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 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.

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

The aim of this study is to identify the linear Fc-binding epitope for IgG1 on bovine IgG Fc receptor Ⅱ(boFcγRⅡ)to understand the molecular basis of IgG-Fcγ interaction.The boFcγRⅡ molecules were expressed on cell surface of the boFcγR Ⅱ-transfected COS-7 cells.The extracel-lular domain of boFcγRⅡ was expressed in NS0 cells,and the boFcγRⅡ recombinant protein was purified from ascites by Ni-chelation chromatography.Peptides derived from the membrane-distal extracellular domain(EC2)of boFcγR Ⅱ were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin(BSA).Binding of bovine IgG1 to the different peptides was tested by dot-blot assay,and the IgG-binding peptide was further modified by truncation and mutation to identify the Fc-binding epitope as well as its key amino acids for Fc-binding.The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay,re-spectively.The results showed that boFcγR Ⅱ molecules were stably expressed on surface of the transfected COS-7 cells,which showed about 90％rosetting with IgG1-RBCs.The soluble boFcγRⅡ recombinant protein specifically bound to bovine IgG1.The minimal effective peptide of 122FYQDRKSKIF131 of boFcγRⅡ was able to bind bovine IgG1 specifically,suggesting it repre-sents a linear Fc-binding epitope located in the putative C-C'loop of the EC2 domain on the recep-tor.The Ala-substitution of Phe122,Tyr123,Arg126,Lys127,Ser128,Lys129 or Phe131 within the linear epitope led to a complete loss of its IgG1-binding capability,indicating those residues are critical for IgG1-binding on boFcγRⅡ.The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRⅡ with IC50 of 20.05 μmol/L,and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRⅡ transfected cells with IC50 of 80.15 μmol/L.The re-sults indicate that boFcγRⅡ possesses the linear epitope for Fc-binding,and the Fc-binding pep-tide showed well capability of regulating boFcγR Ⅱ-IgG1 interaction on cell surface,thereby provi-ding a research foundation for understanding the IgG-Fcγ interaction.

The aim of this study is to identify the linear Fc-binding epitope for IgG1 on bovine IgG Fc receptor Ⅱ(boFcγRⅡ)to understand the molecular basis of IgG-Fcγ interaction.The boFcγRⅡ molecules were expressed on cell surface of the boFcγR Ⅱ-transfected COS-7 cells.The extracel-lular domain of boFcγRⅡ was expressed in NS0 cells,and the boFcγRⅡ recombinant protein was purified from ascites by Ni-chelation chromatography.Peptides derived from the membrane-distal extracellular domain(EC2)of boFcγR Ⅱ were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin(BSA).Binding of bovine IgG1 to the different peptides was tested by dot-blot assay,and the IgG-binding peptide was further modified by truncation and mutation to identify the Fc-binding epitope as well as its key amino acids for Fc-binding.The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay,re-spectively.The results showed that boFcγR Ⅱ molecules were stably expressed on surface of the transfected COS-7 cells,which showed about 90％rosetting with IgG1-RBCs.The soluble boFcγRⅡ recombinant protein specifically bound to bovine IgG1.The minimal effective peptide of 122FYQDRKSKIF131 of boFcγRⅡ was able to bind bovine IgG1 specifically,suggesting it repre-sents a linear Fc-binding epitope located in the putative C-C'loop of the EC2 domain on the recep-tor.The Ala-substitution of Phe122,Tyr123,Arg126,Lys127,Ser128,Lys129 or Phe131 within the linear epitope led to a complete loss of its IgG1-binding capability,indicating those residues are critical for IgG1-binding on boFcγRⅡ.The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRⅡ with IC50 of 20.05 μmol/L,and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRⅡ transfected cells with IC50 of 80.15 μmol/L.The re-sults indicate that boFcγRⅡ possesses the linear epitope for Fc-binding,and the Fc-binding pep-tide showed well capability of regulating boFcγR Ⅱ-IgG1 interaction on cell surface,thereby provi-ding a research foundation for understanding the IgG-Fcγ interaction.

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Animals ; Antibodies, Viral ; Chickens ; HN Protein/metabolism* ; Newcastle Disease/prevention & control* ; Newcastle disease virus/metabolism* ; Oryza/genetics*

