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

OBJECTIVES: To investigate the therapeutic mechanism of Danzhi Jiangtang Capsule (DZJTC) for repairing renal vascular endothelial injury in rats with diabetic nephropathy (DN). METHODS: Fifty male SD rat models of DN, established by left nephrectomy, high-sugar and high-fat diet and streptozotocin injection, were randomized into DN model group, low-, medium-, and high-dose DZJTC treatment groups, and DAPT (a γ-secretase inhibitor) treatment group, with 10 rats with normal feeding as the control group. DZJTC was administered by daily gavage at 0.315, 0.63, or 1.26 g/kg, and DAPT (20 mg/kg, dissolved in 50% CMC-Na solution) was given by gavage every other day for 4 weeks; normal saline was given in the control and model groups. After treatment, the levels of creatinine (CRE), blood urea nitrogen (BUN), and microalbuminuria (mALB) were detected with ELISA, and renal pathologies were observed by transmission electron microscopy. Renal expressions of vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were measured by immunohistochemistry, and the protein expressions of CD31 and Notch signaling pathway components were detected using Western blotting. RESULTS: The rat models of DN showed significantly increased CRE, BUN, and mALB levels, obvious renal pathologies under electron microscopy, increased renal VEGF, ET-1 and CD31 expressions, and upregulated Notch1, NICD, and MAML1 protein levels. Treatment with DZJTC at the 3 doses and DAPT significantly reduced CRE, BUN, and mALB levels, improved renal pathology, decreased VEGF, ET-1 and CD31 expressions, and lowered Notch1, NICD and MAML1 levels, and the effects were the most pronounced with high-dose DZJTC. CONCLUSIONS DZJTC ameliorates hyperproliferation and dysfunction of renal vascular endothelium in DN rats possibly by regulating renal VEGF and ET-1 levels via inhibiting NICD- and MAML1-mediated Notch signaling pathway.
Animals ; Male ; Drugs, Chinese Herbal/therapeutic use* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Diabetic Nephropathies/drug therapy* ; Receptor, Notch1/metabolism* ; Kidney/blood supply* ; Diabetes Mellitus, Experimental ; Down-Regulation ; Endothelium, Vascular/metabolism* ; Nuclear Proteins/metabolism*

Objective:To evaluate the long-term protective efficacy of the recombinant Chinese hamster ovary cell-derived hepatitis B vaccine（CHO-HepB）26 years post-vaccination in the rural China.Methods:Zhengding county，Hebei province was designated as a rural monitoring site for CHO-HepB efficacy. Study participants included individuals born between 1997 and 1999 who had completed the three-dose CHO-HepB primary series without booster doses. A cross-sectional survey was conducted in late 2024 using random sampling. Demographic and vaccination history data were collected via questionnaires，and hepatitis B virus（HBV）serological markers were detected using chemiluminescence. Historical surveillance data were integrated to infer infection statuses of HBsAg-positive individuals and evaluate longitudinal trends in anti-HBs seropositivity and antibody titers.Results:Among 178 participants（mean time since vaccination：26.2 years），the seroprevalence rates were 0.6% for HBsAg（95% CI：0.0%-1.6%），64.6% for anti-HBs（95% CI：57.6%-71.6%），and 1.1% for anti-HBc（95% CI：0.0%-2.7%）. Compared to the pre-vaccination baseline HBsAg positivity of 11.3% in children under 10 years of age，the estimated vaccine protection rate was 95%. Two notable cases were identified：one with concurrent HBsAg and anti-HBc positivity and one with anti-HBs and anti-HBc positivity，suggestive of transient HBV exposure（1999—2009）without chronicity. Natural immune boosting was inferred for the latter case based on anti-HBs titer dynamics. Longitudinal analysis of four prior cross-sectional surveys（2005，2009，2013，and 2017）revealed no significant upward trends in HBsAg and anti-HBc positivity（both P>0.05）over 26 years，while anti-HBs seropositivity declined significantly（ P<0.05）from 6 to 26 years post-vaccination. Conclusion:The CHO-HepB vaccine demonstrates sustained immunological persistence and robust long-term protection up to 26 years post-immunization. Continued emphasis on rigorous implementation of mother-to-child transmission prevention strategies is critical for future hepatitis B control.

