Objective To understand the infection status and distribution characteristics of human papillomavirus (HPV)in fe-male genital tract in Fengxian district of Shanghai City in order to provide preliminary recommendations for prevention and treat-ment of HPV in this area.Methods The women aged over 1 6 years old receiving HPV testing in the Southern branch Hospital of Shanghai Sixth People′s Hospital From January 2011 to December 2015 were taken as the research subjects and their cervical secre-tions were performed the HPV 21 subtypes detection,among them,1 374 cases underwent colposcopic fixed point biopsy of cervix for determining the pathological grade.Then the data were recorded and analyzed by the SPSS20.0 software.Results Twenty-one HPV subtypes were detected,in which the HPV positive rate was 17.54% (6 313/35 977),and types with high HPV infection rate in these five years were in turn HPV18,HPV52,HPV16,HPVCP8304,HPV58,HPV53,HPV53,HPV53 and HPV58;the HPV positive rate in 1374 cases of cervical lesions was 82.75% (237/1 374),the common types distributions in cervical lesions were HPV16,HPV58,HPV52,HPV18 and HPV31 respectively.Conclusion The HPV infection situation in Fengxian district of Shang-hai City has certain regional characteristics.Then the HPV prevention and treatment strategy suitable for this region may be formu-lated according to the research results.

Objective To establish a system for detecting integration frequency of antibiotic resist-ante integron.Methods We cloned integron and aadA2 gene cassette into different sites of plasmid pACYC 184,and the plasmid was transformed into E.coli BL21(DE3)containing plasmid overexpressing integrase.The positive clone was cultured overnight and then was spread on LB agar plate with or without streptomycin respectively,and with appropriate amount of bacteria.Clones after cultured overnight were counted to detect the integration frequency.Meanwhile we used positive clones in LB agar plate containing streptomycin as templates to carry out PCR.The purified PCR products were sequenced to identify the integration sites.Re-suits The integration frequency of integron capturing aadA2 gene cassette in BL21(DE3) host was 1.1 x 10-3 mainly at attI site.Conclusion This system can be used to detect integration frequency.

Objective To establish a high resolution melting based method for the rapid identifica-tion of functional class 2 integron.Methods Nighty-nine non-repetitive Proteus spp.strains positive for genes encoding class 2 integrase were isolated from August, 2011 to August, 2012.The genomic DNAs were extracted and used as templates to amplify 60 base pair fragments containing the mutated point in class 2 in-tegrase gene by PCR.The high resolution melting analysis was conducted to identify the functional class 2 in-tegrons that were further compared by sequence analysis.Results There were remarkable differences with the high resolution melting curves between the functional class 2 integrons and the ordinary class 2 integrons. The results of high resolution melting analysis were consistent with those by using sequence analysis.Con-clusion High resolution melting analysis could be used for the rapid and accurate identification of functional class 2 integron.

Objective To develop a simple high-resolution melting ( HRM) analysis method for differentiation of Pc and P2 variants in class 1 integron.Methods DNA fragments containing Pc and P2 variants were amplified from plasmids pACW ( PcW ) and pACWP2 ( PcW-P2 ) respectively , then these purified PCR products and P 2 promoters were analyed full-length amplicon by HRM .Eight DNA fragments containing different Pc promoters were amplified and site-specific mutated from plasmids pACS ( PcS ) , pACH2 ( PcH2 ) , pACH1 ( PcH1 ) , pACW ( PcW ) , genomic DNA of Klebsiellar pneumonia HS07-68 (PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively.The purified PCR products and eight Pc variants were characterized by HRM analyses of an unlabeled probe and full-length amplicon.This assay was applied to the differentiate Pc and P2 variants in 109 class 1 integrons from 95 urine clinical Escherichia coli isolates in Huashan Hospital during 2004 -2007.The differentiation results were compared with that determined by direct sequencing .Results P2 promoter with a significant higher melting temperature ( Tm ) can be identified by HRM analysis clearly .P2 promoters were identified in 2 class 1 integrons and consistent with direct sequencing results .Eight Pc variants were classified into three groups: PcS, PcSTGN-10 , PcW, PcWTGN-10, PcH1, PcH1TGN-10.Using direct HRM analysis.PcH2, PcH2TGN-10 were classified into four groups:PcS, PcH1, PcH2, PcW, PcSTGN-10 , PcH1TGN-10 , PcH2TGN-10 , PcWTGN-10 according to the melting curves of the unlabeled probe .Combined the HRM analyses of the whole amplicon and unlabeled probe , the eight Pc variants can be differentiated from each other .Five different Pc variants, PcS, PcW, PcH1, PcH2TGN-10 and PcWTGN-10 , were identified and consistent with direct sequencing results .Conclusions This developed a simple Pc and P 2 variants differentiation method via simultaneous HRM analyses of an unlabeled probe and full-length amplicon .This method is cost-effective and accurate , could be used in differentiation of Pc and P2 variants of class 1 integrons in clinical isolates .

