A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.
Bacterial Proteins/*genetics ; DNA Fragmentation ; DNA, Bacterial/genetics ; Mutagenesis, Site-Directed ; Neisseria gonorrhoeae/*genetics ; Neisseria gonorrhoeae/metabolism ; Recombination, Genetic ; Repressor Proteins/*genetics ; Sequence Deletion ; Transfection ; Transformation, Bacterial

OBJECTIVETo set up random amplified polymorphic DNAs (RAPD) method in genotyping Neisseria gonorrhoeae on DNA level, and to explore its use to trace the source of infection.

METHODSFour different pretreatments were used to extract the Neisseria gonorrhoeae genomic DNA with its advantages and disadvantages compared. Arbitrary sequence was then used to amplify the genomic DNA of Neisseria gonorrhoeae and RAPD fingerprint maps was applied to distinct the Neisseria gonorrhoeae strains. Finally, RAPD fingerprint of Neisseria gonorrhoeae strain between patient and his/her sexual partner was compared.

RESULTSCetyltrimethylammonium bromide method was classical in extracting genomic DNA, and could get integrated genomic DNA and good fingerprint maps, since main segments were common to all the Neisseria gonorrhoeae but some were different among strains so that the fingerprint of different Neisseria gonorrhoeae were distinctive. However, fingerprint maps of Neisseria gonorrhoeae collected from sex partners were quite similar.

CONCLUSIONBased on genomic levels, effective fingerprint maps could be identified and to classify the Neisseria gonorrhoeae into different genotypes. RAPD fingerprint maps could be used to trace the source of infection.

DNA Fingerprinting ; DNA, Bacterial ; analysis ; Genotype ; Humans ; Neisseria gonorrhoeae ; classification ; genetics ; Random Amplified Polymorphic DNA Technique

OBJECTIVEIn order to study the epidemiology of TEM-1 genes of Neisseria gonorrhoeae (Ng) in Wuxi area.

METHODSIn the light from foreign literature that positive Ng strains of beta-lactamase contain plasmid sequences of TEM-1 genes, heminested PCR for TME-1 genes of Ng detection was self-designed. Ng of 195 strains were detected, that were isolated in Wuxi area from Jan to the Oct, 2002.

RESULTSThere were 138 TEM-1 genes positive in 195 isolated strains with a positive rate of 70.8%. There was one case of heminested PCR product of TEM-1 genes which showed the homogeneity was 99% in Wuxi area, when comparing with pFA7 sequences of positive Ng plasmid of beta-lactase from register GenBank.

CONCLUSIONData showed that the Ng strains with TEM-1 genes were the prevalent ones in Wuxi area.

China ; epidemiology ; Drug Resistance, Bacterial ; genetics ; Gonorrhea ; epidemiology ; microbiology ; Humans ; Neisseria gonorrhoeae ; enzymology ; genetics ; Polymerase Chain Reaction ; beta-Lactamases ; genetics

