[Establishment and application of TaqMan Real-Time PCR in detection and serogrouping of Neisseria meningitidis].

OBJECTIVE: To establish TaqMan Real-Time PCR method for detection and identification of Neisseria meningitidis. METHODS: Seven sets of primers and FAM-labeled probes targeting different genes of Neisseria meningitidis were designed and synthesized. ctrA gene was used for identification of N. meningitidis species. Six serogruops (A, B, C, X, Y, W135) of N. meningitidis were detected with following genes: sacB (A), siaD (B), siaD (C), xcbB (X), synF (Y) and synG (W135) respectively. Sensitivity and specificity of Real-Time PCR were assessed for different primers and probes. 121 cerebrospinal fluid (CSF) specimens from suspected N. meningitidis invasive meningitis cases were detected by latex agglutination test and Real-Time PCR assay simultaneously. RESULTS: 79 N. meningitidis isolates of different serogroups could be detected and identified by seven sets of primers and probes in this study. Real-Time PCR seemed more sensitive than standard PCR by 10(1)-10(3) times. The respective sensitivities for ctrA, sacB, siaD (B), siaD (C), xcbB, synF and synG were 8, 8, 80, 8, 8, 80, 8 genome DNA copies in each reaction. Of the 121 CSF specimens, 11 were positive for Real-Time PCR and 6 for latex agglutination test. CONCLUSION: Real-Time PCR could rapidly detect and identify N. meningitidis of different serogroups and seemed more sensitive. It could be widely used for diagnose of invasive meningitis caused by N. meningitidis.

[Establishment of fluorescent amplified fragment length polymorphism in Vibrio cholerae and evaluation in molecular typing].

OBJECTIVE: To develop fluorescent amplified fragment length polymorphism (AFLP) method and to evaluate the its typing capability with pulsed-field gel electrophoresis (PFGE) in molecular typing of Vibrio cholerae. METHODS: Forty-seven strains of V. cholerae, with different PFGE patterns, were selected as the reference group to optimize the selective primers of AFLP analysis. Eighty-three strains including 20 strains from one epidemic episode, isolated from different provinces during 1961 and 2005, were used to compare the typing abilities of AFLP and PFGE. LI-COR4300 DNA sequencing system was used for AFLP electrophoresis. The images were recorded by Saga(MX) software and transferred to BioNumerics for clustering analysis. A standard protocol for V. cholerae from PulseNet was used in PFGE. RESULTS: When comparison was made with different selective primers on AFLP based on the 47 strains, results showed that the optimized selective primer pair was EcoR I-G/Mse I-T, and the reproducibility of the tests was 99.2%. Eighty-three isolates showed 52 AFLP patterns and 44 PFGE patterns, with D values as 0.9545 (AFLP) and 0.9251 (PFGE) respectively. CONCLUSION: The protocol of fluorescent AFLP on V. cholerae typing was established. AFLP was higher than PFGE in discrimination of V. cholerae which could be used for molecular typing. When combined with PFGE, AFLP became a more insightful tool to identify genome difference of different isolates.

[Sequence analysis of housekeeping genes recA, dnaE, and mdh in different serogroups or biotypes of Vibrio cholerae isolated from China].

OBJECTIVE: To analyze sequences of the housekeeping genes including recA, dnaE, and mdh in different serogroups or different biotypes of Vibrio cholerae (VC) strains isolated from China. METHODS AND RESULTS: recA, dnaE, and mdh genes of Vibrio cholerae were obtained by PCR, sequenced and analyzed. Forty-four variable bases were identified in the 500 bases of recA gene of 18 VC strains, and the mutation of only 3 variable bases could result in changes of 2 amino acids. In the 600 bases of dnaE genes of 18 strains, 32 variable bases were found and only 1 contributed to an amino acid change. In the 367 bases of mdh genes of 18 strains, only 1 variable base was identified whose mutation involved an amino acid convertion. Toxic EI Tor biotype (EVC) strains and toxic O139 serogroup strains were closely related. Non-toxic strains of different serogroups or types were lowly related. Non-toxic and toxic strains of different serogroups or types were lowly related. CONCLUSION: Toxic EVC and toxic O139 serogroup strains isolated from different areas of China may evolve from a common original strain, and toxic O139 VC strains may come from toxic EVC strains.

