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

Objective:To establish a rapid detection method for pathogenic microorganisms by combining loop-mediated isothermal amplification(LAMP)and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 12a(Cas12a)(CRISPR-Cas12a)system,and to evaluate its efficacy for detecting the thermolabile hemolysin(tlh)gene of Vibrio parahaemolyticus(Vp).Methods:Using the tlh gene of Vp as the target gene,LAMP primers and CRISPR RNA(crRNA)were designed to construct and optimize the optimal concentration ratio of each component in the LAMP-CRISPR detection system.Bacillus cereus,Staphylococcus aureus,and Escherichia coli were used as control groups,and the specificity,sensitivity,reproducibility and positive conformity rate were verified to establish a rapid LAMP-CRISPR/Cas12a method for detecting the tlh gene of Vp.Results:The method specifically detected Vp,while Bacillus cereus,Staphylococcus aureus,and Escherichia coli yielded negative results.The DNA extraction concentration of Vp was 190.67 mg·L-1 with an A(260)/(A280)ratio of 1.84.Under the reaction conditions of 37℃ with 80 cycles for 40 min using quantitative PCR(qPCR)method,when the concentrations of Cas12a protein and crRNA in the LAMP-CRISPR/Cas12a system were 50 nmol·L-1,the visual brightness and relative fluorescence intensity peaks were high.The sensitivity of LAMP CRISPR/Cas12a for detecting Vp DNA concentration could reach 10-6 mg·L-1.The reproducibility test results showed that different experimenters had consistent results in different experimental environments and times.Conclusion:The established LAMP-CRISPR/Cas12a method can rapidly detect the tlh gene of Vp with high sensitivity and specificity,and can achieve short-term visual detection in the field.

Objective To investigate the ultrasonic atomization anesthesia time guided needle aspiration biopsy for endobronchial ultrasound (EBUS-TBNA) effect of anesthesia in patients. Methods A total of 60 patients of EBUS-TBNA were divided into A group, B group and C group by random digits table method, each group of 20 cases. Three groups of patients were treated with topical anesthesia with 2%lidocaine aerosol inhalation, atomizing inhalation time:A group 20 min;B group 25 min;C group 30 min. Fiberoptic bronchoscopy was performed in accordance with operating procedures after anesthesia. The changes of vital signs were observed. The patients were investigated by questionnaire. Results A, B, C three groups of the highest blood pressure were (141.90±3.24), (132.30±3.06), (131.15±3.42) mmHg (1 mmHg=0.133 kPa), heart rate was (98.25 ± 3.39), (90.50 ± 3.85), (92.20 ± 5.12) beats/min, breath respectively was (20.45±1.43), (19.15±1.23), (19.00±1.38) beats/min, blood oxygen saturation was 0.9150± 0.0248, 0.9750±0.0128, 0.9735±0.0114, the B and C group was more stable than the A group (P<0.05), B, C two groups were not statistically significant (P>0.05). A, B, C three groups of patients with bronchoscopy insert a successful number of cases were 55%(11/20), 90%(18/20), 90%(18/20), A group was lower than B, C two group (χ2=6.144, P<0.05). The subjective comfort of the three groups were (28.40± 1.82), (37.10 ± 2.02), (36.15 ± 1.42)points, A group and B, C two groups were statistically significant (P<0.05), B, C two groups were not statistically significant (P>0.05). There was significant difference in the effect of anesthesia among three groups (Z=2.105, P<0.05). Conclusions Using 2%lidocaine ultrasonic atomization inhalation anesthesia for patients with local anesthesia, the effect of anesthesia is 25 min and the patient′s tolerance is the best.