OBJECTIVE: To investigate the value of clinical application of simultaneous amplification and testing of RNA (SAT-RNA) for detecting Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) by comparing with the polymerase chain reaction testing of DNA (PCR-DNA) method. METHODS: Specimens from both urethra swab and the first avoid urine which should be at least one hour after the previous urination were collected from 163 men who were scheduled for in vitro fertilization and embryo transfer (IVF-ET) treatment due to female factors at Center for Reproductive Medicine, Shengjing Hospital of China Medical University during the period of April 2016 to April 2017. Among the 163 men, 109 simultaneously provided semen that was collected after 3-7 days of sexual abstinence for the testing. Urine and semen specimens were detected for CT and UU with SAT-RNA, while urethra swab specimens were detected for CT and UU with standard PCR-DNA. Detection results of the SAT-RNA were compared with those of the PCR-DNA method. RESULTS: The positive rate of UU in the urethra swab detected with PCR-DNA and that of UU in the urine with SAT-RNA were 47.24% and 47.85%, respectively, and the coincidence rate was 93.25%. In addition, the positive and negative coincidence rates were 93.51% and 93.02%, respectively, and the concordance between the two methods was very good (Kappa=0.865). On the other hand, the positive rate of CT in the swab specimen tested with PCR-DNA was 3.07% and that of CT in urine with SAT-RNA was 4.29%, and the coincidence rate was 97.55%. Moreover, the positive and negative coincidence rates were 80.00% and 98.10%, respectively, and the concordance between the two methods was good (Kappa=0.654). Regarding SAT-RNA detection of UU in the urine and semen specimen of the 109 patients, the positive rates of UU in the urine and semen specimens were 50.46% and 44.95%, respectively; and the coincidence rate between the two specimens was 88.99%. In addition, the positive coincidence rate and the negative coincidence rate was 93.88% and 85.00%, respectively, and the concordance between the two specimens was good (Kappa=0.780). Similarly, SAT-RNA detection of CT in the urine and semen specimens showed the positive rate was 5.50% and 3.67%, respectively; and the two specimens showed 98.17% coincidence rate. The positive and negative coincidence rates were 100.00% and 98.10%, respectively, and the concordance was also good (Kappa=0.791). CONCLUSION SAT-RNA detection of CT and UU in the urine specimen showed good concordance with the PCR-DNA detection of CT and UU in the urethra swab specimen. In addition, the concordance was also good between the urine and semen specimens detected with SAT-RNA. These results indicate that, as a less invasive and equally accurate procedure, SAT-RNA may be more suitable for clinical application.
Chlamydia Infections/epidemiology* ; Chlamydia trachomatis/genetics* ; Female ; Humans ; Infertility, Male ; Male ; Neisseria gonorrhoeae/genetics* ; Polymerase Chain Reaction ; Ureaplasma urealyticum/genetics*

The detection method of gene chip based on SPR principle is a potential high-throughput microanalysis method without labelling. With the use of Self-assembled monolayer (SAM) technology, the gene chip of Neisseria gonorrhoeae probe lattice has been prepared, detected and analyzed using the Surface plasmon resonance (SPR) and SPR imaging (SPRI) gene chip detection system here-in provided for research in the hybridizatin reaction on the probe lattice of gene chip. The result indicates that there is an obvious resonance assimilate peak on the SPR resonance curve. And after hybridization, the refractive index and resonance as well as molecular weight of the probe have increased. So whether a hybridization takes place or whether the wanted ingredient is in the sample under examination can be determined by using SPR to watch the detecting interface or the resonance curve. The SPRI detection system is available for observing the happening of a hybridization on the probe of gene chip in real-time and straighforwardly. The SPR and SPRI system can do analysis qualitatively and quantitatively.
DNA Probes ; genetics ; Neisseria gonorrhoeae ; genetics ; Oligonucleotide Array Sequence Analysis ; instrumentation ; methods ; Surface Plasmon Resonance ; methods ; Surface Properties

OBJECTIVETo detect the genes of Neisseria spp. isolated from patients with male genitourinary tract infections, and to study the pathogenicity of non-gonococcal strains of Neisseria and the laboratory diagnosis for the infections caused by Neisseria spp.

METHODSUsing polymerase chain reaction and nucleotide sequencing, we amplified and sequenced 4 genes of Neisseria spp. isolated from patients with male genitourinary tract infections, including 16S rRNA, orfl, cppB and nspA.

RESULTSFourteen Neisseria strains were identified through analysis of the 16S rRNA gene, including 3 N. mucosa strains, 3 N. cinerea strains, 2 N. gonorrhoea strains, 2 N. sicca strains, 2 N. subflava strains, 1 N. lactamica strain, and 1 N. polysaccharea strain. Among them, 9 showed positive results in gonococcal fluorescence-labeled multiplex-PCR detection, 1 in cppB gene reaction, 5 in orfl gene reaction, and 3 in nspA gene reaction. The consistency rate was 85.7% between the above results from our gene detection and those from the routine bacteriological methods.

