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

A 11-year old female patient with severe thalassemia, receipt a lentivirus-based cell and gene therapy (CGT) therapy in Shenzhen Children′s Hosptial on July 27th, 2021. At the two follow-up visits after discharge, patient were continuously tested positive for HIV screening through HIV Ag/Ab Combo assay (chemiluminescence Immunoassay), and the viral load results of HIV-1 nucleic acid testing (NAT) were both>5 000 copies/ml. The patient can be diagnosed with HIV infection according to the National Guideline for Detection of HIV/AIDS(2020 Revised Edition). The thorough investigation findings and supplementary experiment results indicated that the false-positive HIV-1 NAT results was caused by cross-reactivity between the target sites detected by conventional HIV-1 NAT reagents and the lentiviral vectors fragments integrated into the genome of patient′s hematopoietic stem/progenitor cells. In conclusion, it is important for laboratories to select appropriate HIV-1 NAT testing platforms which won′t cause cross-reactivity for the testing of samples from patients who have been treated with HIV-derived vectors. It is also recommended to design and develop NAT testing platforms with multiple target regions labeled by different fluorescents for HIV NAT supplementation experiment to reduce the risk of false-positive diagnoses of HIV infection.

Objective:To establish a method for rapid detection of OXA and par C resistance genes of Acinetobacter baumannii(Ab)by double nucleic acid colloidal gold strip and to develop kit.Methods:DNA of Ab was extracted by heating and boiling method.OXA and par C genes sequences of Ab were selected as target gene fragments based on NCBI.Primers were designed and labeled with 6-FAM,digoxin and biotin,respectively.Drug resistance gene detection reagents were developed,and nucleic acid gold test strips were used for rapid and visual detection.Molecular cloning and sequencing techniques were used to clone positive control samples and evaluate specificity,sensitivity and stability of kit.Results:DNA concentration and purity of Ab extracted by boiling method were good.Homology between cloned and sequenced plasmid DNA and gene sequence in GenBank database was 100%,respectively.Speci-ficity of kit was good,with only Ab showing positive results and other bacterial genera showing negative results;DNA concentration of Ab in double nucleic acid colloidal gold test strip decreased to 10-3 ng/μl,a red line still appeared,whose sensitivity was 10 times higher consistent with minimum detection limit of electrophoresis 10-2 ng/μl;test kits were tested at 3rd,6th and 9th months,and showed good stability.Conclusion:Double resistance detection kit established in this study can simultaneously detect OXA and par C resis-tance of Ab,who has advantages of high sensitivity,strong specificity,rapid and simple,and provides a new method for detection of carbapenem and quinolone antibiotic resistance of Ab.

We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.
African Swine Fever Virus/genetics* ; Animals ; DNA ; Immunity, Innate ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine

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*

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.
African Swine Fever/diagnosis* ; African Swine Fever Virus/genetics* ; Animals ; Antibodies, Viral ; Enzyme-Linked Immunosorbent Assay ; Recombinant Proteins/genetics* ; Swine

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 investigate the intervention effect of Shenfu i njection（SFI）on the nuclear translocation of high mobility group box 1（HMGB1） in lipopolysaccharide （LPS）-induced RAW 264.7 cells. METHODS ： Using LPS-induced RAW264.7 cells as objects ，the histone deacetylase inhibitor RGFP 966 as positive control ，CCK-8 assay was used to screen drug dosage，and the effects of low ，medium and high doses （3，6，12 μL/mL）of SFI on HMGB 1 nuclear translocation in RAW 264.7 cells were observed by immunofluorescence method ；mRNA expression of HMGB 1 in RAW 264.7 cells were detected by real time fluorescent PCR. Western blotting assay was used to determine protein expression of HMGB 1 and Toll-like receptor 4（TLR4）；the expression of HMGB 1 were compared between nucleus and cytoplasm. The levels of HMGB 1，IL-1β and TNF-α in supernatant of cells were detected by ELISA. RESULTS ：In blank control group ，HMGB1 was mainly located in the nucleus ；after LPS induction， HMGB1 migrated from nucleus to cytoplasm. Compared with blank control group ， mRNA and protein （No.81760738） expression of HMGB 1， protein expression of TLR 4 in RAW264.7 cells as well as the levels of HMGB 1，IL-1β and TNF-α in supernatant of cells were increased significantly in LPS group （P＜0.01）. The protein expression of HMGB 1 was decreased significantly in nucleus while was in creased significantly in cytoplasm （P＜0.01）. After SFI treatment ，the nuclear translocation and secretion of HMGB 1 were inhibited in different degrees ；compared with LPS group ，mRNA and protein expression of HMGB 1 in administration groups ，protein expression of TLR 4 in RAW 264.7 cells of positive control group ，SFI medium- and high-dose groups as well as the levels of HMGB 1，IL-1β and TNF-α in supernatant of cells in administration groups were decreased significantly （P＜0.01）. In positive control group ，SFI medium- and high-dose groups ，the protein expressions of HMGB1 in nucleus were increased significantly ，while protein expressions of HMGB 1 in cytoplasm were decreased significantly （P＜0.01）. CONCLUSIONS ：SFI may inhibit the nuclear translocation and secretion of HMGB 1 in RAW 264.7 cells，thus avoiding the activation of inflammatory pathways and the production of inflammatory factors ，so as to reduce the inflammatory response induced by LPS.

OBJECTIVE： To study the improvement and anti-inflammation mechanism of Shenfu injection on lung tissue of endotoxin shock model rats. METHODS： Totally 48 rats were randomized into control group，model group，dexamethasone group （positive control，1 mg/kg） and Shenfu injection low-dose，medium-dose and high-dose groups （5，10，15 mL/kg），with 8 rats in each group. Except for normal group， other groups were given intraperitoneal injection of lipopolysaccharide （LPS） to induce endotoxin shock model. After modeling， each group was given relevant medicine once intraperitoneally. 24 h after medication， HE staining was used to observe pathological changes of lung tissue in rats and pathological scoring was conducted. RT-PCR was used to determine mRNA levels of P65 and P50 proteins related to NF-κB signaling pathway. Western blot assay was used to determine the expression levels of P65 and P50 proteins in lung tissue， and the expression levels of P65 protein in nucleus and cytoplasm of lung tissue were also determined. The level of TNF-α in plasma in rats were determined by ELISA. RESULTS： Compared with control group， alveolar septum became thicker， obvious vascular engorgement was found， and a large number of neutrophils infiltrated the interstitium in model group. Histopathological score， mRNA and protein expression levels of P65 and P50 in lung tissues were increased significantly （P＜0.01 or P＜0.001）； the protein expression of levels P65 in nucleus and cytoplasm and level of TNF-α in plasma were increased significantly （P＜0.001）. Compared with model group， alveolar structure of rats in dexamethasone group and Shenfu injection medium-dose and high-dose groups was complete， no obvious bleeding was observed， and the degree of inflammatory cell infiltration was improved significantly. Histopathological score， mRNA and protein expression levels of P65 and P50 in lung tissue and level of TNF-α in plasma were decreased significantly （P＜0.05 or P＜0.01 or P＜0.001）. The protein expression level of P65 in nucleus and cytoplasm of lung tissue were decreased significantly in dexamethasone group and Shenfu injection low-dose and medium-dose groups were decreased significantly （P＜0.05 or P＜0.01 or P＜0.001）. CONCLUSIONS： Shenfu injection can decrease mRNA and protein expression levels of P65 and P50 in lung tissue， level of TNF-α in plasma， and protect lung tissue of endotoxin shock rats.