Characterization and phylogenetic evolution of mitochondrial genome in Tibetan chicken.

The objective of this study was to characterize mitochondrial genome and investigate phylogenetic evolution in Tibetan chicken. In this study, four haplotypes were identified based on D-loop sequencing in Tibetan chicken (n = 40), and each representative of four haplotypes was selected for total mitochondrial genome sequencing and analyzed together with published mitochondrial genome data of red jungle fowl. Four haplotypes belonged to three previously published clades, i.e., Clade A, clade B and clade E. Based on D-loop sequencing data, the average haplotype diversity and nucleotide diversity were 0.658 ± 0.065 and 0.00442 ± 0.00094, respectively. The mitochondrial genome of Tibetan chicken is 16,785 bp in size, consisting of 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, 13 protein-coding genes and one non-coding control region (CR). Compared with the mitochondrial genome, a phylogenetic tree based on the D-loop sequence had a messy distribution, and no breed cluster pattern was observed in Tibetan chicken. The results indicate that Tibetan chicken populations in our study have relatively low nucleotide and haplotype diversity and likely share multiple maternal lineages. The D-loop sequence has limited power for the resolution of phylogenetic relationships in comparison with the complete mitochondrial genome.

Rapid and sensitive detection of Listeria monocytogenes by loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) was designed for detection of Listeria monocytogenes, which is an important food-borne kind of pathogenic bacteria causing human and animal disease. The primers set for the hlyA gene consist of six primers targeting eight regions on specific gene. The LAMP assay could be performed within 40 min at 65°C in a water bath. Amplification products were visualized by calcein and manganous ion and agarose gel electrophoresis. Sensitivity of the LAMP assay for detection of L. monocytogenes in pure cultures was 2.0 CFU per reaction. The LAMP assay was 100-fold higher sensitive than that of the conventional PCR assay. Taking this way, 60 chicken samples were investigated for L. monocytogenes. The accuracy of LAMP was shown to be 100% when compared to the "gold standard" culture-biotechnical, while the PCR assay failed to detect L. monocytogenes in two of the positive samples. It is shown that LAMP assay can be used as a sensitive, rapid, and simple detection tool for the detection of L. monocytogenes and will facilitate the surveillance for contamination of L. monocytogenes in food.

[Comparison of pretreatment methods for the simultaneous determination of diclazuril and toltrazuril residues in chicken tissues].

The effects of four pretreatment methods (acetonitrile extraction-evaporation concentration, acetonitrile extraction-solid phase extraction (SPE), matrix solid-phase dispersion (MSPD) extraction and MSPD-SPE) for the simultaneous analysis of diclazuril and toltrazuril residues in chicken tissues were compared. The average recovery of 70% for the former three methods as achieved. In comparison with other methods, the MSPD method saved more than 60% in time and solvent. So, MSPD as the sample pretreatment method, an MSPD-high performance liquid chromatography with ultraviolet detection (MSPD-HPLC/UV) method was established for the analysis. Under the optimal chromatographic conditions, the linear range was between 50 and 1,000 microg/kg. At the added levels of 50, 500, 1,000 ng/g, the recoveries of diclazuril and toltrazuril in chicken tissues ranged from 71.13% - 84.02% with the relative standard deviations (RSD) in the range of 3.76% - 12.11%, and the RSDs of intra- and interday analyses ranged from 3.70% - 6.77%. The detection limits of diclazuril and toltrazuril were less than 10 microg/kg. The quantitative limits of diclazuril and toltrazuril were less than 20 microg/kg. The method meet the requirements of the residue analysis on accuracy and precision.