CONCLUSIONThe cppB gene is absent in the non-gonococcal strains of Neisseria spp. that can cause male genitourinary tract infection. Most of the strains not only lack virulence-associated orfl and nspA genes, but also show positive results in gonococcal fluorescence-labeled multiplex-PCR detection, which is one of the important reasons for the misdiagnosis and missed diagnosis of gonorrhea infection. The combination of routine bacteriological methods and gene detection in laboratory examinations may help improve the accuracy rates of Neisseria species identification and clinical diagnosis of the infections caused by Neisseria spp.

Genes, Bacterial ; Gonorrhea ; diagnosis ; microbiology ; Humans ; Male ; Neisseria gonorrhoeae ; classification ; genetics ; Polymerase Chain Reaction ; Urinary Tract Infections ; microbiology

The surface plasmon resonance (SPR)-based gene chip was prepared according to the following processes: First, a film of nanogold, which was synthesized by using Frens' method, was plated on chip by Chlorauric acid/hydroxylamine method. Then probes were fixed on nanogold film by Self-assembled monolayer (SAM) technology. Subsequently, the fixing time and concentration of probes, the sensitivity and the specificity of the chip were optimized. Our results suggested that the chip plated with 2.5 nm nanogold film has a better SPR reflection, and when fixed by probes for 4.5 h at the concentration of 1 500 nmol/L, the gene chip also shows a fine performance of detection and can identify accurately the mismatch between bases in SPR detection system. The gene chip constructed in the research can be used for SPR sensor detection.
Gold ; chemistry ; Nanoparticles ; chemistry ; Neisseria gonorrhoeae ; genetics ; Oligonucleotide Array Sequence Analysis ; Sensitivity and Specificity ; Surface Plasmon Resonance ; methods

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E. coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a (+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E. coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD= 0.992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Neisseria gonorrhoeae ; genetics ; Plasmids ; biosynthesis ; genetics ; Porins ; biosynthesis ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection ; Vaccines, Synthetic

OBJECTIVESTo investigate the correlation between mutation of GyrA and ParC genes and quinolone resistance in Neisseria Gonorrhoeae.

METHODSThe gene fragments of quinolone resistance-determining region (QRDR) in GyrA and ParC genes in 20 Neisseria gonorrhoeae strains clinically isolated in Wuxi, China, were sequenced, and the susceptibility of the 20 strains to quinolone was examined by agar diffusion method.

RESULTSThe mutations at the Asp95 point in GyrA gene were found in 20 strains. Of the 19 stains examined, 16 had mutations at the 86, 87, 88, 91 points in ParC genes.

CONCLUSIONSThe mutations of Asp95 in GyrA gene and Asp86, Ser87, Ser88, Glu91 in ParC gene contribute to quinolone resistance in Neisseria Gonorrhoeae.

Base Sequence ; DNA Gyrase ; genetics ; DNA Topoisomerase IV ; genetics ; Drug Resistance, Bacterial ; genetics ; Humans ; Molecular Sequence Data ; Mutation ; Neisseria gonorrhoeae ; drug effects ; genetics ; isolation & purification ; Quinolones ; pharmacology

OBJECTIVETo establish a new nucleic acid hybridization detection technique which may be used in medical genechips.

METHODSThe specific DNA fragment was detected by sequential two hybridization of fluorescence probe with template DNA and fixed DNA probe.

RESULTSFluorescence probe two-hybridization (FPTH) was applied to genechips for the detection of sex-transmitted pathogens from culture strains, and the results showed that the values of fluorescence density of the positive groups decreased remarkably when compared with those of the negative group. Both the sensitivity and specificity for detecting clinical samples are higher than 90%. There is no need of any additional reagent in hybridization procedure, and the hybridization detection can be accomplished in 40 minutes.

CONCLUSIONThe FPTH technique is rapid, simple and reliable, it can also make the clinical detection process completely automatic and integrative.

DNA Probes ; chemistry ; genetics ; DNA, Bacterial ; genetics ; Fluorescent Dyes ; chemistry ; Humans ; Neisseria gonorrhoeae ; genetics ; Nucleic Acid Hybridization ; methods ; Ureaplasma urealyticum ; genetics