatology Referral Center 1st Department of PediatricsAristotle UniversityThessalonikiGreece

Background:To evaluate the performance of theQuantiferon(R)-TB Gold In-Tube (QFT-IT) interferon (IFN)-7 assay for the detection of latent tuberculosis infection (LTBI)in children receiving anti-rheumatic treatment in a tertiary referral hospital of Northern ***:A total of 79 consecutive children receiving anti-rheumatic treatment [of which 18 screened prior to antitumor necrosis factor (TNF)-α treatment] were tested using Mantoux tuberculin skin test (TST) and *** of both tests with risk factors for latent tuberculosis and Bacillus Calmette-Guerin immunization was *** of age,TNF-α inhibitors,systemic corticosteroids,conventional disease modifying anti-rheumatic drugs (DMARDs) and total duration of therapy on the QFT-IT mitogen-induced response was ***:Agreement between TST and QFT-IT results was moderate (k=0.38).Frequency of QFT-IT indeterminate results was low (2.5%).In patients with risk factors for LTBI,the odds of a positive IFN-7 assay was increased by a factor of 27.6 (P=0.002),whereas there was no positive *** was a significant difference in the mitogeninduced IFN-7 secretion among various treatments (P=0.038).TNF-α inhibitors were associated with increased mitogen-induced IFN-7 secretion compared to monotherapy with conventional DMARDs (P=0.008).All children screened prior to anti-TNF-a treatment exhibited a negative QFT-IT and no active TB disease was detected during a 2-year ***:QFT-IT may be a more reliable test than TST for detection of LTBI in children with rheumatic diseases receiving anti-rheumatic *** regimen might influence the mitogen-induced IFN-γ secretion and the effect of TNF-α inhibitors might vary according to the specific agent administered.

关键词： anti-tumor necrosis factor-α interferon-γ release assay latent tuberculosis rheumatic disease tuberculin