Detecting Mycobacterium tuberculosis complex and rifampicin resistance via a new rapid multienzyme isothermal point mutation assay.

Simple, rapid, and accurate detection of the Mycobacterium tuberculosis complex (MTBC) and drug resistance is critical for improving patient care and decreasing the spread of tuberculosis. To this end, we have developed a new simple and rapid molecular method, which combines multienzyme isothermal rapid amplification and a lateral flow strip, to detect MTBC and simultaneously detect rifampin (RIF) resistance. Our findings showed that it has sufficient sensitivity and specificity for discriminating 118 MTBC strains from 51 non-tuberculosis mycobacteria strains and 11 of the most common respiratory tract bacteria. Further, compared to drug susceptibility testing, the assay has a sensitivity, specificity, and accuracy of 54.1%, 100.0%, and 75.2%, respectively, for detection of RIF resistance. Some of the advantages of this assay are that no special instrumentation is required, a constant low temperature of 39 °C is sufficient for the reaction, the turnaround time is less than 20 min from the start of the reaction to read out and the result can be seen with the naked eye and does not require specialized training. These characteristics of the new assay make it particularly useful for detecting MTBC and RIF resistance in resource-limited settings.

[Three quantitative methods to continuously monitor Legionella in spring water].

OBJECTIVE: To compare the detection effect of Legionella pollution in spring water by three methods, namely traditional plating method, fluorescent quantitation PCR method and ethidium monoazide (EMA) fluorescent quantitation PCR method. METHODS: Every month (except May), we collected 11 water samples from the 5 selected hot spring pools in one hot spring resort in Beijing in 2011. A total of 121 water samples were collected, and then were detected by the above three methods qualitatively and quantitatively. RESULTS: In our study, the Legionella pollution rate was separately 74.4% (90/121), 100.0% (121/121) and 100.0% (121/121) by the above three methods. The quantitative value of Legionella in the 121 water samples detected by the three methods were around 0.10-216.00 colony-forming units (CFU)/ml, 1.47-1557.75 gene units (GU)/ml and 0.20-301.69 GU/ml, respectively. The median (25th and 75th percentiles) was 75.30 (32.51-192.10) GU/ml, 36.46 (16.08-91.21) GU/ml and 5.30 (0.00-33.70) CFU/ml, respectively. The difference in the quantitative value of Legionella detected by the three methods showed statistical significance (χ(2) = 187.900, P < 0.01). The quantitative value of Legionella detected by fluorescent quantitation PCR method was the highest, followed by the value Legionella detected by EMA-fluorescent quantitation PCR method and traditional plating method. CONCLUSION: The sensitivity of the PCR methods was higher than traditional plating method, in detecting Legionella pollution in spring water, especially the EMA- fluorescent quantitation PCR method, which was more suitable for detecting Legionella in water.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

rpoB Mutations are Associated with Variable Levels of Rifampin and Rifabutin Resistance in Mycobacterium tuberculosis.

Objective: To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). Methods: We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. Results: The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified. Conclusion: This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.

rpoB Mutations and Effects on Rifampin Resistance in Mycobacterium tuberculosis.

OBJECTIVE: To investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure. METHODS: We sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed. RESULTS: Results revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance. CONCLUSION: Some mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.

Serotype distribution and clinical characteristics associated with streptococcus pneumoniae among Chinese children and adults with invasive pneumococcal disease: a multicenter observational study.

Few studies in China focused on serotypes of Streptococcus pneumoniae in patients with invasive pneumococcal disease (IPD). We aimed at investigating the serotype distribution for IPD-causing S. pneumoniae and vaccine coverage among Chinese children and adults. This was a multicenter, observational study to collect S. pneumoniae isolates from normal sterile sites and IPD-related clinical information among children and adults. Serotyping was performed by a Capsule-Quellung reaction test using type-specific antisera. The study collected a total of 300 eligible isolates (pediatric = 148, adult = 152) were serotyped in a central laboratory. The most prevalent serotypes were 19A (20.9%) and 23 F (20.3%) in the pediatric group; 3 (21.7%) and 19 F (11.8%) in the adult group. PCV10 had low-to-moderate serotype coverage rates for children (60.8%) and adults (34.2%). PCV13 and PPV23 had high coverage rates for children (89.9%, 93.2%) and adults (70.4%, 82.9%), respectively, Investigational PCVs including PCV15 and PCV20 had high estimated coverage rates in children (89.9%, 93.9%). The study identified 269 subjects with IPD reported as the primary diagnosis in the medical records. Sepsis (48/136, 35.3%) and pneumonia (48/133, 36.1%) had the highest occurrence in the pediatric and adult groups, respectively. Study findings showed that non-PCV7 S. pneumoniae 19A and 3 were the most prevalent serotypes in Chinese children and adults, respectively. High-valent vaccines had similar coverage rates and may have a greater potential in preventing IPD.

Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M. intracellulare and M. tuberculosis.

OBJECTIVES: To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M. intracellulare and M. tuberculosis. METHODS: Protein extracts from M. intracellulare were used to immunize BALB/c mice. The antigens were evaluated using cellular and humoral immunoassays. The common genes between M. intracellular and M. tuberculosis were identified using genome-wide comparative analysis, and cross-reactive proteins were screened using immunoproteome microarrays. RESULTS: Immunization with M. intracellulare proteins induced significantly higher levels of the cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-6 (IL-6) and immunoglobulins IgG, IgG1, IgM, and IgG2a in mouse serum. Bone marrow-derived macrophages isolated from mice immunized with M. intracellulare antigens displayed significantly lower bacillary loads than those isolated from mice immunized with adjuvants. Whole-genome sequence analysis revealed 396 common genes between M. intracellulare and M. tuberculosis. Microchip hybridization with M. tuberculosis proteins revealed the presence of 478 proteins in the serum of mice immunized with M. intracellulare protein extracts. Sixty common antigens were found using both microchip and genomic comparative analyses. CONCLUSION: This is the advanced study to investigate the immunogenicity of M. intracellulare proteins and the cross-reactive proteins between M. intracellulare and M. tuberculosis. The results revealed the presence of a number of cross-reactive proteins between M. intracellulare and M. tuberculosis. Therefore, this study provides a new way of identifying immunogenic proteins for use in tuberculosis vaccines against both M. intracellulare and M. tuberculosis in future.

Accuracy of a reverse dot blot hybridization assay for simultaneous detection of the resistance of four anti-tuberculosis drugs in Mycobacterium tuberculosis isolated from China.

BACKGROUND: Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide. Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M. tuberculosis isolated in China. METHODS: In this study, we applied a RDBH assay to simultaneously detect the resistance of rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) in 320 clinical M. tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing (DST) and sequencing. The RDBH assay was designed to test up to 42 samples at a time. Pearson's chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method, and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing. RESULTS: The results showed that the concordances between phenotypic DST and RDBH assay were 95% for RIF, 92.8% for INH, 84.7% for SM, 77.2% for EMB and the concordances between sequencing and RDBH assay were 97.8% for RIF, 98.8% for INH, 99.1% for SM, 93.4% for EMB. Compared to the phenotypic DST results, the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5% for RIF, 90.3 and 97.3% for INH, 77.4 and 91.5% for SM, 61.4 and 85.7% for EMB, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 97.7 and 97.9% for RIF, 97.9 and 100.0% for INH, 97.8 and 100.0% for SM, 82.6 and 99.1% for EMB, respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples. CONCLUSIONS: Our data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF, INH, SM and EMB, enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.

Detecting Ethambutol Resistance in Mycobacterium tuberculosis Isolates in China: A Comparison Between Phenotypic Drug Susceptibility Testing Methods and DNA Sequencing of embAB.

With the increasing incidence of drug-resistant tuberculosis (DR-TB), determining a rapid and accurate drug susceptibility testing (DST) method to identify ethambutol (EMB) resistance in Mycobacterium tuberculosis has become essential for patient management in China. Herein, we evaluated the correlation between three phenotypic DST methods, namely, proportion method (PM), MGIT 960 system, and microplate alamar Blue assay (MABA), and DNA sequencing of embAB in 118 M. tuberculosis isolates from China. When the results of the phenotypic DST methods were compared with those of DNA sequencing, the overall agreement and kappa values of the PM, MGIT 960 system, and MABA were 81.4% and 0.61, 77.1% and 0.55, and 84.7% and 0.67, respectively. The agreement for EMB resistance between MABA and PM was significantly higher than that between the MGIT 960 system and PM (P = 0.02). Moreover, among the isolates with detectable embAB mutations, 97.2% (70/72 isolates) harbored mutations in embB. The analysis of embB mutations predicted EMB resistance with 81.3% sensitivity, 86.8% specificity, and 83.1% accuracy. Thus, MABA may be a better phenotypic DST method for detecting EMB resistance. DNA sequencing of embB may be useful for the early identification of EMB resistance and the consequent optimization of the treatment regimen.

