More than 100 serotypes of Streptococcus pneumonia have been identified, which has been one bottleneck problem for pneumococcal disease diagnosis, surveillance, development of pneumococcal vaccine and effectiveness evaluation of pneumococcal vaccines. Three categories of approaches for pneumococcal serotyping will be discussed including phenotyping based on anti-serum, biochemical typing based on pneumococcal capsular characteristics and genotyping based on pneumococcal capsular locus sequences. We reviewed the development and applications of different serotyping of pneumococcus to provide guidance for pneumococcal disease prevention and control.
Humans ; Serotyping/methods* ; Pneumococcal Infections/prevention & control* ; Pneumococcal Vaccines ; Streptococcus pneumoniae/genetics* ; Pneumonia

OBJECTIVETo develop a protocol for the rapid detection of Salmonellae.

METHODSA mono-antibody-based direct-ELISA and PCR methods for the detection of Salmonella were developed previously. This study assessed the accuracy of both direct-ELISA and PCR methods for the rapid detection of Salmonella and set up a new detection protocol.

RESULTSThe sensitivity of the PCR method was higher than that of direct-ELISA method. In the 2002 spring physical examination for employees, 1 546 human fecal samples were examined by the combination of direct-ELISA and PCR method. Compared with the results of national standard method, the sensitivity and specificity of direct ELISA was 100% and 97.14%, respectively, while those of PCR method reached both 100%. It also indicated that combination use of two methods could give positive report within 40 hrs, and also achieve high sensitivity and specificity.

CONCLUSIONSBased on the results obtained, a protocol for the rapid detection of Salmonella was developed. The first step is to us direct-ELISA method to screen the large number of samples, and then use PCR method to validate the ELISA positive samples, and the final step is, if needed, is to use the national standard method to determine the serotypes of Salmonellae.

Enzyme-Linked Immunosorbent Assay ; methods ; Feces ; microbiology ; Humans ; Polymerase Chain Reaction ; methods ; Salmonella ; classification ; genetics ; isolation & purification ; Serotyping

OBJECTIVETo establish a specific, sensitive and practicable method for detection and typing of dengue virus.

METHODSBased on the genomic sequence analysis of dengue virus types 1-4, 4 pairs of primers were designed. The specific capture probes of dengue virus types 1-4 were amplified using RT-PCR, cloned and sequenced before using them for precoating the microwell plate. The samples were amplified using biotin-labeled forward primer and reverse primer, and microwell plate hybridization was carried out for detection and typing of dengue virus types 1-4.

RESULTSThe absorbance of the positive samples were higher than 0.5, while the average absorbance of the negative samples was lower than 0.1, with the S/N higher than 10.

CONCLUSIONThe method of PCR-ELISA we established for early detection and typing of all 4 dengue viruses seretypes.

Dengue Virus ; classification ; genetics ; Enzyme-Linked Immunosorbent Assay ; methods ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Serotyping ; methods

OBJECTIVETo analyze the characteristics of Sequence-based Typing (SBT) of the Serotype 1 Legionella pneumophila (Lp1) isolated from environmental water in China, and then create a preliminary database.

METHODSA total of 82 strains of Lp1 isolated from environmental water in 9 provinces of China between 2005 and 2008 were genotyped by SBT method and Pulsed-field Gel Electrophoresis (PFGE) method. The results of the two different typing methods were then compared by cluster analysis, adopting BioNumerics version 5.1 software.

RESULTSBy SBT method, the 82 strains of Lp1 were divided into 22 ST types, of which 17 new types and one new allele was discovered. The dominant type was ST-1 type, found in 8 provinces, accounting for 46.3% (38/82). ST-1, ST-150, ST-154, ST-159, ST-160 and ST-630 types were found in more than 2 isolated-sites; while more than 2 different ST types were found in 5 isolated-sites, as site B4, B5, B6, S3 and S8. In cluster analysis, 15 ST types were grouped into three complexes (ST-1 complex, ST-154 complex and ST-149 complex); and the other 7 ST types were not assigned complex. By PFGE method, 46 banding patterns were observed. As a result of the combination of the two methods, the 82 isolates strains could be divided into 54 molecular types, which showed a reliable accordance in the cluster analysis between the two methods.

CONCLUSIONThe SBT of the Lp1 in environmental water in China was unique. From the study, a preliminary SBT database was set up.

