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 method of dual nueleic acid test strips for rapid detection of Bacillus cereus cytK and nhe toxin genes based on polymerase chain reaction(PCR)and colloidal gold technique,and to evaluate its specificity,sensitivity,repeatability and stability.Methods:Bacillus cereus DNA was extracted by boiling method.Specific primers were designed with Bacillus cereus cytK and nhe as the target genes.Clonal transformation was used to identify the PCR products.The optimal labeling amounts of colloidal gold-labeled streptavidin,quality control line(C line),cytK detection line(T1)and nhe detection line(T2)were determined.The nucleic acid test strips were assembled and its specificity,sensitivity,reproducibility and stability were evaluated.Results:The DNA concentration of Bacillus cereus was 248 mg·L-1,and the purities were 1.8-2.0.After cloning and plasmid sequencing,the similarities between the PCR products and the sequences of cytK and nhe registered in the GenBank database were 100％.Under the condition of pH 7.0,the optimal amount of streptavidin labeling per 200 μL of colloidal gold solution was 6.0 μL;the optimal marking amount was 2.00 g·L-1 for the quality control line(C line),0.550 g·L-1 for cytK gene detection line(T1)and 0.2 g·L-1 for nhe gene detection line(T2).In the specificity test,positive result on the test strips was seen only for Bacillus cereus,and no cross-reactivity was observed for Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Bacillus subtilis,which were consistent with the electrophoresis results.Sensitivity assay showed that even when DNA concentration was reduced to 10-2 mg·L-1,three bands(C line,T1 line and T2 line)could be observed,and the detection limit of the test strip was one-tenth of agarose gel electrophoresis(10-1 mg·L-1).The nucleic acid test strips were verified by different operators in different laboratories,and the results were consistent.The stability of the test strips was verified at the 6th,9th and 12th months,and the results showed good stability.Conclusion:The dual nucleic acid test strip method established in this study can simutaneously detect the cytK and nhe toxin genes of Bacillus cereus with high sensitivity and specificity,achieving short-term visual detection.

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 form the expert consensus on the limb management of patients with transvenous temporary cardiac pacing,standardize the limb management of patients with transvenous temporary cardiac pacing,and reduce complications related to the limb.Methods Using evidence-based methods,the evidence in this field was searched,evaluated and summarized,and relevant recommendations and research conclusions were extracted and classified by the level of evidence quality,and then the first draft of the consensus was formed.From December 2023 to January 2024,through 2 rounds of expert consultation and 4 rounds of expert meetings,the content was adjusted and the consensus was reached.Results Totally 16 experts participated in the consultation.The positive coefficient is 100%;the authoritative coefficient is 0.847 and 0.836;the average value of each index is more than＞3.8;the coefficient of variation is less than 0.21.The Kendall's harmony coefficient of the 2 rounds of expert consultation is 0.372 and 0.314,respectively,which were statistically significant.The consensus covers the preoperative,intraoperative and postoperative on limb management of patients with transvenous temporary cardiac pacing.Totally 11 themes were involved,including the preoperative preparation,position and catheter fixation in operation,position and catheter fixation in postoperative,activity,turn and transfer,duty shift on limb,nursing care after withdrawal of the catheter,prevention of deep vein thrombosis of the operative limb and prevent infection.Conclusion The consensus is highly scientific,and it is helpful to standardize the limb management of patients with transvenous temporary cardiac pacing.

Based on the five core indicators (the case mix index, the proportion of level-4 surgery, the proportion of technical service income in medical income, the proportion of personnel expenditure in business expenditure, and the proportion of fixed part in personnel salary), the authors discussed the guiding significance of " paying attention to the medical level, strengthening the functional orientation, reflecting the technical value, highlighting people-orientation, and ensuring sustainable development" in the high-quality development of public hospitals. In combination with the current situation of a pilot hospital of high-quality development with the corporate supports of National Health Commission and Province Government, the authors explained the common bottlenecks of public hospitals, such as inaccurate functional positioning, weak innovation ability, poor finance structure, poor salary system and insufficient attention to talents. In the last, the authors pointed out that high-quality development was the development of deepening reform, innovation driven, structural optimization, talent oriented, people-oriented and under the leadership of the Communist Party of China, and put forward targeted implementation paths to provide reference for the pilot work.