Prevalence, risk and genetic characteristics of drug-resistant tuberculosis in a tertiary care tuberculosis hospital in China.

OBJECTIVES: To explore the prevalence, risk and genetic characteristics of drug-resistant tuberculosis (TB) from a tertiary care TB hospital in China. PATIENTS AND METHODS: We carried out a retrospective study including isolates from 189 patients with pulmonary TB at Fuzhou Pulmonary Hospital. All isolates from these patients were subjected to drug susceptibility testing and genotyping. For drug-resistant isolates, DNA sequencing was used to investigate mutations in 12 loci, including katG, inhA, oxyR-ahpC, rpoB, rpsL, rrs 1 (nucleotides 388-1084 of rrs), embB, tlyA, eis, rrs 2 (nucleotides 1158-1674 of rrs), gyrA and gyrB. RESULTS: Among 189 isolates, 28.6% were resistant to at least one of the seven anti-TB drugs, including isoniazid (INH), rifampin (RIF), streptomycin (STR), ethambutol (EMB), capreomycin (CAP), kanzmycin (KAN) and ofloxacin (OFX). The proportion of multidrug-resistant TB and extensively drug-resistant TB isolates was 9.5% and 1.1%, respectively. Patients in rural areas as well as previously treated patients showed a significantly increased risk of developing drug resistance. In addition, among these isolates, 111 (58.7%) were Beijing genotype strains, 84 (75.7%) of which belonged to modern Beijing sublineage. There was no association between genotype and drug resistance. The most common mutations were katG315, rpo B531 rpsL43, embB306, rrs1401 and gyrA94. CONCLUSION: These findings provided additional information of drug-resistant TB in China. Previously treated patients and patients in rural areas should receive greater attention owing to their higher risk of developing drug resistance.

Beijing genotype of Mycobacterium tuberculosis is less associated with drug resistance in south China.

Mycobacterium tuberculosis Beijing genotype strains are widespread globally. However, there has been no systematic study on the association between Beijing genotype and the characteristics of drug resistance. In this study, 359 M. tuberculosis isolates from south China were collected and their background information, genotype diversity and drug resistance was investigated. The results revealed that 66.0% of strains (237/359) were categorised as Beijing genotype. There was no statistical difference between Beijing and non-Beijing genotype strains in terms of patient sex, age, place of residence and treatment history. Drug resistance testing showed that 34.8% (125/359) of isolates were resistant to at least one of the seven drugs tested. The proportions of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis were 17.0% and 1.4%, respectively. Previously treated patients presented a significantly higher risk of developing drug resistance than new cases. Although the prevalence of drug resistance was higher in Beijing genotype than in non-Beijing genotype strains, there was no significant difference between these two genotypes in the multivariate analysis. Even in re-treated patients, the association of Beijing genotype with drug resistance was not significant. This study provides an insight into genotype diversity and demonstrates the characteristics of drug resistance in Beijing genotype strains, which will be useful in generating efficient tuberculosis prevention and control strategies in China.

Immunological Evaluation of a Novel Mycobacterium tuberculosis Antigen Rv0674.

OBJECTIVE: This study aimed to characterize the diagnostic and vaccine potential of a novel Mycobacterium tuberculosis antigen Rv0674. METHODS: To evaluate the diagnostic potential and antigenicity of Rv0674, IgG was evaluated using ELISA and interferon (IFN)-γ was done by using ELISpot assay among TB patients and healthy donors. For immunogenicity evaluation, BALB/c mice were immunized with Rv0674. Cytokine production was determined by cytokine release assay using an ELISA kit, and the antibodies were tested using ELISA. RESULTS: The results of serum Elisa tests showed that Rv0674 specific immunoglobulin G (IgG) response was higher in TB patients than negative controls. And Rv0674 had good performance in serological test with sensitivity and specificity of 77.1% and 81.1%, respectively. While it shows poor sensitivity and specificity of 26.23% and 79.69% for IFN-γ tests. In BALB/c mice, Rv0674 adjuvant by DDA/Poly I:C could also induce a high level of IFN-γ, interleukin-2 and interleukin-6 as well as a high IgG titer in both high- and low-dose groups indicating that Rv0674 is essential in humoral and cellular immunity. Moreover, the cytokine profile and IgG isotype characterized Rv0674 as a Th1/Th2-mixed-type protective immunity with the predominance of Th1 cytokines. CONCLUSION: Rv0674 may be a good potential candidate for the development of TB serological diagnosis and a new TB vaccine.

