Objective To analyze and summarize the experiences and problems in diagnosis of anthrax in outbreaks, and to provide a basis for anthrax prevention and control. Methods Lesion exudates were collected from suspected anthrax patients using slides and sterile cotton swabs and venous blood samples were also collected from suspected cases. Smear microscopy, direct culture, and broth enrichment were used to isolate and culture Bacillus anthracis. Real-time PCR was used to amplify the genes such as pagA in the virulence plasmid, rpoB in the chromosome, and cap in the capsule plasmid. Enzyme-linked immunosorbent assay (ELISA) was used to detect double serum anthrax antibodies. Results A total of 17 specimens were collected in the laboratory of the Center for Disease Control and Prevention of Tongliao, Inner Mongolia, China, in August 2018. The time window between disease outbreak and sample collection was 1 day at least and 16 days at most, with a mean of 6 days. In the 17 suspected patients, 15 (88.24%) received antibiotic treatment before sample collection. Bacillus anthracis was isolated from 1 specimen, yielding a positive rate of 5.88% (1/17). The real-time PCR analysis of all the specimens gave a positive rate of 41.18% (7/17). The results of ELISA showed a positive rate of 83.33% (10/12) for the protective antibody. Conclusion The optimal time to collect samples is the key to the early identification of outbreak. Real-time PCR, which is a fast and sensitive approach for diagnosis, is recommended for application in anthrax outbreak. Double serum specimens should be collected from all the suspected patients to increase the diagnosis rate.

Objective To acquire the purified recombinant lethal factor (rLF) of Bacillus anthracis and investigate its use in the rapid diagnosis of anthrax and in the study of the mechanism of the disease. Methods The lethal factor gene was cloned into the Escherichia coli expression vector pET-30a(+) and expressed as a fusion protein in E. coli BL21(DE3). rLF was purification of rLF by Ni-NTA affinity chromatography. The purified rLF was subjected to immunogenicity and biological activity analysis by Western blot assay and MTT assay. Results rLF was expressed and purified. Western blot results suggested that recombinant proteins reacted specifically with anti-B. anthracis serum. The cytotoxicity analysis showed the rLF was fully functional. And rLF combined with PA could significantly decrease cytotoxicity compared with previous study. Conclusion rLF protein has tremendous potential both for the diagnosis of B. anthracis infection and for the study of the mechanism of anthrax toxin.

　　【摘要】　基因预测一般指预测DNA序列中编码蛋白质的部分。其方法主要有两大类：一类是基于相似性的预测方法，即利用已知的mRNA或蛋白质序列为线索在DNA序列中搜寻所对应的片段，达到基因预测的目的；另一类是基于统计学模型的从头预测方法，即利用统计学模型训练出相应参数，再对基因进行预测，这种方法可不依赖已知的DNA序列进行预测。现就基因预测的方法、基因预测中存在的一些问题等做一概述。

Objective To evaluate the allele specific polymerase chain reaction(AS-PCR) method applied to screen point mutagenesis in lef gene of Bacillus anthracis.Methods AS-PCR amplification.Results The false positive rate of AS-PCR was high,its accuracy was not correlative to the base type at the 3′ end of primers,and was correlative to annealing temperature and template concentration.The Taq DNA polymerase with proofreading activity couldn't improve the false positive.Conclusion AS-PCR amplification could be affected by many factors and is not adapt to be a screen method for point mutagenesis.