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 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.

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.

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 determine the impact of inflammatory reaction levels and the culprit plaque characteristics on preprocedural Thrombolysis in Myocardial Infarction (TIMI) flow grade in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). Methods: The is a retrospective study. A total of 1 268 STEMI patients who underwent pre-intervention optical coherence tomography (OCT) examination of culprit lesion during emergency PCI were divided into 2 groups by preprocedural TIMI flow grade (TIMI 0-1 group (n =964, 76.0%) and TIMI 2-3 group (n =304, 24.0%)). Baseline clinical data of the 2 groups were collected; blood samples were collected for the detection of inflammatory markers such as high sensitivity C-reactive protein (hsCRP), myocardial injury marker, blood lipid, etc.; echocardiography was used to determine left ventricular ejection fraction; coronary angiography and OCT were performed to define the lesion length, diameter stenosis degree of the infarct-related arteries, presence or absence of complex lesions, culprit lesion type, area stenosis degree and vulnerability of culprit plaques. Multivariable logistic regression analysis was performed to identify independent correlation factors. The receiver operating characteristic (ROC) curve of continuous independent correlation factors was analyzed, and the best cut-off value of TIMI 0-1 was respectively determined according to the maximum value of Youden index. Results: The mean age of 1 268 STEMI patients were (57.6±11.4) years old and 923 cases were males (72.8%). Compared with TIMI 2-3 group, the patients in TIMI 0-1 group were older and had higher N-terminal-pro-B-type natriuretic peptide level, lower cardiac troponin I (cTnI) level, lower left ventricular ejection fraction, and higher hsCRP level (5.16(2.06, 11.78) mg/L vs. 3.73(1.51, 10.46) mg/L). Moreover, the hsCRP level of patients in TIMI 0-1 group was higher in the plaque rupture subgroup (all P<0.05). Coronary angiography results showed that compared with TIMI 2-3 group, the proportion of right coronary artery (RCA) as the infarct-related artery was higher, the angiographical lesion length was longer, minimal lumen diameter was smaller, and diameter stenosis was larger in TIMI 0-1 group (all P<0.05). The prevalence of plaque rupture was higher (75.8% vs. 61.2%) in TIMI 0-1 group. Plaque vulnerability was significantly higher in TIMI 0-1 group than that in TIMI 2-3 group with larger mean lipid arc (241.27°±46.78° vs. 228.30°±46.32°), more thin-cap fibroatheroma (TCFA, 72.4% vs. 57.9%), more frequent appearance of macrophage accumulation (84.4% vs. 70.7%) and cholesterol crystals (39.1% vs. 25.7%). Minimal flow area was smaller [1.3(1.1-1.7)mm² vs. 1.4(1.1-1.9)mm², all P<0.05] and flow area stenosis was higher (78.2%±10.6% vs. 76.3%±12.3%) in TIMI 0-1 group. Multivariable analysis showed that mean lipid arc>255.55°, cholesterol crystals, angiographical lesion length>16.14 mm, and hsCRP>3.29 mg/L were the independent correlation factors of reduced preprocedural TIMI flow grade in STEMI patients. Conclusions: Plaque vulnerability and inflammation are closely related to reduced preprocedural TIMI flow grade in STEMI patients.
Aged ; Coronary Angiography ; Humans ; Inflammation ; Male ; Middle Aged ; Myocardial Infarction/diagnostic imaging* ; Percutaneous Coronary Intervention ; Plaque, Atherosclerotic/diagnostic imaging* ; Retrospective Studies ; ST Elevation Myocardial Infarction/surgery* ; Stroke Volume ; Thrombolytic Therapy ; Ventricular Function, Left

Discuss the electromyography and imaging characteristics of Hirayama disease，and improve clinicians’ awareness of the disease. Methods A retrospective analysis of clinical data of five cases of Hirayama disease patients in our hospital，analyze its clinical manifestations，EMG and imaging features. Results 5 patients were male，age 18 to 28 years old，（3 cases） unilateral，bilateral involvement in two cases of asymmetry. Five patients showed the hand and forearm muscle weakness with atrophy，forearm ramp-like changes，with cold paralysis. Motor nerve conduction delay mainly ulnar nerve terminal during latent，median nerve，ulnar nerve compound muscle action potential （the CMAP） amplitude decreased，no motor nerve conduction block；sensory nerve conduction were normal. Needle electrode EMG neurogenic damage，abnormal muscle mainly in the C7-T1 segment dominated area. MRI in the neutral position of the cervical spine in 5 patients showed that the physiological curvature was straightened or the spinal cord was slightly thinned；2 cases showed LOA phenomenon in the cross section；5 cases showed different degrees of compression of the cervical spinal cord forward and the epidural space signal shadow in the flexion position，enhanced scanning Abnormal enhancement in the epidural space can be seen. Conclusion According to the clinical manifestations of Hirayama disease combined with its characteristic electromyography and MRI features，early diagnosis of Hirayama disease can be made.

Objective:To explore the relationship between new biomarkers in donor blood and delayed graft function after kidney transplantation and evaluate the clinical value of new biomarkers in the diagnosis of DGF (delayed graft function).Methods:For recipients of donor kidney transplantation from August 2016 to December 2017, blood samples were collected from operations of donor organ resection within 12 hours of the day. Enzyme-linked immunosorbent assay (ELISA) was employed for detecting neutrophil gelatinase-associated lipocalin (NGAL), L-type fatty acid binding protein (L-FABP), kidney injury molecule-1 (KIM-1) and interleukin-18(IL-18). They were divided into DGF and EGF (early graft function) groups according to the diagnosis of DGF. The inter-group differences of four new biomarkers were calculated. Receiver operating characteristic curve (ROC curve) was plotted for finding the best positive cutoff value and the sensitivity and specificity of new donor blood marker for diagnosing delayed graft function were calculated.Results:Among them, 8 had postoperative DGF and 62 had none. The overall incidence of DGF was 11.43%. The serum concentration of NGAL was 521.01±132.84 ng/ml in DGF group versus (299.99±100.03) ng/ml in EGF group ( P<0.001). The ROC curve was plotted. With a NGAL concentration >425.15 ng/ml, the sensitivity and specificity for diagnosing DGF were 87.5% and 90.3% respectively. The area under the curve (AUC) was 0.891. The serum concentration of IL-18 was (14.10±12.36) ng/ml in DGF group and (4.61±1.83) ng/ml in EGF group ( P=0.047). With a IL-18 concentration of >5.345 ng/ml, the sensitivity and specificity for diagnosing DGF were 100% and 64.5% respectively. The AUC was 0.914. No significant inter-group difference existed in serum L-FABP/KIM-1. The sensitivity of donor creatinine in the diagnosis of DGF was 62.5%, specificity 75.8% and AUC 0.692. Conclusions:With an excellent level of sensitivity and specificity, an elevated concentration of NGAL/IL-18 in donor blood is superior to traditional creatinine in the diagnosis of DGF after renal transplantation.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.
Antibodies* ; Collodion ; Colloids ; Diagnostic Tests, Routine ; Enzyme-Linked Immunosorbent Assay ; Gold Colloid ; Immunochromatography ; Membranes ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; Sensitivity and Specificity ; Staphylococcal Protein A ; Swine