Safety and immunogenicity from a phase I trial of inactivated severe acute respiratory syndrome coronavirus vaccine.

BACKGROUND: Emergence of severe acute respiratory syndrome (SARS) from the winter of 2002 to the spring of 2003 has caused a serious threat to public health. METHODS: To evaluate the safety and immunogenicity of the inactivated SARS coronavirus (SARS-CoV) vaccine, 36 subjects received two doses of 16 SARS-CoV units (SU) or 32 SU inactivated SARS-CoV vaccine, or placebo control. RESULTS: On day 42, the seroconversion reached 100% for both vaccine groups. On day 56, 100% of participants in the group receiving 16 SU and 91.1% in the group receiving 32 SU had seroconverted. The geometric mean titre of neutralizing antibody peaked 2 weeks after the second vaccination, but decreased 4 weeks later. CONCLUSION: The inactivated vaccine was safe and well tolerated and can elicit SARS-CoV-specific neutralizing antibodies.

[Analysis on pneumococcus isolated from part of china using serotype and pulsed-field gel electrophoresis].

OBJECTIVE: To investigate the serotype distribution and molecular features of Streptococcus pneumonia isolates in China. METHODS: 144 strains isolated from 6 provines were selected as the research object. The serotypes were determined with the method of capsular swelling. The molecular characteristics were conducted by optimized pulsed-field gel electrophoresis. RESULTS: A total of 135 Streptococcus pneumoniae isolates were divided into 14 serogroups. 19, 6, 15, 23 serogroups are prevalent types in China. 9 isolates were failed to identify specific serogroups. All isolates could be divided into 92 pulsotypes. Pulsotypes were scattered. Absolutely dominant pulsotype was not found. Pulsotypes were similar among same serogroup isolates. CONCLUSION: The pneumococcus of serogroup 19, 6, 15, 23 were common in China. PFGE had strong discriminatory ability of subtyping of Streptococcus pneumoniae. PFGE showed better epidemiological survey capacity.

[Detection of bacterial meningitis by multiplex polymerase chain reaction].

OBJECTIVE: To develop a rapid method for detecting 8 pathogens which were highly related to bacterial meningitis by multiplex polymerase chain reaction. METHOD: By optimizing the reaction condition and amplification program of single pair polymerase chain reaction, the multiplex pairs polymerase chain reactions (M-PCR) was developed to identify eight pathogens simultaneously including Neisseria Meningitis, Haemophilus Influenzae, Streptococcus Pneumoniae, Cryptococcus Neoformans, Staphylococcus Aureus, Listerisa Monocytogene, Streptococcus Suis and Mycobacterium Tuberculosis. Meanwhile, The sensitivity of M-PCR assay was also studied. RESULTS: M-PCR methods for detecting 8 pathogens which could cause bacterial meningitis have been established. M-PCR showed specific, sensitive and more rapid than conventional culturing method. CONCLUSION: This multiplex polymerase chain reaction method can be used for diagnosis and scanning of suspicious bacterial meningitis cases in order to improve the diagnostic positive rate of bacterial meningitis cases.

[Resistance and serotype of 123 strains of Streptococcus pneumoniae].

OBJECTIVE: To investigate antibiotic resistance and the prevalence of serotype of Streptococcus pneumoniae. METHODS: Totally 123 strains of S. pneumoniae were collected to test the susceptibility of 6 antibiotics by K-B method according to the standard of CLIS. Serotyping of S. pneumoniae was performed by using quelling reaction. RESULTS: Among 123 strains of S. pneumoniae, all strains (100%) were resistant to AZM. There were 65.04%, 78.05%, 19.51%, 0.81%, 0% of S. pneumoniae were resistant against the MH, SXT, C, RA, OFX respectively. 11 serotypes were involved in 123 strains. The prevalent serogroup were 6, 14, 15, 19 and 23.5 strains were unable to serotype. The multi-resistant strains of S. pneumoniae included in serogroup 6 and 19. CONCLUSION: The antibiotic resistance of S. pneumoniae is very serious. Most of them are multi-resistant strains. The prevalent serogroup, especially of the multi-resistant strains, are 6 and 19 serogroup.