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 investigate the relationship between 9 immunoinflammatory indicators derived from complete blood count and the severity of Mycoplasma pneumoniae pneumonia(MPP)in children of different ages.Methods Totally 2 132 children with MPP who were hospitalized in the Department of Pediatrics of The First Affiliated Hospital of Naval Medical University from Jul.1,2023,to Dec.31,2024 were enrolled,and were assigned to severe MPP(SMPP)or non-severe MPP(NSMPP)groups.According to age and gender 1∶1 matching,the children were assigned to 2 subgroups according to age(1-6 years old and＞6-16 years old).The basic data,laboratory examination and immunoinflammatory indicators from complete blood count of each group were collected and compared.The influencing factors of SMPP were analyzed by univariate and multivariate Cox proportional hazards regression models.Receiver operating characteristic curves were used to analyze the predictive value of indicators that showed statistically significant differences for SMPP.Results There were 220 patients with SMPP,accounting for 10.3%of MPP.In children aged 1-6 years,compared with the NSMPP group,the SMPP group had a longer hospital stay,higher platelet(PLT)count,platelet-to-lymphocyte ratio(PLR),neutrophil-to-lymphocyte ratio,derived neutrophil-to-lymphocyte ratio and systemic immune-inflammation index(all P＜0.05).PLR was an independent risk factor for SMPP(odds ratio=1.010,95%confidence interval[CI]1.003-1.018,P=0.007).The area under curve predicted by PLR for SMPP was 0.635(95%CI 0.560-0.711,P＜0.001),the best cut-off value was 125.04,and the corresponding sensitivity and specificity were 57.7%and 70.2%,respectively.All the children were assigned to low PLR group or high PLR group using the best cut-offvalue as the boundary,and the severe disease rate in the high PLR group was significantly higher than that in the low PLR group(65.9%[60/91]vs 37.6%[44/117],P＜0.001).All the children were assigned to Q1-Q4 groups by quartile,and the severe disease rate of the Q4 group(71.2%,37/52)was significantly higher than that of the Q1-Q3 group(all P＜0.05).In children aged＞6-16 years,compared with the NSMPP group,the PLT and PLR in the SMPP group were higher(both P＜0.05),but neither was an independent risk factor.All the children were assigned to low PLR group or high PLR group using the best cut-offvalue(137.03)as the boundary,and the severe disease rate in the high PLR group was significantly higher than that in the low PLR group(57.0%[77/135]vs 40.2%[39/97],P=0.011).All the children were assigned to Q1-Q4 groups by quartile,and the severe disease rate of the Q4 group(65.5%,38/58)was significantly higher than that of the Q1-Q3 group(all P＜0.05).Conclusion The immunoinflammatory indicators derived from complete blood count,especially PLR,have certain application value in predicting the severity of MPP children in different ages.

