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ObjectiveA rapid enrichment and detection method for Escherichia coli O157∶H7 was developed by using multienzyme isothermal rapid amplification （MIRA） fluorescence method combined with metal organic frameworks immunomagnetic beads. MethodsUsing rfbE gene as the target， the primers， probes and reaction system were screened， and the specificity， sensitivity and practical application of this method were investigated. ResultsThe detection limit of Escherichia coli O157∶H7 was 1.18×105 CFU‧mL-1， and the detection limit of DNA concentration was 9 pg‧μL-1. The detection process was completed in 20 minutes. The test results of 47 strains （24 target strains and 23 non-target strains） were consistent with real-time PCR （RT-PCR）. ConclusionA method based on metal-organic framework immunomagnetic beads enrichment combined with MIRA assay is developed in this study. The method is simple， rapid and suitable for rapid enrichment and detection of Escherichia coli O157∶H7 in food.
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Objective To prepare samples of proficiency testing （PT） containing live Staphylococcus aureus with drug matrix in microbial PT program of drug testing， and to evaluate the laboratory capability of microbiology tests in China. Methods Two kinds of PT samples containing living bacteria with drug matrix were prepared and evaluated. The results of laboratories participated in PT program and their response to the survey questionnaire were collected and analyzed. Results The homogeneity and stability of the PT samples complied with the requirements of CNAS-CL03： 2010. Samples could be stored stably for at least 6 months at -20 ℃. Among 63 laboratories participating in PT program， 53 laboratories （84.1%） achieved satisfactory results. The satisfaction rate was 94.1% （16/17） in 17 government laboratories （27.0% of total 63 laboratories）， 81.4% （35/43） in 43 pharmaceutical quality control laboratories （68.3% of total 63 laboratories）， and 66.7% （2/3） in 3 non-government laboratories （4.8% of total 63 laboratories）， respectively. Conclusion The government laboratories performed better than pharmaceutical quality control laboratories in microbiology tests of drug， and the testing abilities of some pharmaceutical laboratories needs to be improved. The preparation and application of microbial samples in drug matrix could provide evaluation tools for drug testing laboratories in microbiology.
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Objective To prepare samples of proficiency testing （PT） containing live Staphylococcus aureus with drug matrix in microbial PT program of drug testing， and to evaluate the laboratory capability of microbiology tests in China. Methods Two kinds of PT samples containing living bacteria with drug matrix were prepared and evaluated. The results of laboratories participated in PT program and their response to the survey questionnaire were collected and analyzed. Results The homogeneity and stability of the PT samples complied with the requirements of CNAS-CL03： 2010. Samples could be stored stably for at least 6 months at -20 ℃. Among 63 laboratories participating in PT program， 53 laboratories （84.1%） achieved satisfactory results. The satisfaction rate was 94.1% （16/17） in 17 government laboratories （27.0% of total 63 laboratories）， 81.4% （35/43） in 43 pharmaceutical quality control laboratories （68.3% of total 63 laboratories）， and 66.7% （2/3） in 3 non-government laboratories （4.8% of total 63 laboratories）， respectively. Conclusion The government laboratories performed better than pharmaceutical quality control laboratories in microbiology tests of drug， and the testing abilities of some pharmaceutical laboratories needs to be improved. The preparation and application of microbial samples in drug matrix could provide evaluation tools for drug testing laboratories in microbiology.
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OBJECTIVE：To compare the difference of microbiological limit test and criteria of TCM decoction pieces among 43 edition of United States Pharmacopeia （USP43），10.0 edition of European Pharmacopeia （EP10.0），17 edition of Japanese Pharmacopeia （JP17）and 2020 edition of Chinese Pharmacopeia （ChP2020），and to provide refernce for the revision and improvement of microbiological standards for TCM decoction pieces in China. METHODS ：The differences in the microbial enumeration tests method （including sampling and sample preparation ，selection of bacteria and culture medium ，count of microorganisms and heat-resistant bacteria ，etc.），tests for specified microorganisms （including sample pretreatment ，enrichment， separation and identification ，etc.）and microbial related limit criteria were compared among USP 43，EP10.0，JP17 and ChP 2020. RESULTS & CONCLUSIONS ：In terms of microbiological examination of TCM decoction pieces ，USP43，EP10.0，JP17 had their own independent provisions. Chp 2020 added“general rule 1108”. In terms of inspection items ，in addition to the total aerobic bacteria count and total combined yeasts and molds count ，ChP2020 and EP 10.0 provided three methods for the inspection of control bacteria （bile-resistant Gram-negative bacteria ， Escherichia coli ， Salmonella）. On the basis ， JP17 supplemented Staphylococcus aureus test；However，USP43 added Clostridium test method and put forward the concept of objectionable microorganisms risk assessment ；ChP2020 also added a new method for counting heat-resistant bacteria. In terms of microbial limit criteria，USP43 was the most detailed in the classification of TCM decoction pieces ，which was more strict than EP 10.0 and JP 17； ChP2020 had not set up a unified limit for the inspection of control bacteria of TCM decoction pieces. ChP 2020 revised the “microbial limit standard for TCM extracts and TCM decoction pieces ”，but it was not perfect compared with the Pharmacopoeia of the United States ，Europe and Japan. It is suggested that according to the current situation of microbial contamination and control of TCM decoction pieces ，the microbial limit test and criteria of TCM related products in Pharmacopoeia should be gradually improved ，and the microbial limit level of corresponding products should be reasonably refined.
