Objective By the method of multiple polymerase chain reaction (PCR),we intend to amplify different regions (DFR) of Yersinia pestis DNA,and to establish a multiple DFR genotyping technique for detection of Yersinia pestis.Methods According to the product size of 23 DFRs and pMT plasmid,24 primers were optimized and combined,then multiple primers in one PCR reaction system were added,and positive template DNA was amplified.Meanwhile,200 wild strain DNAs were amplified by multiple PCR and normal PCR,to verify the coincidence rate of the two methods.Results Totally 24 target segments were amplified through the positive DNA template.Through different permutation and combination,24 primers were optimized and combined into 9 groups.Totally 200 wild strain DNAs were used for verification,the coincidence rate of multiple PCR and normal PCR was 100％.Conclusions Multiple PCR is applicable and feasible for DFR genotyping of Yersinia pestis.It is an efficient,economic and high accuracy experimental method for large quantities of Yersinia pestis DFR genotyping.

Objective To evaluate the value of immunophenotype in diagnosis of myelodysplastic syndrome (MDS).Methods The immunophenotype of bone marrow cells in 27 patients with MDS were detected by monoclonal antibody by flow cytometry.Results As the progression of the disease,CD34 positive cells gradually increased:refractory anemia/ring sideroblasts refractory anemia (RA/ AS) 7.43 %,refractory anemia with excess of blasts (RAEB) 36.81%,refractory anemia with excess of blasts transformed (RAEB-T)56.45 %,and the differences were statistically significant (P ＜0.05); the expressions of CD33+,CD13 and HLA-DR increased gradually,the expressions of CD14 and CD15 antigens gradually decreased,the difference of three groups was statistically significant (P ＜0.05),the differences between RA/RAS and RAEB-T,RAEB and RAEB-T were statistically significant (P ＜0.05); the expression of CD19 and CD10 decreased and the expression of CD7 increased (RA/RAS 2.63 %,RAEB 10.79 % and RAEB-T 11.00 %) with the progression of the disease,the difference of three groups was statistically significant (F =10.439,P ＜0.05),the differences between RA/RAS and RAEB,RA/RAS and RAEB-T were statistically significant (P ＜0.05).Conclusion The detection of immunophenotype of bone marrow cella in patients with MDS contributes to the diagnosis,classification and prognosis of MDS.

Objective To validate the determination method of microbacteria limit of Shuitiaosan powder .Methods Plate counting method was used .The method of counting bacteria and mould was validated by the recovery rates with 5 control strains .The method of checking control bacteria was validated by observing cultivation of Staphylococcus aureus and Pseudomonas aeruginosa in the test group, positive control group and negative control group in the same environment .Results The recovery rate of every trail strains was higher than 70%when centrifugal sedimentation methods were used in the counting bacteria and mould .To the examination of con-trol bacteria , Staphylococcus aureus and Pseudomonas aeruginosa were detected by the centrifugal sedimentation methods .The tested strains were observed in the test group and in the positive control group .No strains were observed in the negative control group .Con-clusion The methods are simple , feasible, reliable and can be used for the examination of microbacteria limit .

Abstract: Objective To understand the phenotypic and genetic characteristics of Yersinia pestis strains isolated from Himalayan marmot natural focus area and domestic rat plague focus area in southern China, and provide reference for mastering the pathogenic characteristics of Yersinia pestis of two plague foci. Methods A total of 412 of Yersinia pestis strains isolated from Himalayan marmot plague focus and domestic rat plague focus of southern China were subjected to to sorbitol fermentation assays, virulence factor, different region (DFR) typing, and clustered regularly interspaced palindromic repeats (CRISPR) typing. Results The biochemical types of Y. pestis from the two plague foci showed distinct regional distribution features. Five biochemical phenotypes were identified in Yersinia pestis isolated from Himalayan marmot natural focus area, while only one biochemical phenotype was identified in strains isolated from the domestic rat plague focus of Southern China. Most of the Yersinia pestis isolated from the two plague foci were capable of producing the virulence factors of Fl and PstI. Among the strains from Himalayan marmot focus, 70.53% (201/285) were VW-positive, 75.09% (214/285) were Pgm-positive, 20.00% (57/285) of the strains were Pgm-negative, and 5.26% (15/285) were Pgm mixed-type strains. Among strains from domestic rat plague focus of southern China, 37.80% (48/127) were VW-positive, 29.13% (37/127) were Pgm-positive, 58.27% (74/127) were Pgm-negative, and 12.60% (16/127) were Pgm mixed-type strains. DFR typing revealed 22 genotypes of Y. pestis from the Himalayan marmot plague focus, with the main genotypes being type 5, 7, 8, 10, 19, 32 and 49. All strains from domestic rat plague focus area in southern China belonged to type 9. CRISPR typing revealed that all strains from the Himalayan marmot natural focus were classified into 7 CRISPR gene clusters and 14 CRISPR genotypes, with the main genotypes being G7, G22, G26-a1'and G22-A1'. All strains from domestic rat plague focus area in southern China belonged to CRISPR genotype G30, with the gene cluster being Ca8. Conclusions The phenotypes and genotypes of the Yersinia pestis of Himalayan marmot plague focus are diverse, with an obvious characteristics of geographical distribution. The phenotype and genotype of the Yersinia pestis of domestic rat plague focus of Southern China are single. DFR and CRISPR genotyping methods with phenotypic characteristics can effectively identify the Yersinia pestis isolated from the two plague foci, thereby meeting the needs of identification and traceability research.