China ; Cluster Analysis ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Legionella pneumophila ; classification ; genetics ; isolation & purification ; Serotyping ; methods ; Water Pollution

BACKGROUNDTo establish a molecular detection and typing assay for identification and typing of human enteroviruses (HEV) which is suitable for clinical detection and epidemiologic research.

METHODSUsing both primers specific for HEV genus and HEV typing primers and reverse transcription polymerase chain reaction (RT-PCR) the authors detected preliminarily HEV by agarose gel electrophoresis and then identified serotype through nucleotide sequence analysis of RT-PCR amplicons. The monospecific antisera neutralization was applied to validate the typing results.

RESULTSThe serotype of 18 suspicious HEV samples was identified: 4 Coxsackievirus type A24 (CVA24), 3 CVB3, 1 CVB2, 1 CVA9, 1 CVA15, 1 Echovirus type 3 (E3), 1 E6, 1 E9, 1 E11, 1 E14, 1 E33 and 1 Rhinovirus type 9. The result was validated by monospecitive antisera neutralization.

CONCLUSIONThis RT-PCR assay for HEV detection and typing may be suitable for clinical detection and epidemic research since this method is sensitive and specific for direct identification and typing of HEV.

DNA Primers ; Enterovirus ; classification ; genetics ; Enterovirus Infections ; diagnosis ; Humans ; RNA, Viral ; genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Serotyping ; methods

PURPOSE: In this study we tried to look at the spreading, duration of colonization, and acquisition of new streptococci which were obtained in one geographical area, as well as the bacteriologic and molecular epidemiology of normal school children carrying group A streptococci and their clonal relationship through the combined application of the serotype of T antigen and Pulsed Field Gel Electrophoresis(PFGE). METHODS: A total of 88 strains of group A streptococci were isolated from 396 normal school children. All isolates were classified in groups by Streptex and serotyped by T. agglutination. Restriction enzyme digestion of DNA was taken using Sma I. DNA fragments were separated by PFGE. RESULTS: A total of 33 strains were allocated their epidemiologic characteristics. Four out of 33 strains were not restricted by enzyme(Sma I). Twenty nine strains out of 33 strains showed 12 subtypes with 8-12 fragments between 40kbp and 500kbp of DNA fragments on PFGE. Eight strains of NT and T6 war same fragment patterns on PFGE, respectively. Three strains out of 4 strains of T8/25 were not restricted and the other one showed different, unique patterns. One strain out of 8 stains of T12 was not restricted, and the others were classified as 5 different subtypes. Two strains of Tl were different patterns from each other, and 2 strains of T4 showed the samefragment pattern CONCLUSION: T serotypes with PFGE will be useful as a screening and molecular epidemiologic method in a country where anti-M antisera is not available, after recognizing the advantages and disadvantages of M and T serotyping.
Agglutination ; Antigens, Viral, Tumor ; Child* ; Colon ; Coloring Agents ; Digestion ; DNA ; Epidemiologic Methods ; Epidemiologic Studies* ; Humans ; Immune Sera ; Mass Screening ; Molecular Epidemiology ; Serotyping* ; Streptococcus pyogenes* ; Streptococcus*

Adeno-associated virus (AAV) has many advantages for gene therapy over other vector systems. However, after the production of recombinant AAV (Raav) vectors, the biological titration of rAAV stocks is still cumbersome. Different investigators used laboratory-specific methods or internal reference standards that may limit preclinical and clinical applications. The inverted terminal repeats (ITR) sequences are the only cis-regulated viral elements required for rAAV packaging and remain within viral vector genomes. ITR is the excellent target sequences for qPCR quantification of rAAV titer. In this study, we developed a novel qPCR strategy to quantify rAAVs' vector genome titer via targeting the ITR2 or ITR2-CMV element. In conclusion, the method is fast and accurate for the titration of rAAV2-derived vector genomes. It will promote the standardization of rAAV titration in the future.
Dependovirus ; classification ; genetics ; Genetic Vectors ; genetics ; Genome, Viral ; genetics ; Polymerase Chain Reaction ; methods ; Recombination, Genetic ; Serotyping ; Terminal Repeat Sequences ; genetics ; Transduction, Genetic

OBJECTIVEWe developed a multiplex polymerase chain reaction (PCR) method for detection, identification and sero-grouping strains of N. meningitidis.

