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 discuss the effect of quorum sensing-related gene expression on biofilm formation and drug resistance in clinically multidrug-resistant Pseudomonas aeruginosa,and to clarify the mechanism of enhacing drug resistance.Methods:A total of 77 strains of Pseudomonas aeruginosa were collected.Based on drug resistance,the strains were divided into multidrug-resistant group and sensitive group.The optimal biofilm formation conditions were determined using the microtiter plate method;biofilm formations of the stains in both groups was observed under an optical microscope;crystal violet staining was used to semiquantitatively detect biofilm formation ability of P.aeruginosa in both groups;microbroth dilution method was used to determine the minimal inhibitory concentration(MIC)values of the quorum sensing inhibitor(C-30)against Pseudomonas aeruginosa in both groups;RNA was extracted from two groups using a commercial kit,while RNA from planktonic state and biofilm state of multidrug-resistant strains was extracted using modified TRIzol method;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of quorum sensing-related genes(lasR/I,RhlR/I,PqsR/A)of the stains in multidrug-resistant group and sensitive group,as well as before and after adding the quorum sensing inhibitor C-30.Results:Among 77 strains of Pseudomonas aeruginosa,56 were multidrug-resistant(multidrug-resistant group)and 21 were fully sensitive(sensitive group).Optimal biofilm formation occurred at a bacterial concentration of 1.5×108 CFU·mL-1 with 48 h incubation.The biofilm positivity rate was 91%,with strongly positive,moderately positive,weakly positive,and negative biofilms accounting for 16%,34%,41%,and 9%,respectively.The biofilm positivity rate in multidrug-resistant strains was 96%,and biofilm formation ability in multidrug-resistant group was higher than that in sensitive group(P<0.05).When the concentration of C-30 was 8 mg·L-1 the biofilm formation in most Pseudomonas aeruginosa was inhibited,with enhanced suppression at higher concentrations.The absorbtion(A)value of both planktonic-state and biofilm-state RNA ranged from 1.8 to 2.0.The RT-qPCR results showed that compared with planktonic state,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA of the stains in biofilm state were significantly increased(P<0.01).Compared with non-inhibitor group,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA in biofilm state of inhibitor-treated group were significantly decreased(P<0.01).Conclusion:High expression of quorum sensing-related genes in multidrug-resistant P.aeruginosa promotes biofilm formation,thereby enhancing drug resistance.

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.

AIM:To investigate the preventive and therapeutic effects of sesamin(SES)on fatty liver disease in rats with hypertension combined with dyslipidemia,and to explore the potential mecha-nisms based on mRNA-seq.METHODS:Spontane-ously hypertensive rats(SHRs)were fed a high-fat,high-cholesterol diet to establish a rat model of hy-pertension combined with dyslipidemia,and then treated with SES for 16 weeks continuously.The ex-periment was divided into four groups:WKY,SHR,Model,and Model+SES(160 mg·kg-1·d-1).Blood pressure was measured using the tail-cuff method.Body weight was monitored,and body mass index was calculated.Liver morphology was detected by ultrasound,and liver thickness was measured.Liver wet weight was weighed,and liver index was calcu-lated.Liver volume was detected by the water dis-placement method.Serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),alanine aminotransferase(ALT),aspartate amino-transferase(AST),and total bile acids(TBA)were de-tected by ELISA.Liver sequencing analysis was per-formed using mRNA-seq.Liver histomorphological changes were observed by HE staining.The degree of hepatic steatosis was observed by Oil Red O stain-ing,and the degree of hepatic fibrosis was observed by MASSON staining.The mRNA expression of Al-dh1a7,Nnmt,Irs2,Pltp,and Scd was detected by q-PCR.The protein expression of Scd,Nnmt,AMPK,p-AMPK,PPARα,and PPARγ was detected by Western blotting.RESULTS:After 16 weeks of continuous SES administration to rats with hypertension combined with dyslipidemia,blood pressure was significantly reduced(P＜0.01),and body weight was decreased.Serum TG,TC,and LDL-C levels were decreased,while HDL-C levels were increased.Serum ALT and AST levels were decreased.Liver weight,organ in-dex,liver thickness,and liver volume were de-creased.The degree of hepatic steatosis and hepat-ic fibrosis was improved.A total of 545 differentially expressed mRNAs were identified in the livers of rats in each group,of which 278 were upregulated and 267 were downregulated.Among the 27 com-monly differentially expressed mRNAs,five mRNAs related to lipid metabolism were screened,namely Aldh1a7,Nnmt,Irs2,Pltp,and Scd.KEGG enrich-ment analysis showed that the enriched pathways were AMPK and PPAR.Further validation revealed that in the SES-treated group,the mRNA expression of Scd in the liver was decreased,while the mRNA expression of Nnmt was increased.The protein ex-pression of Scd was decreased,while the protein ex-pression of Nnmt,AMPK,p-AMPK,PPARα,and PPARγ was increased.CONCLUSION:SES has preven-tive and therapeutic effects on fatty liver disease in rats with hypertension combined with dyslipidemia,and its mechanism of action may be related to the reduction of Scd expression levels in the liver and the increase in the expression of Nnmt,AMPK,p-AMPK,PPARα,and PPARγ.