Objective:To understand whether there are drug resistant and disinfectant resistant Yersinia pestis strains in China, and to provide accurate information for clinical treatment of plague. Methods:A total of 2 753 Yersinia pestis strains isolated from 10 natural plague foci in China from 1943 to 2016 were collected. According to National Center for Biotechnology Information (NCBI) released sequences of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1, a pair of primers of each gene was designed for above-mentioned genes. Genomic DNA of 2 753 strains of Yersinia pestis was extracted, and the 9 target genes of all DNA samples were amplified by PCR. Results:Negative and positive controls of PCR detection were established. No corresponding target bands of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1 were found in the DNA samples of 2 753 strains of Yersinia pestis.Conclusion:The above-mentioned genes of drug resistance and disinfectant resistance have not been detected in Yersinia pestis of China, but the monitoring of drug resistance of Yersinia pestis still needs to be carried out continuously.

Objective:To investigate the drug resistance of Yersinia pestis to 11 kinds of antibiotics in the natural foci of plague in Inner Mongolia Autonomous Region, and to provide a theoretical basis for scientifically and effectively selecting antibiotics for treatment of the plague. Methods:A total of 137 strains of Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region at different times, regions, hosts and vectors were collected. According to Clinical and Laboratory Standard Institute (CLSI), the agar plate dilution method was used to determine the minimum inhibitory concentration (MIC) of the 11 kinds of antibiotics against 137 strains of Yersinia pestis, including ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. The MIC 50 and MIC 90 (the minimum concentration of drug which could inhibit 50% and 90% of bacterial growth) were calculated, and their sensitivity was determined according to CLSI standards. Results:Among 137 strains of Yersinia pestis tested, no strains of Yersinia pestis had single or multiple resistance to ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. According to CLSI standards, 137 strains of Yersinia pestis were all sensitive to the 11 kinds of antibiotics; among them, ofloxacin, ciprofloxacin, ceftriaxone and sulfamethoxazole-trimethoprim had higher antibacterial activity, with MIC 90 < 0.250 μg/ ml; the antibacterial activity of spectinomycin was the lowest, with MIC 90 of 16.000 μg/ml. Conclusions:The Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region is not found to have single or multiple resistance to the 11 kinds of antibiotics. Continuous drug resistance monitoring of Yersinia pestis should be carried out to provide a basis for clinical medication.

Objective To purify native F1 antigen from E pestis EV76 strain and determine its ef-ficacy against Y. pestis. Methods A new purification method was developed by the substitution of physical disruption ( glass beads) for organic solvent ( acetone and toluene) one, followed by a combination of ammo-nium sulfate fractionation and SephacrylS-200HR column filtration chromatography. Groups of mice were im-munized with F1 antigen adsorbed to 25% aluminum hydroxide in PBS by intramuscular route. The immu-nized animals were challenged subeutaneously(s, c. ) with 104 CFU of Y. pestis strain 141 at 18 weeks after the primary immunization. Results There was no IgG titre difference between two groups of mice with one-dose immunization, whereas in the two-dose immunization groups, the group F1-40 μg induced a statistically higher antibody titre than the group F1-20 μg. Complete protection was observed for animals immunized with purified F1 antigen by s.c. route. In contrast, the control mice immunized with aluminum hydroxide suc-cumbed to a same dose of Y. pestis 141 challenge. Conclusion This purification strategy is a simple and ef-fective, and can be operated in a large scale. Native F1 antigen extracted from Y. pestis EV76 is highly im-munagenic, and can be used as a key antigen component to develop sub-unit vaccine of plague.

