Development of a Direct Competitive ELISA Kit for Detecting Deoxynivalenol Contamination in Wheat.

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

Development of an enzyme-linked immunosorbent assay for detection of clopidol residues in chicken tissues.

BACKGROUND: Clopidol is mainly used for the prevention and treatment of coccidiosis, which poses a serious potential hazard to public health, in veterinary medicine. The aim of this study was to prepare monoclonal antibodies (mAbs) against clopidol (CLOP) and develop an immunoassay for detecting CLOP residues in chicken tissues. After derivation, CLOP hapten was conjugated to carrier proteins to synthesize the artificial antigen, and immunized Balb/C mice were employed to screen mAbs. RESULTS: A sensitive hybridoma named C1G3 was screened out and two indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curves were established. For the traditional two-step assay the linear range was from 0.06 to 98 ng mL(-1) , with half-maximal inhibitory concentration (IC50 ) and limit of detection (LOD) values of 2.76 ng mL(-1) and 0.03 ng mL(-1) respectively, while the rapid one-step icELISA had a working range from 0.08 to 102 ng mL(-1) , with IC50 and LOD values of 3.52 ng mL(-1) and 0.03 ng mL(-1) respectively. It was also indicated that a 10-fold dilution in chicken muscles gave an inhibition curve almost the same as that obtained in phosphate-buffered saline. When applied to spiking tests in chicken samples, the correlation coefficient (R(2) ) between concentrations added and measured was 0.9534. CONCLUSION: The results of this study suggest that the immunoassay described is a promising alternative for screening CLOP residues in biological matrices and is suitable for routine diagnostics.

Antimicrobial peptide MPX attenuates LPS-induced inflammatory response and blood-testis barrier dysfunction in Sertoli cells.

Orchitis accounts for a high proportion of male animal reproductive disorders. Hence, it is urgent to identify drugs for the prevention and treatment of orchitis. Antimicrobial peptides (AMPs) are currently recognized as one of the most promising alternatives to antibiotics. However, the protective effects of AMPs on lipopolysaccharide (LPS)-induced orchitis have not been reported. In this study, we developed an LPS-induced orchitis model in which primary bovine Sertoli cells were used as model cells. MPX was indicated to effectively reduce the inflammatory response of Sertoli cells. MPX attenuated the gene expression of the proinflammatory cytokines TNF-α, IL-6 and IL-1ß by suppressing the MAPK pathway, especially the phosphorylation of p38 and ERK. MPX also decreased the oxidative stress response caused by LPS and upregulated Occludin and Claudin-1 expression, thereby maintaining the integrity of the blood-testis barrier. Moreover, we found that MPX inhibited apoptosis in Sertoli cells. In a mouse model, we found that MPX significantly inhibited the disruptive effects of LPS, reducing seminiferous epithelium damage, vacuolations, hyperplasia, and apoptosis in spermatogenic cells and rescuing spermatogenesis. In addition, the expression of inflammatory factors such as IL-1ß, IL-18, IL-6 and TNF-α was decreased after MPX treatment in the mouse testes. MPX had no effect on other organs in mice, indicating its safety. This study was undertaken to investigate how MPX regulates the inflammatory response in Sertoli cells and provide a reference for the clinical prevention and treatment of male animal orchitis.

Characterization of the complete mitochondrial genome of Corvus corone orientalis.

In this study, the complete mitochondrial genome of Corvus corone orientalis was assembled through next-generation sequencing data. This circular mitochondrial genome of C. corone orientalis is 16,947 bp in length and has a base composition of A (30.8%), T (24.7%), C (29.9%), and G (14.5%), demonstrating a bias of higher AT content (55.5%) than GC content (44.5%). The mitochondrial genome contains a typically conserved structure among bird mitogenomes, encoding 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), two ribosomal RNA genes (12S rRNA and 16S rRNA), and a control region (D-loop region). Except ND6, all other PCGs were located on the H-strand. ATP8 gene and ATP6 gene were overlapped by 8 bp. The whole mt genome of C. corone orientalis and other Corvoidea mitogenomes (24 species, in total) were used for phylogenetic analysis. The result indicated C. corone orientalis has the closest relationship with Corvus cornix cornix (NC_024698) and clustered within clade of genus Corvus.

Characterization of the complete mitochondrial genome of Notospermus geniculatus.

In this study, the complete mitochondrial genome of Notospermus geniculatus, was recovered through Illumina sequencing data. This complete mitochondrial genome of N. geniculatus is 15,180 bp in length and has a base composition of A (14.4%), T (41.3%), C (14.6%), G (29.6%), demonstrating a bias of higher AT content (55.7%) than GC content (44.2%). The mitochondrial genome contains a typically conserved structure among Lineidae mitogenomes, encoding 13 protein-coding genes (PCGs), 23 transfer RNA genes (tRNA), 2 ribosomal RNA genes (12S rRNA and 16S rRNA), and a control region (D-loop region). All PCGs were located on the H-strand. ND4L gene and ND4 gene were overlapped by 6 bp. The whole mt genome of N. geniculatus and other Protostomia mitogenomes (17 species, in total) were used for phylogenetic analysis. The result indicated N. geniculatus has the closest relationship with Lineus viridis (FJ839919.1) and clustered within Heteronemertea Clade, which representing a distinct order.

Characterization of the complete mitochondrial genome of band-rumped Storm-petrel, Hydrobates castro.