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Objective To investigate the efficacy of endovascular stenting for aortic arch artery stenosis after nasopharyngeal carcinoma radiotherapy. Methods The clinical data of 8 patients with symptomatic severe aortic arch artery stenosis after nasopharyngeal carcinoma radiotherapy were analyzed retrospectively. The patients were all received endovascular stenting,and their improvement of cerebral ischemic symptoms was observed. They were followed up by cervical color Doppler ultrasound.Results The whole brain vascular DSA confirmed that there were 24 severe arterial stenoses on the aortic arch arteries of extracranial segments in 8 patients,including 11 in internal carotid artery,2 in common carotid artery,10 in vertebral artery and 1 in subclavian artery. The patients were treated with vascular angioplasty and stenting respectively. All the patients were followed up for 1 year;there were no recurrence of cerebral ischemic symptoms.Cervical color Doppler ultrasound did not reveal any obvious restenosis. Conclusion Endovascular stent angioplasty for the treatment of aortic arch artery stenosis after nasopharyngeal carcinoma radiotherapy is relatively safe and feasible.
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Objective To investigate the changes of circulating endothelial progenitor cells (EPCs) in patients with acute cerebral infarction or chronic cerebral ischemia and discuss the related clinical significance.Methods Circulating EPCs were isolated using staining markers of CD34,CD133,and kinase insert domain receptor (KDR).Peripheral venous blood was collected from patients with acute cerebral infarction within 24 hours of onset (infarction group,n =30),with chronic cerebral ischemia (ischemia group,n =20),and without cerebral ischemia (control group,n =10) to quantify circulating level of EPCs using flow cytometry and measure parameters of systolic pressure,glycosylated hemoglobin (HbAlc),total cholesterol (TC),and triglyceride (TG),and low density lipoprotein-cholesterol (LDL-C),and high density lipoprotein-cholesterol (HDL-C).Results CD34-,CD34/CD133-,and CD34/KDR-positive cells counted (14.2 ± 8.1)‰,(7.1 ± 4.1)‰ and (5.0 ± 3.7)‰ in infarction group,(28.5 ± 9.9)‰,(15.2 ± 3.7)‰ and (6.8 ± 2.0)‰ in ischemia group,and (44.8 ± 9.5) ‰,(22.1 ± 6.6) ‰ and (16.7 ± 6.9) ‰ in control group.Taken together,circulating level of EPCs lowered substantially in infarction and ischemia groups compared to control group (P ＜ 0.05) and a far lower level was observed in infarction group (P ＜ 0.05).Circulating level of EPCs in infarction group was in a moderate negative correlation with systolic pressure,TC,TG,and LDL-C (P ＜ 0.05).Conclusions Decreased circulating level of EPCs may be a risk factor to the development of cerebral ischemia in acute cerebral infarction patients.Therefore,level of EPCs is vital for prediction,prevention and treatment of acute cerebral infarction.
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The aim of this study was to establish a fast and accurate method for developing specific DNA sequences and PCR primers for the detection of Staphylococcus aureus. An automatic C++ program for genomic comparison was used to identify specific DNA sequences from the genome of S. aureus MRSA 252. Four primer pairs were obtained from 9 specific target sequences by comparison of 2656 coding sequences with our local genome database, and 2 pairs of primers were confirmed to be specific to S. aureus by PCR evaluation against 137 bacterial strains, including 11 species of Staphylococcus. Furthermore, the DNA detection sensitivity of primer SA3 was 13.7 fg/microL and the cell sensitivity for this primer was 9.25 x 10(2) CFU/mL. This method has overcome the limitations of specific target mining in conventional assays, and it could be easily and widely used for other foodborne pathogens.