Food safety problems caused by foodborne pathogenic bacteria pose a serious threat to public health,creating an urgent need to develop testing methods and techniques with excellent performance and are simple to use and of affordable cost.Traditional testing methods,such as isolation and culture,morphological observation,biochemical identification,and serological tests,have many limitations,including complex procedures,reliance on specialized technical equipment and personnel,and long turnaround time,rendering them inadequate for meeting current and future testing demands.Therefore,it is particularly important to develop simple,rapid,and highly sensitive methods for analyzing pathogenic bacteria.The fusion of nucleic acid aptamers and nanozymes brings new ideas for the rapid testing of pathogenic bacteria.On one hand,aptamers offer specific recognition capability for target bacteria and can be combined with various nucleic acid signal amplification techniques.On the other hand,the enzyme-like catalytic activity and signal amplification effect of many nanomaterials provide a basis for highly sensitive testing.This review highlights the application potential of nanozyme?aptamer coupling systems in the field of microbial analysis by briefly summarizing the latest research progress in the use of nanozymes combined with aptamers for the detection of foodborne pathogenic bacteria.First of all,two main approaches to conjugating nanozymes with aptamers are introduced.Then,the testing mechanisms and typical applications of nanozyme?aptamer coupling systems for foodborne pathogenic bacteria are discussed.Finally,future development trends and existing challenges are disucssed from four perspectives,including specificity,high sensitivity,high throughput,and intelligent detection.This review aims to provide a useful reference for the fusion of nanozymes and aptamers and for the development of on-site rapid testing techniques for foodborne pathogens,and to encourage broader academic interest to further advance this promising research field.

Objective To investigate the therapeutic mechanism of Danzhi Jiangtang Capsule(DZJTC)for repairing renal vascular endothelial injury in rats with diabetic nephropathy(DN).Methods Fifty male SD rat models of DN,established by left nephrectomy,high-sugar and high-fat diet and streptozotocin injection,were randomized into DN model group,low-,medium-,and high-dose DZJTC treatment groups,and DAPT(a γ-secretase inhibitor)treatment group,with 10 rats with normal feeding as the control group.DZJTC was administered by daily gavage at 0.315,0.63,or 1.26 g/kg,and DAPT(20 mg/kg,dissolved in 50%CMC-Na solution)was given by gavage every other day for 4 weeks;normal saline was given in the control and model groups.After treatment,the levels of creatinine(CRE),blood urea nitrogen(BUN),and microalbuminuria(mALB)were detected with ELISA,and renal pathologies were observed by transmission electron microscopy.Renal expressions of vascular endothelial growth factor(VEGF)and endothelin-1(ET-1)were measured by immunohistochemistry,and the protein expressions of CD31 and Notch signaling pathway components were detected using Western blotting.Results The rat models of DN showed significantly increased CRE,BUN,and mALB levels,obvious renal pathologies under electron microscopy,increased renal VEGF,ET-1 and CD31 expressions,and upregulated Notch1,NICD,and MAML1 protein levels.Treatment with DZJTC at the 3 doses and DAPT significantly reduced CRE,BUN,and mALB levels,improved renal pathology,decreased VEGF,ET-1 and CD31 expressions,and lowered Notch1,NICD and MAML1 levels,and the effects were the most pronounced with high-dose DZJTC.Conclusion DZJTC ameliorates hyperproliferation and dysfunction of renal vascular endothelium in DN rats possibly by regulating renal VEGF and ET-1 levels via inhibiting NICD-and MAML1-mediated Notch signaling pathway.