Objective:To explore the value of MR elastography (MRE) and shear wave elastography (SWE) for staging liver fibrosis in a rabbit model.Methods:From March to November 2020, 200 healthy New Zealand white rabbits were randomly divided into control group ( n=40) and liver fibrosis group ( n=160) by random number table method. The volume ratio of CCl 4 and olive oil was 1∶1 to prepare 50% CCl 4 oil solution, and the experimental rabbits in the liver fibrosis group were subcutaneously injected with 50% CCl 4 olive oil solution. It was injected once a week at the dose of 0.1 ml/kg in the first to third weeks, once a week at the dose of 0.2 ml/kg in the 4th to 6th weeks. The dose of 0.1 ml/kg was injected twice a week from week 7 to 16. The control group were subcutaneously injected with an equal dose of normal saline. At the end of the 4th, 8th, 12th, and 16th week, 40 and 10 animals in the liver fibrosis group and the control group were randomly selected by random number table method for MRE and SWE, respectively, to obtain the liver elastic stiffness (LS), which were recorded as LS MRE and LS SWE. After the examination, the experimental rabbits were sacrificed and liver tissue of rabbits were taken for histopathological Scheuer staging, and they were divided into F0-F4 groups. One-way ANOVA was used to evaluate the differences of LS MRE and LS SWE in different stages of liver fibrosis. Spearman correlation was used to analyze the correlation between LS and pathological stages. The receiver operating characteristic curve was used to evaluate the efficacy of LS MRE and LS SWE in diagnosing liver fibrosis staging, and the area under the curve (AUC) was compared using the Z test. Results:Totally 162 rabbits were included, which covered F0 ( n=38), F1 ( n=33), F2 ( n=35), F3 ( n=31) and F4 ( n=25). Significant differences of LS MRE and LS SWE values were found among different stages of liver fibrosis ( F=295.29, 102.40, both P<0.001). LS MRE, LS SWE were both positively correlated with liver fibrosis stage ( r=0.93, 0.81, both P<0.001). The AUC of LS MRE for diagnosing liver fibrosis stages ≥F1, ≥F2, ≥F3, and ≥F4 were 0.955, 0.967, 0.996, and 0.980, respectively; the AUC of LS SWE were 0.856, 0.880, 0.974, and 0.953, respectively. The AUC of liver fibrosis stage ≥ F1, ≥ F2 for LS MRE value were greater than LS SWE value ( Z=2.93, 3.29, P=0.003, 0.001), and the AUC of ≥F3, ≥F4 had no significant differences ( Z=1.58, 1.68, P=0.115, 0.093). Conclusion:Both MRE and SWE can accurately predict the stage of liver fibrosis in experimental rabbits, and MRE is better than SWE in diagnosing early liver fibrosis.

Objective::Microchannels are associated with the progression of atherosclerotic vulnerable plaques. However, in patients with culprit optical coherence tomography (OCT)-defined plaque erosion, the knowledge of microchannels and culprit lesion vulnerability is limited. The aim of this study was to investigate culprit lesion characteristics in patients with ST-segment elevated myocardial infarction (STEMI) caused by plaque erosion with and without microchannels using OCT.Methods::In all, 348 STEMI patients with plaque erosion who underwent OCT of the culprit lesion at the 2 nd Affiliated Hospital of Harbin Medical University (Harbin, China) from August 2014 to December 2017 were included and divided into the microchannel group ( n= 116, 33.3%) and no-microchannel group ( n = 232, 66.7%). The clinical characteristics and OCT-derived plaque features were compared between both groups. Results::Among the 348 STEMI patients with plaque erosion, culprit lesions with microchannels had higher incidence of lipid plaque (59.5% vs. 45.3%, P= 0.012); calcification (41.4% vs. 24.6%, P= 0.002); spotty calcification (30.2% vs. 18.1%, P= 0.014); macrophages accumulation (72.4% vs. 45.7%, P < 0.001); and cholesterol crystals (32.8% vs. 14.2%, P < 0.001) than those without microchannels. In addition, minimal lumen area was smaller ((1.9 ± 0.9) mm 2vs. (2.8 ± 2.3) mm 2, P < 0.001) and lumen area stenosis was greater ((71.3% ± 13.4%) vs. (65.3% ± 19.3%), P= 0.001) in the microchannel group than in the no-microchannel group. Conclusion::In patients with STEMI caused by plaque erosion, one-third manifested typical microchannel characteristics, and those with microchannels were associated with more severe luminal stenosis and more vulnerable plaque features than those without microchannels.

Objective::Microchannels are associated with the progression of atherosclerotic vulnerable plaques. However, in patients with culprit optical coherence tomography (OCT)-defined plaque erosion, the knowledge of microchannels and culprit lesion vulnerability is limited. The aim of this study was to investigate culprit lesion characteristics in patients with ST-segment elevated myocardial infarction (STEMI) caused by plaque erosion with and without microchannels using OCT.Methods::In all, 348 STEMI patients with plaque erosion who underwent OCT of the culprit lesion at the 2 nd Affiliated Hospital of Harbin Medical University (Harbin, China) from August 2014 to December 2017 were included and divided into the microchannel group ( n= 116, 33.3%) and no-microchannel group ( n = 232, 66.7%). The clinical characteristics and OCT-derived plaque features were compared between both groups. Results::Among the 348 STEMI patients with plaque erosion, culprit lesions with microchannels had higher incidence of lipid plaque (59.5% vs. 45.3%, P= 0.012); calcification (41.4% vs. 24.6%, P= 0.002); spotty calcification (30.2% vs. 18.1%, P= 0.014); macrophages accumulation (72.4% vs. 45.7%, P < 0.001); and cholesterol crystals (32.8% vs. 14.2%, P < 0.001) than those without microchannels. In addition, minimal lumen area was smaller ((1.9 ± 0.9) mm 2vs. (2.8 ± 2.3) mm 2, P < 0.001) and lumen area stenosis was greater ((71.3% ± 13.4%) vs. (65.3% ± 19.3%), P= 0.001) in the microchannel group than in the no-microchannel group. Conclusion::In patients with STEMI caused by plaque erosion, one-third manifested typical microchannel characteristics, and those with microchannels were associated with more severe luminal stenosis and more vulnerable plaque features than those without microchannels.