ObjectiveTo determine whether aadA2 gene can be translated from the ATG triplet,which there was no plausible ribosome binding site preceding it,and synthetized a functional protein in class 1 integron.MethodsSite-specific mutagenesis was used to construct aadA2 gene cassette with different start codons,together with their upstreamed promoters of variable regions were cloned into plasmid pACYC184 respective.The constructed plasmids were then transfored into Escherichia coli JM109,Western blot was used to detect the translation products of aadA2 gene with different start codons.Broth microdilution method was used to detect the minimal inhibitory concentrations to streptomycin in Escherichia coli JM109 containing aadA2 gene with different start codons.ResultsaadA2 gene can initiate translation from both ATG and GTG triplets in aminoacyl -3-adenylyltransferase protein synthesis,though there was no plausible ribosome binding site preceding the ATG triplet.Besides GTG and ATG triplets,there was other start codon downstream of the GTG triplet in aadA2 gene.The translated products that initiated from the start codons that described above were all functional AAD(3) proteins that can be detected by anti- aminoacyl -3-adenylyltransferase polyclonal antisera in Western blot and conferred different resistance levels to streptomycin in E.coli.ConclusionWhen inserted as the first gene cassette in class 1 integron,aadA2 gene can initiate translation from ATG triplet and synthetized a functional protein,though there was no plausible ribosome binding site preceding it.This structural characterization of class 1 integron can initiate translation of the open reading frame harbored in gene cassette that integrated into class 1 integron,though there was no plausible RBS preceding the start codon.This make class 1 integron be more convenience to express the genes that capture from environment.

ObjectiveTo prepare antiserum specific to aminoacy1-3″-adenylyltransferase [ AAD (3″) ],and to explore the application value of the prepared antiserum in detecting the expression levels of aadA2 gene that downstream of 8 different promoters (PcS,PcH2,PcH1,PcW,PcS-P2,PcH2-P2,PcH1P2 and PcW-P2 ) of variable regions in class 1 integron.MethodsaadA2 gene was amplified by polymerase chain reaction(PCR) and cloned into the expression plasmid pET19b.After inducing,the recombined aminoacy1-3″-adenylyltransferase[ AAD(3″)] with His-tag was expressed,purified and immunized rabbits to get anti- AAD(3″) specific serum.The prepared antiserum was used to detect the translation levels of aadA2 gene that downstream of different promoters of variable regions in class 1 integron by Western blotting (WB).Broth microdilution method was used to detect the minimal inhibitory concentrations (MIC) to streptomycin in Escherichia coli JM109 with aadA2 gene downstream of different promoters of variable regions.ResultsRecombined AAD (3″) expression plasmid pET19b-aadA2 was constructed successfully and was verified by sequence analysis.After transformed into E.coli BL21 ( DE3 ),a resoluble recombined AAD(3″) high expression strain was obtained.After fermentation,recombined AAD(3″) was purified and immunized rabbits.The anti- AAD(3″) specific serum was obtained with titer ＞ 1∶100 000.WB was used to detect the expression levels of AAD (3″),the translation product of aadA2 gene,that downstream of 8 different promoters of variable regions.The relative expression level of AAD (3″) that downstream of PcW was assumed to be 1,then the relative expression levels of AAD(3″),which all were detected 3 times independently,that downstream of PcS,PcH2,PcH1,PcS-P2,PcH2-P2,PcH1-P2 and PcW-P2 were 12.9±2.3,9.1±1.0,2.0±0.4,16.0±1.3,14.1 ±1.3,10.5±0.7 and 8.9 ±1.7 respective.Very different expression levels of AAD (3″) that downstream of different promoters of variable regions were obtained( F =32.421,P ＜ 0.01 ).The mean values of MIC,which all were detected 3 times independently,to streptomycin in E.coli JM109 with aadA2 gene downstream of PcS,PcH2,PcH1,PcW,PcS-P2,PcH2P2,PcH1-P2 and PcW-P2 were 256,256,64,128,32,128,4 and 64 mg/L respective.These results indicated the different expression levels of aadA2 gene that downstream of different promoters of variable regions can confer their host bacteria different resistance levels to streptomycin.Conclusions Resoluble recombined AAD(3″) is purified successfully and high titer anti- AAD(3″) specific antiserum is obtained from the immunized rabbits.This laid foundation for further investigation on the correlationship between the expressions of intI1 gene and the gene cassettes within variable regions.The expression levels of antibiotic gene cassettes that downstream of different promoters of variable regions are very different,so are the very different antibiotic resistance levels of the host bacteria.Therefore more attentions should be paid to the researches on the classification of promoters of variable regions when molecular epidemiology studies on the class 1 integrons in clinical isolates were conducted.