METHODSThe gene of crgA was selected for detection and identification of N. meningitidis and the 230 bp of crgA fragments were amplified. The genes of siaD and orf-2 were used for sero-grouping. With this technology, N. meningitidis of serogroup A,B,C,Y and W135 can be differentiated from the specific fragment 400 bp,450 bp,250 bp, 120 bp,120 bp.

RESULTSThis multiple PCR method seemed to be more sensitive than latex agglutination. We used this method to detect 61 isolates of N. meningitidis and the 230 bp of crgA fragments were amplified. No positive results were obtained from on 4 strains of non-N. meningitidis. It seemed that the PCR method which was based on crag, was specific in detecting N. meningitidis. The 55 strains of N. meningitidis could be grouped into A,B,C,Y and W135 by this multiplex PCR. No gene fragment was synthesized for the other serogroups of N. meningitidis. No cross-reaction was observed for other bacterial species. 4 samples of meningococcal meningitis patients were detected by the multiplex PCR and two of them were positive with 400 bp band,representing serogroup A of N. meningitidis but all were negative to culture and latex agglutination. 18 samples of Japanese (B) encephalitis patients were also detected by multiplex PCR and no positive result was obtained which was consistent to the finding from culture and latex agglutination.

CONCLUSIONThis multiplex PCR method showed its potential in clinical diagnosis and epidemiological investigation.

Bacterial Proteins ; genetics ; China ; Humans ; Meningitis, Meningococcal ; diagnosis ; epidemiology ; Neisseria meningitidis ; classification ; genetics ; Polymerase Chain Reaction ; methods ; Serotyping ; Transcription Factors ; genetics

OBJECTIVETo investigate the application of pulsed-field gel electrophoresis (PFGE) in food-borne outbreak.

METHODSPathogens were isolated and further characteristics identified by traditional methods. The strains isolated were carried out with molecular typing with using PFGE. PFGE was performed by Laboratory Directions for molecular subtyping of Salmonella by PFGE (CDC, USA) and the results of PFGE were analyzed by BioNumerics soft.

RESULTSTotally 14 Salmonella serotype Muenchen strains were isolated from 19 patients, 3 of 9 suspicious foods were positive for S. muenchen and 7 strains were isolated from 18 cooks. The biochemistry characterization and antimicrobial susceptibility of all the strains isolated were the same. 23 S. muenchen isolates were all shown indistinguishable by PFGE.

CONCLUSIONPFGE should play a key role in identifying the outbreak-associated isolates and distinguishing them from unrelated sporadic isolates. It might also demonstrate that the genetic fingerprints of serotype Muenchen isolates derived from patients were indistinguishable from those derived from drinks. PFGE might provide precise information on bacterial food-borne pathogens, promptly identify the source of infection, and effectively prevent from spreading. It should be one of the early warning method on controlling outbreak of the food-borne disease.

China ; epidemiology ; Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; methods ; Humans ; Microbial Sensitivity Tests ; Salmonella Food Poisoning ; epidemiology ; microbiology ; Salmonella enterica ; classification ; isolation & purification ; Serotyping

OBJECTIVETo study the distribution of serotype and the positive rate of toxins among vibrio cholerae non-O(1) isolated from environmental waters in Foshan city.

METHODSWater specimens were collected from river and cultured for vibrio cholerae non-O(1). The PCR method was used to detect cholerae enterotoxin (CT) gene; the ELISA method was used to detect heat-stable toxin (ST) and heat-labile toxin (LT).

RESULTS478 vibrio cholerae non-O(1) strains were isolated from 1 644 water specimens, with a positive rate of 29.07%. Serological assay showed that the main serotype of vibrio cholerae non-O(1) in Foshan city is VBO(7). Positive rate of CT, ST and LT were 1.91%, 13.14% and 12.17%, respectively.

CONCLUSIONSA few non-O(1) strains were found to have several virulent factors simultaneously, and the results suggest that vibrio cholerae non-O(1) in environmental waters is potentially pathogenic and may affect people's health. It is necessary to pay attention to the prevention of diarrhoea caused by vibrio cholerae.

China ; Enterotoxins ; genetics ; Environmental Monitoring ; methods ; Enzyme-Linked Immunosorbent Assay ; Polymerase Chain Reaction ; Seasons ; Serotyping ; Vibrio cholerae non-O1 ; classification ; genetics ; isolation & purification ; Water ; analysis ; Water Microbiology