In this study, the complete mitochondrial genome of band-rumped Storm-petrel, Hydrobates castro, was determined through sequencing of PCR fragments. The complete mitochondrial genome of H. castro was 17,065 bp in length and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 30.4% A, 24.5% T, 30.9% C, and 14.2% G, with a total G + C content of 45.1%. By phylogenetic analysis using Bayes method, H. castro showed the closest relationship with the white-faced storm petrel (Pelagodroma marina).

Characterization of the complete mitochondrial genome of Cassin's auklet, Ptychoramphus aleuticus.

In this study, the complete mitochondrial genome of Cassin's auklet, Ptychoramphus aleuticus, was determined using Illumina sequencing. The complete mitochondrial genome of P. aleuticuss was 16,524 bp in length and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA genes. The overall nucleotide composition is: 30.8% A, 24.4% T, 30.6% C, and 14.2% G, with a total G + C content of 44.8%. By phylogenetic analysis using ML method, P. aleuticuss clustered with two Synthliboramphus species that belong to Alcidae.

Characterization of the complete mitochondrial genome of cape elephant shrew, Elephantulus edwardii.

In this study, the complete mitochondrial genome of cape elephant shrew, Elephantulus edwardii, was determined through sequencing of PCR fragments. The complete mitochondrial genome of E. edwardii was 16,552 bp in length and and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 32.7% A, 29.5% T, 25.0% C, and 12.9% G, with a total G+C content of 37.9%. By phylogenetic analysis using Bayesian method, E. edwardii showed the closest relationship with another Elephantulus speceis (Genbank accession: AB096867.1).

Characterization of the complete mitochondrial genome of short-tailed field vole, Microtus agrestis.

In this study, the complete mitochondrial genome of short-tailed field vole, Microtus agrestis, was determined through sequencing of PCR fragments. The complete mitochondrial genome of M. agrestis was 16,538 bp in length and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 15.7% A, 27.5% T, 25.5% C, and 31.4% G, with a total G + C content of 56.9%. By phylogenetic analysis using Bayesian method, M. agrestisa showed the closest relationship with the southern vole (Microtus rossiaemeridionalis).

Characterization of the complete mitochondrial genome of three-spined stickleback, Gasterosteus aculeatus.

In this study, the complete mitochondrial genome of three-spined stickleback, Gasterosteus aculeatus, was determined through sequencing of PCR fragments. The complete mitochondrial genome of G. aculeatus was 16,543 bp in length and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 27.0% A, 28.4% T, 27.4% C, and 17.2% G, with a total G + C content of 44.6%. By phylogenetic analysis using ML method, G. aculeatus showed the closest relationship with the blackspotted stickleback (Gasterosteus wheatlandi).

Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk.

Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR), and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curve was established. This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml, with the half maximal inhibitory concentration (IC(50)) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml, respectively. The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested, and the IC(50) values to enoxacin, ciprofloxacin, and pefloxacin were 3.1, 3.4, and 4.1 ng/ml, respectively. It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%. When spiked in milk at 5, 20, and 50 ng/ml, the recoveries for NOR, enoxacin, ciprofloxacin, and pefloxacin ranged 90.5%-98.0%, 84.0%-95.2%, 94.0%-106.0%, and 89.5%-100.0%, respectively. The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.

Analysis of 19-nortestosterone residue in animal tissues by ion-trap gas chromatography-tandem mass spectrometry.

A rapid sample treatment procedure for the gas chromatography-tandem mass spectrometry (GC-MS) determination of 19-nortestosterone (19-NT) in animal tissues has been developed. In our optimized procedures, enzymatic hydrolysis with ß-glucuronidase from Escherichia coli was performed in an acetate buffer (pH 5.2, 0.2 mol/L). Next, the homogenate was mixed with methanol and heated at 60 °C for 15 min, then placed in an ice-bath at -18 °C for 2 h. After liquid-liquid extraction with n-hexane, the analytes were subjected to a normal-phase solid phase extraction (SPE) C18 cartridge for clean-up. The dried organic extracts were derivatized with heptafluorobutyric anhydride (HFBA), and then the products were injected into GC-MS. Using electron impact mass spectrometry (EI-MS) with positive chemical ionization (PCI), four diagnostic ions (m/z 666, 453, 318, and 306) were determined. A standard calibration curve over the concentration range of 1-20 ng/g was reached, with Y=467084X-68354 (R²=0.9997) for 19-NT, and the detection limit was 0.3 ng. When applied to spiked samples collected from bovine and ovine, the recoveries ranged from 63% to 101% with relative standard deviation (RSD) between 2.7% and 8.9%. The procedure is a highly efficient, sensitive, and more economical method which offers considerable potential to resolve cases of suspected nandrolone doping in husbandry animals.

Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin.

Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method was employed to synthesize the artificial antigen of enrofloxacin (ENR), and New Zealand rabbits were used to produce anti-ENR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (ELISA) standard curve was established. This assay was sensitive and had a linear range from 0.6 to 148.0 µg/kg (R(2) = 0.9567), with the half maximal inhibitory concentration (IC(50)) and limit of detection (LOD) values of 9.4 µg/kg and 0.2 µg/kg, respectively. Of all the competitive analogues, the produced pAb exhibited a high cross-reactivity to ciprofloxacin (CIP) (87%), the main metabolite of ENR in tissues. After optimization, the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork. The overall recoveries and coefficients of variation (CVs) were in the ranges of 86%-109% and 6.8%-13.1%, respectively. It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.