Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin （vvhA） Coding System

Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles （Ct value） and fluorescence intensity enhancement （ARn value） were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.     

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction(PCR) and capillary electrophoresis(MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10~(-7) to 10~(-2) ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.     

Performance of the microscopic observation drug susceptibility assay in pyrazinamide susceptibility testing for Mycobacterium tuberculosis

Background Drug susceptibility assay is very important in tuberculosis therapy. Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis （M. tuberculosis） is difficult and time consuming by conventional methods. In this study, we aimed to evaluate the performance of the microscopic observation drug susceptibility （MODS） assay in the detection of pyrazinamide resistance in M. tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen （L J） proportion method. Methods M. tuberculosis clinical isolates （n=132） were tested by the MODS and the Wayne assay： the results were compared with those obtained by the LJ proportion method. Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods. Results Compared to the LJ results, the sensitivity and specificity of the MODS assay were 97.8% and 96.5% respectively; the sensitivity and specificity of the Wayne assay were 87.0% and 97.7% respectively. Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant （3 tests）, in 1 of the 4 strains （LJ only）, in 42 of 48 strains （at least I test）, but no mutations in 1 strain sensitive according to the MODS assay only. The MODS assay, Wayne assay and LJ proportion method provided results in a median time of 6, 7 and 26 days respectively. Conclusions MODS assay offers a rapid, simple and reliable method for the detection of pyrazinamide resistance in M. tuberculosis and is an optimal alternative method in resource limited countries.     

Detection of mitochondrial DNA deletion by a modified PCR method

Objective: To develop a simple and efficient method for detecting small populations of mitochondrial DNA deletion. Methods: Peripheral blood cell DNA was obtained from a victim who was aecidently exposed to a ^60Co radiation source 11 years ago. Using the DNA as template, PCR was performed to generate multiple products including true deletions and artifacts. The full length product was recovered and used as template of secondary PCR. The suspicious deletion product of mtDNA could be confirmed if it was only yielded by first PCR. Using either original primers or their nested primem, the suspicious deletion product was amplified and authenticated as true deletion product. The template was recovered and determined to be a deletion by sequencing directly. Results: A new mtDNA deletion, spanning 889 bp from nt11688 to nt12576, was detected in the peripheral blood cells of the victim. Conclusion: The new PCR-based method is more efficient in detecting small populations of mtDNA deletion than other routine methods. MtDNA deletion is found in the victim, suggesting there is relationship between the deletion and phenotypes of the disease.     

Surveillance of Mycoplasma pneumoniae infection among children in Beijing from 2007 to 2012

Background Mycoplasma pneumonia （M.pneumoniae） is one of the key pathogens of community-acquired pneumonia.A global pandemic of M.pneumoniae has occurred since 2010.The aim of this study was to survey the prevalence of M.pneumoniae in children in Beijing from 2007-2012.Methods A total of 3 073 clinical specimens were obtained from pediatric patients with respiratory tract infections from January 2007 to December 2012,and examined by nested polymerase chain reaction.PCR products were visualized by 2％ agarose gel electrophoresis,positive products sequenced,and compared with reference sequences in GenBank.Macrolide resistance-associated mutations were also detected for some positive samples.Results Of the 3 073 specimens,588 （19.13％） were positive for M.pneumoniae,12.4％ of which were accompanied by viral infections.Positive rates for M.pneumoniae were highest in 2007 and 2012,showing a significant difference when compared with other years.Infections tended to occur in autumn and winter and positive rates were significantly higher for children aged 3-16.The rate of macrolide resistance-associated mutations was 90.7％,and the predominant mutation was an A→G transition （89.92％） at position 2063 in domain V of the 23S rRNA gene.Conclusions M.pneumoniae outbreaks occurred in 2007 and 2012 in pediatric patients in Beijing,which is consistent with the global prevalence of M.pneumoniae.M.pneumoniae can cause multi-system infections in children,and may be accompanied with viral infections.We determined that school-age children are more susceptible to this disease,particularly in autumn and winter.Gene mutations associated with macrolide resistance were very common in M.pneumoniae-positive specimens during this period in Beijing.     

Applied Study on Magnetic Nanometer Beads in Preparation of Genechip Samples

A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe203) were set up.The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.     

Clinical features and management of Crohn’s disease in Chinese patients

Background An increasing incidence of Crohn‘s disease has been found in China in recent years.Our study has been focused on evaluating the diversity of the clinical manifestations of Crohn‘ s disease in order to improve early diagnostic accuracy and therapeutic efficacy.Methods Thirty patients with active Crohn‘s disease were enrolled and their clinical data, including diagnostic and therapeutic results, were analyzed. Endoscopy combined with histological examination of biopsy specimens provided characteristic features of the disease. Transabdominal bowel sonography (TABS) was used for detecting intestinal complications. Nutritional supportive therapy was given to 20 subjects with active cases of the disease.Results Most patients were young adults with a higher proportion of females to males ( ratio: 1.14:1 ). The disease affects any segment or a combination of segments along with the alimentary tract(from the mouth to the anus). In this study, the colon and small bowel were the major sites involved.Recurrent episodes of abdominal pain in the right lower quadrant and watery diarrhea were the most common symptoms. Granulomas were identifiable in nearly one-third (30. 8%) of all biopsy specimens. In moderate cases of the disease, remission was achieved more quickly through the use of oral prednisone therapy than with SASP or 5-ASA. Beneficial effects on the host‘ s nutritional status were observed. Immunosuppressives were used on an individual basis and showed variable therapeutic effects. Sixteen patients had surgery due to intestinal obstruction or failure to respond to drug therapies. Rapid improvement after surgery was reported.Conclusion Endoscopy (with biopsy) and TABS were both crucial procedures for diagnosis. SASP (or 5-ASA) and prednisone were effective as inductive therapies. Azathioprine has demonstrable benefits after induction therapy with prednisone. Surgery, as an alternative treatment, provided another effective choice in selected patients.     

A rapid and sensitive method to detect mycobacterium tuberculosis DNA by fluorescence polarization

Objective: To develop a new high sensitivity, rapid and simple mycobacterium tuberculosis DNA detection method using fluorescence polarization technology. Methods: In our asymmetric PCR protocol, 100 times mole of TB-R primer was added than in the usual symmetric PCR to get enough single strands PCR product. The probe TB-5‘-TAMRA and PCR products were mixed in a tube and incubate for 5 - 15 min at 46℃.The polarization (mp) was measured using victor2 Multilabel Plate Reader. Results: Asymmetric and symmetric PCR products were analyzed by the FP method. Asymmetric PCR products are detected more sensitively than symmetric ones. The polarization values of probe associated with asymmetric products were much higher than with symmetric products. Conclusion: This fluorescence polarization assay in conjunction with asymmetric PCR is a powerful and widely applicable method for the rapid and sensitive detection of micro-organisms in clinical laboratories.     

Identification of E. coli K12 chromosomal insertion sites of bacteriophage φ297

Objective：To identify the specific integration site of prophage φ297 in the host of E. coli K12 chromosome. Methods：Using molecular techniques such as Siebert PCR for walking from the int gene of prophage 297, which is similar to that of phage 933W to an unknown region in genomic DNA. A special adaptor is ligated to the ends of DNA fragments generated by digestion of genomic DNA with restriction enzymes that generates blunt ended fragments. Clone and subclone of PCR products, DNA sequencing and data analysis were used in this study. Results：The attL, attR and the core sequences were determined. The bacterial attachment site of phage φ297 was located in the yecE gene of E. coli K12. Conclusion：The phage φ297 integrates into the yecE gene of the E. coli K12 genome.