OBJECTIVES: To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. METHODS: L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. RESULTS: The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. CONCLUSIONS The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.
Listeria monocytogenes/isolation & purification* ; Gold Colloid ; Bacterial Toxins/genetics* ; Polymerase Chain Reaction/methods* ; Hemolysin Proteins/genetics* ; Bacterial Proteins/genetics* ; DNA, Bacterial/genetics* ; Food Microbiology ; Heat-Shock Proteins

BACKGROUND:PA6 cells are bone marrow stromal stem cells from the mouse skul , and scientists have found that PA6 cells co-cultured with some kinds of stem cells have shown neural differentiation, based on which, PA6 cells can be used for repair of nerve injury. Therefore, researchers give more and more attentions to PA6, but few studies have addressed isolation and culture methods of bone marrow stromal stem cells from the skul . OBJECTIVE:To isolate and culture bone marrow stromal stem cells from the skul of Sprague-Dawley rats and to observe cellular morpholopy in vitro and perform immunofluorescence identification. METHODS:Under sterile conditions, newborn Sprague-Dawley rat’s skul was cut into pieces. Smal skul pieces were washed using Dulbecco’s modified Eagle’s medium containing 10%fetal bovine serum. Single cellsuspension was made, and placed in culture flask. The culture medium was changed many times for cellpurification. The second passage of cells was obtained for morphology observation under an inverted microscope. cellsurface markers were detected by using immunofluorescence staining. RESULTS AND CONCLUSION:After the primary culture for 24 hours, cells exhibited adherent growth;after 3 days, cells were increased in number, and presented with irregular shapes, such as polygon, triangle, spindle and flat shape. After passage, cells were uniform in morphology, arranged in radial and bunch patterns, and exhibited strong adherent ability. Some cells grew in cluster, and proliferated faster than primary cells. Immunofluorescence staining showed that cells were positive for CD105, CD73, CD44, CD90, but negative for CD45, D34,CD14, HLA-DR. Results indicate that this method can obtain bone marrow stromal stem cells.

Objective To evaluate the relationship between melatonin and development of post-herpetic neuralgia (PHN) in rats.Methods Sixty adult Wistar rats of both sexes,aged 3 months,weighing 180-230 g,were randomly divided into A-F 6 groups (n =10 each) using a random number table.Group A served as sham operation group,and inactivated herpes simplex virus type-1 (HSV-1) was inoculated.Group B served as sham operation group,and HSV-1 was inoculated.In group C,pinealectomy was performed and inactivated HSV-1 was inoculated.In group D,pinealectomy was performed,and HSV-1 was inoculated.In group E,pinealectomy was performed,inactivated HSV-1 was inoculated,and melatonin was injected intraperitoneally everyday.In group F,pinealectomy was performed,HSV-1 was inoculated,and melatonin was injected intraperitoneally everyday.HSV-1 or inactivated HSV-1 was inoculated on the left hind paw of the rats at 1 h after pinealectomy,and the dosage of melatonin was 120 mg/kg once a day.The mechanical pain response score (MPRS) was determined before inoculation and on days 1,5,10,20 and 30 post-inoculation.The development of PHN was recorded on day 30 post-inoculation.Results MPRS was significantly higher in group B than in group A,and in group D than in group B (P ＜ 0.05).MPRS was significantly lower in group F than in group D (P ＜ 0.05).PHN was not found in A,C and E groups,and the incidence of PHN was 40％,90％ and 40％,respectively.Compared with group B,the incidence of PHN was significantly increased in group D (P ＜ 0.05),and no significant change was found in group F (P ＞ 0.05).Conclusion Reduction of melatonin secretion is related to the development of PHN in rats.