AIM:To investigate the preventive and therapeutic effects of sesamin(SES)on fatty liver disease in rats with hypertension combined with dyslipidemia,and to explore the potential mecha-nisms based on mRNA-seq.METHODS:Spontane-ously hypertensive rats(SHRs)were fed a high-fat,high-cholesterol diet to establish a rat model of hy-pertension combined with dyslipidemia,and then treated with SES for 16 weeks continuously.The ex-periment was divided into four groups:WKY,SHR,Model,and Model+SES(160 mg·kg-1·d-1).Blood pressure was measured using the tail-cuff method.Body weight was monitored,and body mass index was calculated.Liver morphology was detected by ultrasound,and liver thickness was measured.Liver wet weight was weighed,and liver index was calcu-lated.Liver volume was detected by the water dis-placement method.Serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),alanine aminotransferase(ALT),aspartate amino-transferase(AST),and total bile acids(TBA)were de-tected by ELISA.Liver sequencing analysis was per-formed using mRNA-seq.Liver histomorphological changes were observed by HE staining.The degree of hepatic steatosis was observed by Oil Red O stain-ing,and the degree of hepatic fibrosis was observed by MASSON staining.The mRNA expression of Al-dh1a7,Nnmt,Irs2,Pltp,and Scd was detected by q-PCR.The protein expression of Scd,Nnmt,AMPK,p-AMPK,PPARα,and PPARγ was detected by Western blotting.RESULTS:After 16 weeks of continuous SES administration to rats with hypertension combined with dyslipidemia,blood pressure was significantly reduced(P＜0.01),and body weight was decreased.Serum TG,TC,and LDL-C levels were decreased,while HDL-C levels were increased.Serum ALT and AST levels were decreased.Liver weight,organ in-dex,liver thickness,and liver volume were de-creased.The degree of hepatic steatosis and hepat-ic fibrosis was improved.A total of 545 differentially expressed mRNAs were identified in the livers of rats in each group,of which 278 were upregulated and 267 were downregulated.Among the 27 com-monly differentially expressed mRNAs,five mRNAs related to lipid metabolism were screened,namely Aldh1a7,Nnmt,Irs2,Pltp,and Scd.KEGG enrich-ment analysis showed that the enriched pathways were AMPK and PPAR.Further validation revealed that in the SES-treated group,the mRNA expression of Scd in the liver was decreased,while the mRNA expression of Nnmt was increased.The protein ex-pression of Scd was decreased,while the protein ex-pression of Nnmt,AMPK,p-AMPK,PPARα,and PPARγ was increased.CONCLUSION:SES has preven-tive and therapeutic effects on fatty liver disease in rats with hypertension combined with dyslipidemia,and its mechanism of action may be related to the reduction of Scd expression levels in the liver and the increase in the expression of Nnmt,AMPK,p-AMPK,PPARα,and PPARγ.

