The purpose of this study was to develop a membrane filtration method to isolate uninuclei conidium of Aspergillus oryzae, then the method was adopted to develop a transformation system of A. oryzae. A. oryzae 3.951 contained 1–4 nuclei in each conidium. The percentages of uninucleate and binucleate conidia were approximately 16.15 and 74.22%, respectively. Conidia suspension was filtrated with a 5-μm membrane to overcome the bottleneck caused by multinucleate conidia and to remove excess multinucleate conidia before UV mutagenesis. Uninucleate conidia of 5-fluoroorotic acid (5-FOA)-resistant strains were enriched by filtration with a 3-μm membrane. The pyrG mutant strain AS11 was obtained and GFP-pyrG was successfully transformed into AS11.

The development of polyvinylidene fluoride (PVDF) membrane-based dot immunoassay for rapid and simultaneous detection of multi-mycotoxins in cereal samples is described. To facilitate the simultaneous identification of multiple mycotoxins in a single test, membrane reaction zones were constructed using PVDF membrane, rubber fences and scotch tape. The cut-off level for this method, assessed visually, were 20, 60, 1000, 20, and 250 μg kg−1 for Aflatoxin B1, zearalenone, deoxynivalenol, ochratoxin A, and fumonisins B1, respectively, and the final results can be obtained within 10 min. This simultaneous method was designed to be simple, with no time-consuming cleanup procedure, and good accuracy and reproducibility were obtained in recovery experiments. These results suggest that this method could be a useful on-site screening tool for the rapid detection of multiple mycotoxins in cereal samples without special instrumentation.Highlights► To the best of our knowledge, PVDF membrane-based dot immunoassay for multiple mycotoxins has not yet been reported. ► The method can simultaneously detect five different mycotoxins in a single test. ► The method do not require time-consuming sample clean-up and enrichment steps. ► It is suitable for rapid screening of multiple mycotoxins under field conditions.

A method for the quantitative monitoring of sulfadiazine (SD) residues in pork was established by molecular imprinted column coupling with high performance liquid chromatography (HPLC). The molecular imprinted column was selected as an extraction device. To obtain the optimum extraction efficiency, several parameters related to the molecular imprinted column, including column solvent, flow-rate, eluent of the sample matrix and eluent volume, were investigated. The sample solution was directly injected into the device for the extraction after simple extraction. Under the optimum conditions, the relative standard deviations (RSD) was ≤ 6.1%, and the recoveries for SD were higher than 75.6%. In comparison with the AL-SPE column, the MIP-SPE column had good reusability and extraction efficiency. This method was successfully applied to the determination of sulfadiazine residues in pork.

A reversed-phase HPLC method with fluorescence detection for the determination of two Monascus metabolites, monasfluore A (MFA) and monasfluore B (MFB), in red yeast rice was carried out. Optimum conditions for the extraction and chromatographic separation were investigated. The method was validated through the following performance criteria: linearity, stability, limit of detection (LOD), quantification (LOQ), etc. This assay was successfully used for determination of the MFA and MFB in 20 samples inoculated from different Monascus sp. The results revealed that significant variations were demonstrated in the contents of the MFA and MFB in these samples. The high contents of both MFA and MFB in sample 13 were found to be 81.400 and 26.300 mg g−1, respectively. The low contents of both MFA and MFB in sample 14 were also found to be 0.010 and 0.003 mg g−1, respectively.Research highlights► A HPLC method has been established for the quantitative analyses of two Monascus metabolites MFA and MFB in red yeast rice. The method is simple, sensitive and reliable. ► The high contents of both MFA and MFB in red yeast rice (sample 13) were found to be 81.400 and 26.300 mg g−1, respectively. ► The low contents of both MFA and MFB in red yeast rice (sample 14) were also found to be 0.010 and 0.003 mg g−1, respectively. ► It will be interesting in future research to investigate the factors led to the variation in the MFA and MFB content of different the red yeast rice samples.

Monoclonal antibody 7G5 (McAb 7G5), which recognises the zearalenone (ZEN), was used to select for peptides that mimic the ZEN by employing a library of filamentous phages that have 7-mer peptides on their surfaces. After three rounds of panning, two mimotope peptides were obtained to be able to mimic ZEN in binding with the McAb 7G5, their amino acid sequences were DAVILLM and HHCHWWH. Enzyme-linked immunosorbent assay (ELISA) for detect ZEN was established with the phages. In the most sensitive assay, the linear range of the inhibition curve was 100–10,000 pg/ml, the detection limit was 100 pg/ml. The results demonstrated that those phage peptides could be used as the surrogate of ZEN to establish the immunoassay.Research highlights► Monoclonal antibody 7G5 (McAb 7G5), which recognizes the zearalenone (ZEN), was used to select for peptides that mimic the ZEN by employing a library of filamentous phages that have 7-mer peptides on their surfaces. ► Two mimotope peptides were obtained to be able to mimic ZEN in binding with the McAb 7G5, their amino acid sequences were DAVILLM and HHCHWWH. ► In the most sensitive phage ELISA , the linear range of the inhibition curve was 100-10,000 pg/ml, the detection limit was 100 pg/ml. ► The paper results demonstrated that those phage peptides could be used as the surrogate of ZEN to establish the immunoassay.

An anti-DON monoclonal antibody (Mab) was produced from a stable hybridoma cell line, 12D1, generated by the fusion of SP2/O myeloma cells with spleen cells isolated from a Balb/c mouse immunized with DON–cationic bovine serum albumin (CBSA) conjugate. The 12D1 Mab belongs to the immunoglobulin G1 (κ-chain) isotype.A colloidal gold immunochromatographic strip (ICS) test for rapid detection of deoxynivalenol (DON) in wheat and maize samples was also developed using this Mab. The ICS test, which has a detection limit of 50 ng mL−1 for DON and can be completed in 10 min. Analysis of DON in wheat and maize samples revealed that data obtained from ICS test were in a good agreement with those obtained from ELISA and GC/MS. The results demonstrate that the ICS test can be used as qualitative tool for screening technique of DON on-site.

Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5′-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.

Red yeast rice obtained as cultures of Monascus AS3.4444 on rice was extracted and analyzed by high-performance liquid chromatography (HPLC). Two new Monascus metabolites with similar fluorescence spectra (λ ex = 396 nm, λ em = 460 nm) and UV absorption spectra (λ max = 386 nm) were detected. They were isolated by rechromatography on a silica gel column and semipreparative HPLC, and two strong blue fluorescent compounds were obtained. Their structures were elucidated by electrospray ionization mass spectrometry (ESI−MS), electrospray ionization tandem mass spectrometry (ESI−MS/MS), intensive ESI−MS, and nuclear magnetic resonance spectroscopy ( 1H NMR, 13C NMR, COSY, and HMBC) studies. High-resolution mass spectrometry indicated the molecular formulas C 21H 24O 5 and C 23H 28O 5. The two new compounds, named monasfluore A and monasfluore B, respectively, contain a alkyl side chain, γ-lactone, and propenyl group, whereas the more lipophilic compound, monasfluore B, is a higher homologue of monasfluore A, with the more lipophilic octanoyl instead of the hexanoyl side chain.