[Study on infection of different strains of Vibro cholerae O1 by El Tor CTXPhi].

To study the horizontal transfer efficiencies of filamentous bacteriophage CTXPhi in different V. cholera O1 strains and the phage immunities of these strains. The infectious El Tor CTXPhi particles genetic marked by chloramphenicol resistance gene were used to infect four different V. cholerae O1 strains in vivo and in vitro. Selected the infected clones based on its character of chloramphenicol resistance and identified and judged the exist form of CTXPhi genome through Southern bolt and other hybridization methods. Calculated the infection rates of different strains and compared each other. Then we analyzed the mechanism of infection and phage immunity. The infection rate of classic strain 1119 with the genetic marked CTX(ET)Phi in vivo is much higher than that in vitro. In vitro experiment, the rate of 1119 is higher than other three El Tor strains. And in El Tor strains, the infection frequency of IEM101 that had no rstR gene is 100 to 1000 times higher than other two strains containing rstR. Classical biotype strain is more susceptible to CTX(ET)Phi particles than El Tor strains. Expression of TCP and the phage immunity mediated by rstR gene affect the horizontal transfection of CTXPhi in V. cholerae strains.

[Sequence analysis of the mutated genes in CTXEVC Phi and nct-CTXnew Phi genomes in Vibrio cholerae JS94484 strain].

OBJECTIVE: To analyze the sequences of the mutated genes in CTX(EVC)Phi and nct-CTX(new)Phi genomes in Vibrio cholerae JS94484 strain. METHODS: The mutated genes in CTX(EVC)Phi and nct-CTX(new)Phi genome were obtained by PCR, sequenced and analyzed. RESULTS: ig1, rstR, ig2, and ctxAB genes in CTX(EVC)Phi genome of V. cholerae strain JS94484 were highly homologous with those of standard EVC strain N16961, while ig1, rstR and ig2 genes in nct-CTX(new)Phi genome of the strain JS94484 shared low homology with those of the other 3 biotypes of V. cholerae. Considerable difference was detected in the last 60 bp of zot genes between CTX(EVC)Phi and nct-CTX(new)Phi genomes, indicating possible difference in the amino sequences of the Zot proteins encoded by these two genes. The sequence of toxin-coregulated pilus A subunit gene (tcpA) of the strain JS94484 was identical with that of strain N16961. CONCLUSION: ig1, rstR and ig2 genes of nct-CTX(new)Phigenome are of a novel type, and their functions await further investigation.

[Functional analysis of promoters of Vibrio cholerea typing phage VP1 with reporter system].

Phage VP1 infects and lyses Vibrio cholerae. The VP1 genome is a circular double-strand DNA and its size is 32176 base pairs. Analysis of the sequence of the VP1 genome revealed the presence of 15 putative promoter sequence. The activities of these putative promoters in V. cholerae were assayed by transformation of reporter gene plasmid and phage infection together. Promoter regions were ligated into pRS1274/BamH I/EcoR I. Then transformed into E. coli JM109 and all of clone display blue. The recombinant plasmids were transformed into V. cholerae 7743 deltaZ by electroporation, then bacteriophage VP1 infect transformant. The time-course expressing lacZ gene and detecting change of beta-galactosidase enzyme activity in V. cholerae transformants at latent period, indicated P17 probably is a early promoter; P2 and P3 and P9 etc are medium-term promoters; P18 is a late promoter.

[Analysis of gene cluster of Tat-dependent protein export system of Vibrio cholerae and its function].

The Tat (Twin-arginine translocatin) system is a recently defined protein export pathway that serves to translocate folded proteins. The substrates of the Tat pathway contain specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif-S/T-R-R-x-F-L-X. Here is the report of knocked out the tatA, tatB and tatC genes of the V. cholerae by suicide plasmid homologous recombination technology. Mutant strains showed obvious changes of growth characteristics. The transport of a typical Tat pathway substrate, trimethylamine N-oxide reductase (molybdoenzyme), was completely blocked. The physiochemical reactions of the parent and mutant strains were also analyzed. Four physiochemical reactions using D-galactose, L-asparagine, glyl-L-aspartic acid and D-L-alpha-glycerol phosphate as substrates were negative in mutant strains, which might be affected by the inactivation of the Tat-dependent system.