Objective: To study the efficacy and safety of sofosbuvir and daclatasvir regimens for patients who received kidney transplantation (KT) with hepatitis C virus (HCV) infection. Methods: This study enrolled a prospective cohort of consecutive KT patients with HCV infection from March 2016 to January 2018 in the hepatology Department of the Second Hospital of Shandong University. They were given sofosbuvir combined with daclatasvir, with or without ribavirin. The course of treatment was 12 weeks or 24 weeks. Clinical assessment, conventional liver and kidney biochemical parameters, hemoglobin, serum HCV RNA, as well as the types of immunosuppressive drugs and their doses were assessed routinely as follows: at the beginning of treatment; 2, 4, and 8 wk post treatment; at the end of treatment (EOT); and at 12, 24 wk after the therapy was completed. Adverse events and adjustment of anti-rejection drugs were surveilled during the treatment period. Results: A total of 13 patients were enrolled. All patients were naive to treatment. Their mean age was 46.84±7.79 years. There were 10 males and 3 females, 3 patients had cirrhosis (1 cases had decompensated cirrhosis), 10 patients had no cirrhosis. They were infected with HCV genotype 1 (6/13 GT1b), genotype 3 (2/13 GT3a) and genotype 6 (3/13 GT6a), and genotype 2 (2/13 GT2a). Twelve patients′ estimated glomerular filtration rate (eGFR) was > 30 ml/min per 1.73 m² at the beginning of treatment, 1 patient′s eGFR was <30 ml/min·1.73 m^2; 9 patients received 12 wk therapy, 4 patients received 24 wk therapy. Twelve patients had undetectable viral load by week 4 of treatment. All patients had undetectable HCV viral load at the end of treatment. Sustained virological response (SVR) 12 rate was achieved in 100% (13/13) of the recipients. The basic renal function remained stable during the course of treatment. No serious adverse events were observed during the treatment. Antiviral therapy was not discontinued due to side effects in any patient. Conclusions Sofosbuvir and daclatasvir for treatment of KT patients with HCV infection are highly effective and safe.

Objective To study the alterations of renal function in patients with acute ischemic stroke and diabetes and to analyze the risk factors of contrast-induced nephropathy (CIN) after digital subtraction angiography (DSA).Methods Eight hundred and seventy-one cerebral infarction patients with diabetes who underwent DSA were selected in the Third Affiliated Hospital of Chongqing Medical University and Nanjing Brain Hospital Affiliated to Nanjing Medical University from August 2012 to August 2016.The patients were divided into diabetic group (n =178) and non-diabetic group (n =693).The alterations of renal function and the incidence rate of CIN were observed between two groups 3 days after DSA.Univariate analysis and multi-factor Logistic regression analysis were used to analyze the independent risk factors of CIN.Results The levels of estimated glomerular filtration rate (eGFR,ml · min-1 · 1.73 m-2) in diabetic group at 1,2 and 3 days after DSA(81.94 ±9.38,75.36 ±8.21,84.43 ±9.72) were lower than that in non-diabetic group (84.62 ± 10.06,79.08 ±9.84,87.62 ± 10.15,t =3.213,4.645,3.772,all P ＜ 0.05).The levels of serum creatinine (Scr) and cystatin (CysC) in diabetic group at 1,2 and 3 days after DSA (Scr:85.63 ±9.83,92.37 ±10.07,83.43 ±9.07;CysC:1.08 ±0.12,1.35 ±0.14,0.95 ±0.10) were higher than that in non-diabetic group (Scr:81.36 ± 8.98,87.84 ± 9.85,80.31 ± 8.64,t =5.548,5.448,4.253;CysC:0.97 ±0.11,1.21 ±0.12,0.88 ±0.09;t =11.677,13.400,9.043;all P ＜ 0.05).The incidence rate of CIN in diabetic group (25.84％ (46/178)) was higher than that in nondiabetic group (7.07％ (49/693),x2 =51.358,P =0.001).Multi-factor Logistic regression analysis showed allergies,plasma brain natriuretic peptide,heart failure,the original renal insufficiency,NIHSS score,contrast agent dosage,preoperative eGFR,preoperative Scr and preoperative CysC were the independent risk factors of CIN in cerebral infarction patients with diabetes.Conclusions The renal function decreased significantly and the incidence rate of CIN was high in cerebral infarction patients with diabetes after DSA.Allergies,plasma brain natriuretic peptide,heart failure,the original renal insufficiency,contrast agent dosage,preoperative eGFR,preoperative Scr and preoperative CysC were the independent risk factors of CIN in cerebral infarction patients with diabetes.