Objective:To evaluate the cosmetic outcomes of non-eyebag associated tear trough deformities (TTDs) using the transconjunctival lower eyelid blepharoplasty with release of the tear trough ligament (TTL) and orbital septum fat reposition.Methods:Non-eyebag associated TTDs patients who received the transconjunctival lower eyelid blepharoplasty with release of TTL and with reposition of the orbital septum fat flap internally fixed to the premaxillary-prezygomatic space were retrospectively reviewed at the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from September 2019 to April 2020. The "Hirmand" types of TTDs were classified preoperatively based on the digital photography. Tear trough rating scale (TTRS) introduced by Sadick was used to assess TTDs before and after surgeries based on depth of the trough, hyperpigmentation, prolapse of nasal fat pads/pockets, and lower eyelid skin rhytidosis. "Barton" grading system was used to evaluate demarcation of orbit-cheek junction area preoperatively and postoperatively. Ultimate surgical outcomes of TTDs were assessed by TTRS, Barton and patient self-assessment scores. Patients were followed up postoperatively and the complications were filed. Matched samples t-test (SPSS 20.0 software) was used for statistical analysis to compare TTRS scores pre- and post-surgery. Results:A total of 79 patients with bilateral TTDs were enrolled, including 24 men and 55 women. Their age ranged from 19 to 38 years old (average, 25.7 years old). Edema or congestion in conjunctiva presented in all patients within 3 days after surgery. Slight eyelid skin ecchymosis, lower eyelid edema or asymmetry was presented in 11 cases, and they all recovered in 1-2 weeks. No other complications occurred. The follow-up period ranged from 5 to 13 months (average, 8.5 months). TTRS scores between pre- and post-surgeries demonstrated no significant difference in Hirmand type Ⅰ ( P>0.05), but presenting significant difference both in Hirmand type Ⅱ and Hirmand type Ⅲ ( P<0.01). All cases (33) in Hirmand type Ⅰ graded as Barton 1 pre-surgery were improved to grade 0 post-surgery. There were 35 cases categorized as Hirmand type Ⅱ. Among them, 7 (20%), 25 (71.4%) and 3 cases (8.6%) were graded as Barton 1, 2 and 3 pre-surgery, respectively. Postoperatively, they were improved to Barton 0 (30 cases, 85.7%) and 1 (5 cases, 14.3%), accordingly. There were 11 cases categorized as Hirmand type Ⅲ, which included 6 cases graded as Barton 2 and 5 cases graded as Barton 3. After surgery, they were improved to Barton 0 (2 cases, 18.2%), Barton 1 (5 cases, 45.5%) and Barton 2(4 cases, 26.3%). As to overall cosmetic satisfaction, 78.5% (62/79), 17.7% (14/79) and 3.8% (3/79) were self-assessed as good, moderate and poor by the patients, respectively. Conclusions:Transconjunctival lower eyelid blepharoplasty with release of tear trough ligament and fat reposition can achieve satisfactory lower eyelid rejuvenation in tear trough deformities not associated with eyebags.

Male accessory nipple is a special manifestation of accessory breast with or without presenting areola and mammary tissues. It has been recognized as a rare disease with a low prevalence. Male accessory nipples might result in embarrassing circumstances, psychological concerns, and even potential malignancy. The present study reports a case of male accessory nipples in chest wall and was treated with radical surgical resection in outpatient center at the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in November 2019. Pathology revealed pilosebaceous units in lesions without mammary tissue. 13 months after surgery, surgical areas were observed minimal cicatrix with slight pigmentation. No relapse was filed. Patient was satisfied with the cosmetic result.

