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Zhu P.,China Agricultural University | Fu W.,Chinese Academy of Inspection and Quarantine | Wang C.,Chinese Academy of Inspection and Quarantine | Du Z.,Guangxi Entry Exit Inspection and Quarantine Bureau | And 3 more authors.
Analytica Chimica Acta | Year: 2016

The possibility of the absolute quantitation of GMO events by digital PCR was recently reported. However, most absolute quantitation methods based on the digital PCR required pretreatment steps. Meanwhile, singleplex detection could not meet the demand of the absolute quantitation of GMO events that is based on the ratio of foreign fragments and reference genes. Thus, to promote the absolute quantitative detection of different GMO events by digital PCR, we developed a quantitative detection method based on duplex digital PCR without pretreatment. Moreover, we tested 7 GMO events in our study to evaluate the fitness of our method. The optimized combination of foreign and reference primers, limit of quantitation (LOQ), limit of detection (LOD) and specificity were validated. The results showed that the LOQ of our method for different GMO events was 0.5%, while the LOD is 0.1%. Additionally, we found that duplex digital PCR could achieve the detection results with lower RSD compared with singleplex digital PCR. In summary, the duplex digital PCR detection system is a simple and stable way to achieve the absolute quantitation of different GMO events. Moreover, the LOQ and LOD indicated that this method is suitable for the daily detection and quantitation of GMO events. © 2016 Elsevier B.V. Source


Chen D.,Wenzhou University | Lin J.,Wenzhou University | Liu Y.,Wenzhou University | Li X.,Guangxi Entry Exit Inspection and Quarantine Bureau | And 5 more authors.
Acta Tropica | Year: 2016

Autophagy is a catabolic process in eukaryotic cells involved in the targeted degradation of cellular organelles and the cytoplasm. Recent works in Toxoplasma gondii suggest that the autophagy processes may serve as an important pathway in modulating parasite survival or death. As an important modulator of Atg8 lipidation and autophagy, Atg8-Atg3 interaction has been attracting increasing attention. However, there is no direct evidence that TgAtg8-TgAtg3 interaction occurs in the parasite. In this study, we firstly found TgAtg8 partially colocalized with TgAtg3 in GFP-TgAtg8 transgenic strains using IFA. Then, lysates from GFP-TgAtg8 tachyzoites were directly subject to large-scale tandem affinity purification with anti-GFP antibody. Western blot and tandem mass spectrometry (MS/MS) analysis determined the interaction between TgAtg8 and TgAtg3. Additionally, we performed real-time interaction analysis with a surface plasmon resonance biosensor using BIAcore system. As expected, the result demonstrated a concentration-dependent increases in resonance signals and indicated the TgAtg8 could bind directly TgAtg3 in vitro. Noteworthily, A KD of 34.9. nM obtained from TgAtg8-TgAtg3 interaction indicate a high-affinity between Atg8-Atg3 in Toxoplasma. Furthermore, homology modeling and sequence alignment showed that TgAtg8 has greatest sequence and structural conservation. Within TgAtg3, this protein possesses the core E2 enzymatic activity structure and a truncated handle region which may contain AIM sequence. Taken together, our findings would help elucidate the formation mechanism of autophagosome in Toxoplasma and provide a possibility for looking into parasitic drug targets. © 2015 Published by Elsevier B.V. Source


Zheng L.,Guangxi Entry Exit Inspection and Quarantine Bureau | Wu Y.,Guangxi Entry Exit Inspection and Quarantine Bureau | Li Y.,Guangxi Entry Exit Inspection and Quarantine Bureau | Li L.,Guangxi Entry Exit Inspection and Quarantine Bureau
Chinese Journal of Chromatography (Se Pu) | Year: 2012

A high performance liquid chromatography-tandem mass spectrojaetry (HPLC-MS/ MS/ method was developed for the simultaneous quantitative determination of 3-methyl-quinox-aline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA) as the marker residues for carbadox (CBX) and olaquindox (OLA) respectively in the muscles and livers of porcine and chicken and in the muscles of fish and shrimp. The MQCA and QCA were depro-teinated with 5% metaphosphoric acid in 10% methanol followed by liquid-liquid extraction. Further clean-up was performed by solid phase extraction (SPE) through mixed mode anion-exchange columns (Oasis MAX SPE). The separation of the compounds was carried on a Waters Xterra MS C18 column (l50 mm × 2. 1mm, 5 μm) by a gradient elution using methanol and 0. 2% formic acid as mobile phases. The analytes were detected by tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive electrospray ionization. The MQCA and OCA were quantified by internal standard method. The linear ranges were 1. 0-20. 0μg/L and the correlation coefficients were not less than 0. 999 6. The average recoveries and relative srandard deviations ranged from 62. 4% - 118% and 1. 48% -28. 1% respectively at the spiked levels of 0. 1 0. 2 and 1. 0μg/kg for the both markers. The limit of quantitation (LOQ) was 0. 1μg/kg. The method is sensitive accurate and suitable for the determination and confirmation of MQCA and QCA in animal origin foods. Source


Zheng L.,Guangxi Entry Exit Inspection and Quarantine Bureau | Wu Y.,Guangxi Entry Exit Inspection and Quarantine Bureau | Zhao Y.,Guangxi Entry Exit Inspection and Quarantine Bureau | Li L.,Guangxi Entry Exit Inspection and Quarantine Bureau | Ma Y.,Guangxi Entry Exit Inspection and Quarantine Bureau
Chinese Journal of Chromatography (Se Pu) | Year: 2014

A multi-residue method was developed for the simultaneous determination of 18 β-agonist residues (clenbuterol, ractopamine, penbutolol, tulobuterol, etc) in feed by using QuEChERS sample preparation and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The feed samples were dispersed by water, then the analytes were extracted with acetonitrile containing 4% (v/v) ammonia and cleaned up by QuEChERS method with 25 mg octadecylsilyl (C18) and 50 mg primary secondary amine (PSA) adsorbents. The separation of compounds was carried on an Agilent ZORBAX Eclipse XDB-C18 column (50 mm× 4. 6 mm, 1.8 μm) by a gradient elution using methanol-0. 1% (v/v) formic acid aqueous solution as mobile phase. The analytes were detected by tandem mass spectrometry under multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+) and quantified by the matrix-matched external standard method. The results showed that the calibration curves of the 18 β-agonists were linear in the range of 5-200 μg/L with correlation coefficients of 0. 991 2-0. 999 5. The average recoveries of the 18 analytes at three spiked levels of 0. 05, 0. 1 and 0. 5 mg/kg ranged from 78. 4% to 107. 1% with the relative standard deviations (RSDs) of 3. 50/0-12. 3o/o. The limit of quantification (LOQ, S/N≥10) was 0. 05 mg/kg for each analyte. The developed method is simple and sensitive, and can be applied as a screen and confirmatory method for the analysis of β-agonists in feed. Source

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