Objective To understand the epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae occurred between 2006 and 2009 in a university hospital of Shanghai, China. Methods A total of 57 panresistance K. pneumoniae isolates were collected from August 2006 to December 2009.Antibiotic susceptibility of the isolates were determined by Kirby-Bauer disc diffusion method and microbroth dilution (MBD). ESBLs-producing initial screen test and phenotypic confirmatory test and carbapenemase-producing modified Hodge test ( MHT) were performed to detect the resistance phenotype of the isolates. Be-ta-lactamases were studied by IEF, PCR and the product sequencing. While conjugation assay were conducted to understand the transferability of these genes. The genetic relationship between isolates was established by ERIC-PCR and multilocus sequence typing (MLST). Except for the antibiotics recommended by CLSI guideline in the routine test, the other antibiotics were added to find out the effective drugs to treat the infection. Results All 57 isolates were highly resistant to all examined antibiotics. All isolates produced ESBLs and carbapenemase. IEF revealed that each isolate produced four beta-lactamases. All isolates carried blaKPC-2,blaCTX-M-14,blaSHV12,blaTEM-1,qnrB and aac(6') - I b-cr. Forty-four of the 57 (77.2% ) isolates were successful to transfer their resistance genes to E. coli recipient J53 by conjugation assay. By RAPD, all 57 isolates were grouped into two genotypes that were further identified as members of MUST types 423 and 11.Sequence types 423(ST423) only occurred before May 2008 and ST11 occurred (52 isolates) after May 2008. Most of isolates of the outbreak were ST11 (91. 2% ). A part of isolates were susceptive to added antibiotics. Conclusion The outbreak of panresistance K.pneumoniae was caused by those isolates which carried multiple resistant genes. There is a different ability of dissemination between different ST types K. pneumoniae isolate. It was necessary to add the antibiotics to find out the effective drugs to treat the infection.

Objective To establish a new method, applying high resolution melt, to discriminate the VEB-3 hypotype from the clinical gram negative isolates. Methods From January to December 2003,292 consecutive and non-repetitive gram-negative bacteria producing VEB extended spectrum β-lactamase (ESBL) were collected. Extract the DNA of clinical gram negative isolates with phenol-chloroform. PCR was performed to amplify the VEB gene with the DNA being template. After that, we amplify the fragment of VEB gene containing the position 168. Then we detect the high resolution melt curve and analyze them. At last, we analyze the results of sequence and high resolution melt( HRM ). Results VEB-1 and VEB-3 gene are markedly different through HRM analysis. Conclusion It is accurately and quickly for us to identify the VEB-3 from other hypotype through the technology of HRM.

Objective To investigate the prevalence of VRE in Huashan hospital of Shanghai from 2007 to 2009, and to examine the molecular characteristics of the VRE isolates.Methods A total of 890 non-repetive clinical isolates of Enterococcus were screened by the agar screening method ( ADSP method).Broth dilution susceptibility test was performed to determine the antimicrobial susceptibility of Enterococcus isolates to vancomycin and teicoplanin.The resistant genes and virulent genes of VRE isolates were investigated by PCR and sequencing methods.VRE isolates were classified by MLST and six isolates of VRE from 2007 to 2008 were analyzed by PFGE.Results Thirteen VRE isolates were identified by ADSP method and broth dilution susceptibility test. Six of them were resistant to vancomycin but sensitive to teicoplanin ( vancomycin MICs were from 64 to 256 μg/ml).The sequencing data of PCR products indicated these isolates might harbor a potential novel vancomycin resistant gene, which was different from the one reported in previous studies. The rest 7 isolates harbored vanA gene. The MICs of these isolates to vancomycin and teicoplanin were 32 - 64 μg/ml and 16 - 32 μg/ml, respectively.MLST results revealed 4 STs were identified in 13 VRE isolates.Eleven isolates belonged to clonal complexes(CC) 17.The positive rates of esp gene and hyl gene were 69.2% and 30.8%, respectively.Conclusions This study suggests that the most common VRE clone in Huashan Hospital was CC17.A potentially novel vancomycin resistance gene was identified, and further work needs to be done to investgate the function and the location of this novel gene.

Objective To detect quantitatively AKAP12 methylation and evaluate its clinical significance in peripheral blood in colorectal cancer. Methods MS-HRM technology was used to detect quantitatively AKAP12 methylation in peripheral blood from 80 colorectal cancer patients and 20 healthy volunteers. They also validated the reproducibility and compared with MSP. Results Thirty-eight of the 80 colorectal cancer samples (47. 5% ) were found to be methylated at the AKAP12 promoter region by MS-HRM (the methylation levels of 24 cancer samples ranged between 1 % and 20% , the methylation levels of 12 cancer samples ranged between 20% and 60% , the methylation levels of 2 cancer samples ranged between 60% and 100% ). The methylation levels of 2 health samples were less than 10% . They also compared the results generated by MS-HRM with a traditional MSP assay. The AKAP12 MS-HRM assay was able to reproducibly detect 1% AKAP12 methylated DNA, whereas the MSP method was unable to detect less than 10% methylation. No significant correlation was observed between the AKAP12 methylation levels and patients' age and gender. However, AKAP12 methylation was significantly higher in DNA from colorectal cancer patients with high Dukes stage and differentiation (x2 =5. 93 or 8. 41, P = 0.01). Conclusions The authors demonstrate here for the first time, the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods have many promising applications in the detection of colorectal cancer.