Objective:To evaluate the cosmetic outcomes of non-eyebag associated tear trough deformities (TTDs) using the transconjunctival lower eyelid blepharoplasty with release of the tear trough ligament (TTL) and orbital septum fat reposition.Methods:Non-eyebag associated TTDs patients who received the transconjunctival lower eyelid blepharoplasty with release of TTL and with reposition of the orbital septum fat flap internally fixed to the premaxillary-prezygomatic space were retrospectively reviewed at the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from September 2019 to April 2020. The "Hirmand" types of TTDs were classified preoperatively based on the digital photography. Tear trough rating scale (TTRS) introduced by Sadick was used to assess TTDs before and after surgeries based on depth of the trough, hyperpigmentation, prolapse of nasal fat pads/pockets, and lower eyelid skin rhytidosis. "Barton" grading system was used to evaluate demarcation of orbit-cheek junction area preoperatively and postoperatively. Ultimate surgical outcomes of TTDs were assessed by TTRS, Barton and patient self-assessment scores. Patients were followed up postoperatively and the complications were filed. Matched samples t-test (SPSS 20.0 software) was used for statistical analysis to compare TTRS scores pre- and post-surgery. Results:A total of 79 patients with bilateral TTDs were enrolled, including 24 men and 55 women. Their age ranged from 19 to 38 years old (average, 25.7 years old). Edema or congestion in conjunctiva presented in all patients within 3 days after surgery. Slight eyelid skin ecchymosis, lower eyelid edema or asymmetry was presented in 11 cases, and they all recovered in 1-2 weeks. No other complications occurred. The follow-up period ranged from 5 to 13 months (average, 8.5 months). TTRS scores between pre- and post-surgeries demonstrated no significant difference in Hirmand type Ⅰ ( P>0.05), but presenting significant difference both in Hirmand type Ⅱ and Hirmand type Ⅲ ( P<0.01). All cases (33) in Hirmand type Ⅰ graded as Barton 1 pre-surgery were improved to grade 0 post-surgery. There were 35 cases categorized as Hirmand type Ⅱ. Among them, 7 (20%), 25 (71.4%) and 3 cases (8.6%) were graded as Barton 1, 2 and 3 pre-surgery, respectively. Postoperatively, they were improved to Barton 0 (30 cases, 85.7%) and 1 (5 cases, 14.3%), accordingly. There were 11 cases categorized as Hirmand type Ⅲ, which included 6 cases graded as Barton 2 and 5 cases graded as Barton 3. After surgery, they were improved to Barton 0 (2 cases, 18.2%), Barton 1 (5 cases, 45.5%) and Barton 2(4 cases, 26.3%). As to overall cosmetic satisfaction, 78.5% (62/79), 17.7% (14/79) and 3.8% (3/79) were self-assessed as good, moderate and poor by the patients, respectively. Conclusions:Transconjunctival lower eyelid blepharoplasty with release of tear trough ligament and fat reposition can achieve satisfactory lower eyelid rejuvenation in tear trough deformities not associated with eyebags.

Male accessory nipple is a special manifestation of accessory breast with or without presenting areola and mammary tissues. It has been recognized as a rare disease with a low prevalence. Male accessory nipples might result in embarrassing circumstances, psychological concerns, and even potential malignancy. The present study reports a case of male accessory nipples in chest wall and was treated with radical surgical resection in outpatient center at the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in November 2019. Pathology revealed pilosebaceous units in lesions without mammary tissue. 13 months after surgery, surgical areas were observed minimal cicatrix with slight pigmentation. No relapse was filed. Patient was satisfied with the cosmetic result.

Objective:To analyze the clinical features, diagnosis and treatment plan, clinical outcomes of pregnancy with cervical cancer.Methods:The clinical data of 10 pregnant women with cervical cancer from January 2008 to October 2018 in Dalian Obstetrics and Gynecology Hospital Affiliated to Dalian Medical University, the First Hospital of Dalian Medical University, Dalian Central Hospital, Dalian Women and Children′s Medical Center and Wafangdian Central Hospital of Liaoning Province were retrospectively analyzed.Results:The incidence of pregnancy with cervical cancer was 0.004% (10/238 128). Among the 10 cases of pregnancy with cervical cancer, the gestational weeks ≤ 20 weeks at the time of diagnosis was in 6 cases, and they all chose to terminate the pregnancy; the gestational weeks 20 +1 to 30 weeks at the time of diagnosis was in 1 case, and the patient chose to terminate the pregnancy; the gestational weeks >30 weeks at the time of diagnosis was in 2 cases, and they all chose to continue the pregnancy; 1 case was diagnosed after delivery. There were 3 newborns, including 1 premature infants, and they all survived. Conclusions:It is helpful to the diagnose of the disease to strengthen cervical cancer screening before pregnancy and improve the examination of patients with abnormal symptoms during pregnancy. The treatment plan should be individualized according to the pregnancy status, stage of the disease, and wishes of the patient and family.