Objective To investigate any potential and independent demographic and serologic risk factors contributing to bone destruction in patients with rheumatoid arthritis ( RA) . Methods A total of 445 patients with RA were recruited in this study. Three autoantibodies including rheumatoid factor ( RF) , anti-cyclic citrullinated peptide antibody ( anti-CCP antibody) and anti-citrullinated alpha-enolase peptide 1 antibody ( anti-CEP-1 antibody) were quantified by using specific ELISA kits. The hand radiographs of all subjects were graded by using the modified Sharp/van der Heijde score ( Sharp score) . The potential and in-dependent risk factors were assessed by using univariate linear regression analyses and the stepwise multiple regression analysis, respectively. Results Based upon the univariate regression analyses, 7 covariates were identified as the potential risk factors for bone destruction in patients with RA, which were female (β=0. 100, P=0. 035), longer disease duration (β=0. 498, P=3. 26×10-29), RF (β=0. 096, P=0. 042), younger age at onset (β=-0. 312, P=1. 60 × 10-11 ), anti-CCP antibody positive (β=0. 202, P=1.74×10-5), anti-CEP-1 antibody positive (β=0.148, P=0.017) and positive for either anti-CCP or anti-CEP-1 antibodies (β=0. 157, P=1. 42×10-3). However, smoking (β=-0. 121, P=0. 018) were identi-fied as the potential protective factors. The multiple regression analysis indicated that the longer disease du-ration (P=2. 24×10-15) and anti-CCP antibody positive (P=0. 012) were independent risk factors for bone destruction. Conclusion Female, longer disease duration, younger age at onset, RF, anti-CCP and anti-CEP-1antibodies are potential risk factors for bone damage in patients with RA. Moreover, longer disease du-ration and anti-CCP antibody are two independent risk factors contributing to bone destruction in RA.

OBJECTIVE:To observe the clinical efficacy and safety of Bromfenac sodium eyed drops in the treatment of xe-rophthalmia. METHODS:80 patients with xerophthalmia were randomly divided into observation group and control group,with 40 cases in each group. Control group was given 0.1% Sodium hyaluronate,one drop,qd;observation group was given 0.1% Sodi-um hyaluronate eye drops,one drop,bid. Both groups received 14 d of treatment. The subjective symptom and sign,the time of lacrimal film break-up,fluorescent staining score and schemer test were observed in 2 groups before treatment,3,7,14 d after treatment. Clinical efficacy and the occurrence of ADR were observed in 2 groups. RESULTS:There was no statistical significance in subjective symptom and sign,the time of lacrimal film break-up,fluorescent staining score and tear between 2 groups before treatment,3,7 d after treatment(P>0.05). 14 d after treatment,subjective symptom and sign and fluorescent staining score of 2 groups were decreased significantly,and the time of lacrimal film break-up was prolonged and secretion was increased significant-ly;the observation group was significantly better than the control group,with statistical significance (P<0.05). The cure rate of observation group was 60.0%,which was significantly higher than that of control group(35.0%),with statistical significance(P<0.05). There were no statistical significance in ADR between 2 groups(P>0.05). CONCLUSIONS:Sodium hyaluronate eye drops is effective for xerophthalmia,and can relieve the symptoms with good safety.

Objective:Selected virulence factors more than and high virulent Aeromonas hydrophia strain made into inactivated vaccine,to study the immunization effect of inactivated vaccine.Methods:Crucian were vaccinated with formalin-killed vaccine via in-traperitoneal injection.Controls were injected with the same volumes of saline.Then the antibody titres, histopathology and relative percent survival were analyzed from samples of both groups.Results: The antibody in the indirect agglutination reaction could be detected in vaccinated fish once a weeks after immunization and reached highest level 6 weeks after immunization.The histopathology analysis indicated that the vaccine had a good protective effect on crucian target organs.Vaccinated fish showed 100%relative percent survival and the immune period would be 6 month.Conclusion:The vaccine in this study has a significant protective effect on crucian and may be used as effective fish vaccines against bacterial septicemia.