Developing Qualitative Plasmid DNA Reference Materials to Detect Mechanisms of Quinolone and Fluoroquinolone Resistance in Foodborne Pathogens.

The aim of this study was to develop homogeneous and stable plasmid DNA reference materials for detecting the mechanisms of resistance to quinolones and fluoroquinolones in foodborne pathogens. The DNA fragments of 11 target genes associated with quinolone and fluoroquinolone resistance were artificially synthesized, inserted into plasmid vectors, and transferred into recipient cells. PCR and sequencing of DNA were performed to assess the genetic stability of the target DNA in recombinant Escherichia coli DH5α cells during subculturing for 15 generations. The limit of detection (LOD) of the target DNA was determined using PCR and real-time qualitative PCR (qPCR). The homogeneity and storage stability of plasmid DNA reference materials were evaluated in terms of plasmid DNA quantity, PCR-measured gene expression, and qPCR threshold cycle. All 11 target DNAs were successfully synthesized and inserted into vectors to obtain recombinant plasmids. No nucleotide mutations were identified in the target DNA being stably inherited and detectable in the corresponding plasmids during subculturing of recombinant strains. When the target DNA was assessed using PCR and qPCR, the LOD was ≤1.77 × 105 and 3.26 × 104 copies/µL, respectively. Further, when the reference materials were stored at 37 °C for 13 days, 4 °C for 90 days, and -20 °C for 300 days, each target DNA was detectable by PCR, and no mutations were found. Although the threshold cycle values of qPCR varied with storage time, they were above the LOD, and no significant differences were found in the quantity of each plasmid DNA at different timepoints. Further, the homogeneity and stability of the materials were highly consistent with the requirements of standard reference materials. To summarize, considering that our plasmid DNA reference materials conformed to standard requirements, they can be used to detect the mechanisms of quinolone and fluoroquinolone resistance in foodborne pathogens.

[Transplantation of free latissimus dorsi myocutaneous flaps for craniomaxillofacial reconstruction].

OBJECTIVE: This clinical study was to improve the surgical treatment to craniomaxillofacial tissue defects. METHODS: Since 1997, eight cases with severe craniomaxillofacial defects were treated using free latissimus dorsi myocutaneous flaps. In the operation, nerve anastomosis was performed. Of the 8 cases, 7 were treated in one stage, 1 was treated in 3 steps. The craniomaxillofacial defects ranged from 10 cm x 8 cm to 30 cm x 12 cm. The flaps was 12 cm x 10 cm to 32 cm x 16 cm in size. RESULTS: Postoperative follow-up for 6 months to 4 years demonstrated satisfactory results in all the cases. There was neither necrosis nor ulcer after the operation. The sensation recovery of the flap was also satisfactory. CONCLUSION: Free transfer of the latissimus dorsi myocutaneous flap is an ideal treatment to severe craniomaxillofacial defects as it possesses the advantages of reliable blood supply, ability against infections, large size, concealed donor site, and functional restoration of sensation and movement.

[Method on PCR detection of bovine materials in animal feedstuffs].

The reliable, simple and easy to operate methods were established to extract DNA from animal feedstuffs. A set of PCR primers were designed for bovine specific mitochondrial DNA sequence. With this method, PCR can be successfully utilized to evaluate the presence of bovine materials in animal feedstuffs. A set of commonly used primers of 18Sr-RNA were designed as endogenuous reference gene, and check on the quality of template extraction from animal feedstuffs, and avoid the result of false negative. PCR amplification condition was one cycle of 96 degrees C for 2 min, then 35 cycle of (94 degrees C for 40s, 60 degrees C for 50s, 72 degrees C for 60s), and finally one cycle of 72 degrees C for 5 min. PCR amplified specific gene sequence 271 bp for bovine materials from animal feedstuffs.