Objective To investigate the biological characteristics and epidemiological significance of Yersinia pestis strains in Qilian County,Qinghai Province,in order to provide a scientific basis for plague prevention and control.Method Totally 67 strains were separated from kinds of host in Qilian County,Qinghai Province from 1958 to 2011,to do biochemical test,toxicity test,virulence factors evaluation,plasmid analysis and different region (DFR) genotyping.Results According to biochemical typing,48 of the 50 strains tested were Qing-Tibet Plateau ecotype,15 were Qilian Mountain ecotype,and the remaining 4 were different ecotypes from the plague foci in Qinghai plateau.The strains had 8 genomovars,and were given priority to genomovar8 (42 strains),secondly,genomovar44 (15 strains),genomovar5 (4 strains),genomovar7 (2 strains),genomovar19 (1 strain),genomovar30 (1 strain),genomovar32 (1 strain),and genomovar34 (1 strain).A proportion of 95.52％ (64/67) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 52 × 106;85.07％ (57/67) contained all the four virulence factors,and 96.00％ (48/50) were velogenic strains.Conclusion The strains separated in Qilian County,Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity,so we should enhance the plague monitoring and give more publicity to plague prevention to prevent animal plague spreading to human.

Objective To understand the epidemic trend of Tibetan sheep plague in Guoluo Prefecture,Qinghai Province,we detected the plague F1 antibody in Tibetan sheep serum in this area.Methods Indirect hemagglutination test (IHA) and colloidal gold immunochromatography (GICA) were applied to test serum samples of Tibetan sheep which were separated from 5 ml whole blood drew from jugular vein in Maqin County,Maduo County,Gande County,Banma County,Jiuzhi County and Dari County in 2014 and 2015.Results We collected 1 481 serum samples,566 from Maqin County,315 from Maduo County,150 from Gande County,150 from Banma County,150 from Jiuzhi County and 150 from Dari County.Totally 14 serum samples showed F1 antibody positive,the positive rate was 0.95％ (14/1 481),and they were all from Maqin County.Conclusions This area has the prevalence of Tibetan sheep plague.Therefore,the monitoring work of Tibetan sheep plague should be strengthened.

Objective To study the biological characteristics and epidemiological significance of Yersinia pestis in Chengduo County of Qinghai Province,in order to provide scientific basis for plague prevention and control in this area.Methods Thirty one strains of Yersinia pestis isolated from Chengduo County of Qinghai Province from 1980 to 2011 were selected as study subjects.Biochemical test,virulence factors evaluation [Fra1 (F1),pesticin Ⅱ (Pst Ⅱ),virulence antigen (VW),pigmentation (Pgm)] and different region (DFR) genotyping were carried out.Nineteen of the 31 strains Yersinia pestis were selected according to different time,different areas and different hosts to determine their toxicity in mice,MLD ≤ 10 000 was strong toxic strain,10 000 ＜ MLD ＜ 100 000 was moderate toxic strain.Results Among thirty one strains of Yersinia pestis,23 strains were isolated from human,the Himalaya marmot and its fleas and lice,and their biological type was classical,biochemical type was Qinghai-Tibet plateau;21 strains genotype was type 5,1 was type 16,1 was type 32,and they contained all four kinds of virulence factors (F1,Pgm,Pst Ⅱ,VW),and toxicity test showed all strains (14) were strong toxic strains.The rest 8 strains of Yersinia pestis isolated from the Microtus fuscus and its fleas,and their biological type was Microtus,biochemical type was Chuanqing plateau;they could produce F1 and Pgm,of which 87.5％ (7/8) strains could produce Pst Ⅱ,but could not produce VW antigen factor,the genotype was 14,and the toxicity results showed that they were strong (3)and moderate (2) toxic strains.Conclusion The strains separated in Chengduo County of Qinghai Province from 1980 to 2011 have the pathogen characteristics of Qinghai-Tibet plateau plague,they are mainly strong toxic strains;the work on prevention and control of plague should not be neglected.