Objective To analyze the value of actin-related protein 2(ACTR2)and DEAD-box RNA helicase 3X-linked(DDX3X)expression in evaluating the severity of Rotavirus gastroenteritis(RVGE)in children.Methods A total of 153 children with rotavirus gastroenteritis admitted to Handan Maternal and Child Health Hospital from September 2022 to September 2024 were selected as the research objects.According to the severity of RVGE,the children with RVGE were divided into mild group(n=60),moderate group(n=71)and severe group(n=22).In addition,60 healthy children were randomly selected as the healthy group.The clinical data of the light group,the medium group and the heavy group were compared.The expression levels of ACTR2 and DDX3X in serum of each group were detected by real-time fluorescence quantitative PCR.ROC curve was used to analyze the evaluation value of combined detection of serum ACTR2 and DDX3X in children with severe RVGE.Results The age of children with RVGE in the severe group was significantly lower than that in the mild group and the moderate group,and the differences were statistically significant(Z=8.307,5.885,all P＜0.001).There were statistically significant differences in dehydration and diarrhea among the three groups(F=9.434,126.080,all P＜0.05).The expression levels of ACTR2(1.20±0.28)and DDX3X(1.22±0.37)in RVGE group were significantly higher than those in control group(1.01±0.02,1.04±0.09),and the differences were statistically significant(t=5.071,3.584,all P＜0.05).The expression levels of ACTR2[1.11±0.23,1.23±0.21,1.42(1.25,1.57)]and DDX3X[1.14±0.22,1.25±0.24,1.32(1.23,1.62)]in the serum of the mild group,the moderate group and the severe group increased in turn,and the differences were statistically significant(Z=27.196,18.013,all P＜0.05).The ROC curve analysis results showed that the combined detection of serum ACTR2 and DDX3X predicted the AUC(95%CI)of critically ill RVGE patients,with higher sensitivity and specificity than the detection of the two alone(Z=2.573,2.101,P=0.014,0.034).Conclusion The expression levels of serum ACTR2 and DDX3X are closely related to the severity of RVGE.The combined detection of the two has a high predictive value for the diagnosis of the severity of RVGE.

Objective To explore the relationship between ribonuclease A3(RNase3)gene polymorphism and its expression level with airway inflammation and glucocorticoid efficacy in children with bronchial asthma(BA).Methods A total of 110 children with bronchial asthma admitted to Handan Maternal and Child Health Hospital June 2022 to June 2024 were selected as the study objects.According to the severity of the condition at admission,they were divided into mild group(n=64),moderate to severe group(n=46),and children who underwent physical examination in the hospital during the same period were selected as the control group(n=82).Molecular weight array gene analysis(MassArray)was used to detect the genotyping of RNase3 gene rs2073342 locus.Quantitative real-time PCR(RT-PCR)was used to detect the expression level of RNase3 mRNA.The clinical data of the children were collected,and the differences of genotype and allele frequency were compared.The relationship between RNase3 gene polymorphism and BA susceptibility was analyzed by unconditioned Logistic regression.Results Compared with the mild group,IL-6(21.49±3.01ng/L vs 13.21±2.84ng/L)and PCT(19.16±4.02μg/L vs 9.94±3.15μg/L)in the moderate and severe group were significantly increased(t=-14.568,-12.952),while FVC,PEF and FEV1 were significantly decreased(t=2.534,3.304,2.011),and the differences were statistically significant(all P<0.05).The genotype distribution of RNase3 gene rs2073342 in both control and study groups was consistent with Hardy-Weinberg balance law(χ2=0.402,0.689,all P>0.05),indicating population representation.Compared with the control group and the mild group,the CC,CA genotype frequencies were higher in moderate to severe group(χ2=35.008,23.079),Compared with the control group,the frequency of CC and CA genotypes in mild group was higher(χ2=7.325),the differences were statistically significant(all P<0.05).Compared with mild group,RNase3 mRNA expression in peripheral blood mononuclear cells of children with C gene BA at rs2073342 in moderate to severe group was higher,and the difference was statistically significant(t=-19.622,P<0.05).In the same group,the mRNA expression level of RNase3 in peripheral blood mononuclear cells of the children with C gene BA at rs2073342 was higher than that of the children without C gene(t=4.169,22.608,all P<0.05).Unconditional Logistic regression results showed that RNase3 gene carrying allele C or dominant model(CC vs CA+AA)was a risk factor for severe disease in BA children(P<0.05).The total effective rate of genotype AA carriers was the highest after glucocorticoid therapy,and the therapeutic effect of genotype AA carriers was significantly better than that of genotype AC and CC,and the difference was statistically significant(χ2=11.858,P<0.05).Conclusion The expression of RNase3 mRNA in peripheral blood of BA children increased significantly with the exacerbation of the disease.Carrier of allele C or dominant model(CC vs CA+AA)is a risk factor for severe disease in BA children.Genotype AA carriers had better therapeutic effect.