[Establishment and application of PCR method in serotyping of Streptococcus pneumonia.].

OBJECTIVE: To establish a method based on PCR for serotyping of Streptococcus pneumonia isolates. PCR serotyping method was applied for investigating the serotypes of S. pneumonia strains. METHODS: 12 pairs of primers targeting different serotypes of S. pneumonia were designed and synthesized. After optimizing the PCR amplification reaction, sensitivity and specificity of each pair was performed. We applied the PCR methods for testing the serotypes of the isolated S. pneumonia strains. RESULTS: Each pair of primers showed satisfied PCR sensitivity and specificity. Of all 119 S. pneumonia strains tested by PCR serotyping method, 113 isolates were identified (3, 5, 6A/B, 9A/V, 14, 18, 19A, 19F, 23F) with 6 isolates were unable to be serotyped. CONCLUSION: We developed a simple, reliable and economic method for S. pneumonia serotyping which could be used for testing the serotypes of S. pneumonia that had been prevailed among general population.

[Molecular typing of Klebsiella pneumonia by pulse-field gel electrophoresis in combination with multilocus sequence typing].

OBJECTIVE: To type Klebsiella pneumonia through methods including pulse-field gel electrophoresis (PFGE) in combination with multilocus sequence typing. METHODS: Four selected different EPs, referring to the Standard Operating Procedure of PulseNet China, were used. The single colony of Klebsiella pneumonia was quantified after enriched culture. Embedding organisms in agarose and genome DNA were lysed with Proteinase K and then digested by restriction endonuclease XbaI, to produce agarose gel. Fingerprint was obtained through PFGE and bands were marked with their molecular weights and then analyzed by BioNumerics software. Using MLST to analyze the strains that were highly similar, by PFGE typing RESULTS: By comparing the four results from each EPs, fk3 (switch time from 6s to 36s, total run time is 18.5 hours) seemed to be better than the others. 59 strains of Klebsiella pneumonia were divided into 47 PFGE types and 19 PFGE clusters. The highly similar strains could be typed into ST-340, ST-342, ST-343, ST-344, ST-345 by MLST. Among them, ST-342, ST-343, ST-344, ST-345 types were all new MLST types that were reported in China. CONCLUSION: Highly similar Klebsiella pneumonias typed by PFGE could also be typed by MLST.

[Research on the relationship between variable number of tandem repeats sequences and serogrouping of Neisseria meningitis].

OBJECTIVE: To research the relationship between variable number of tandem repeats sequences (VNTRs) in genome and serogrouping of Neisseria meningitides (N. meningitidis). METHODS: 118 meningococcal strains which were isolated from invasive patients and carriers in China were serogrouped by antiseria agglutination testing. 38 strains were serogroup A. 80 were serogroup C. And then four variable number of tandem repeats sequences (VNTRs) loci in the DNA of 118 meningococcal strains were amplified by PCR. A dendrogram was constructed on the basis of DNA length of PCR productions by the Unweighted Pair Group Method with Arithmetic Averages (UPGMA). The two pair primers-specific were designed based on the nucleotide variant afterward the VNTR01 locus (T01-A and T01-AC, T01-C and T01-AC) to amplify 118 meningococcal strains. RESULTS: 118 strains were classified into five VNTR groups. 98.3% (116/118) strains were identical between serogrouping and VNTRs genotyping. 97.3% (36/37) strains of serogroup A were categorized into group I of VNTRs genotyping. 98.7% (75/76) strains of serogroup C were categorized into group II of VNTRs genotyping. According to the presence or absence of PCR products using two pair primers-specific, 118 strains were classified into two groups which correspond to serogroup A and C. The sensitivity of primers-specific T01-A and T01-AC was 89.5% (34/38). The specificity of primers-specific T01-A and T01-AC was 100% (38/38). The sensitivity of primers-specific T01-C and T01-AC was 100% (80/80). The specificity of primers-specific T01-C and T01-AC was 95.2% (80/84). CONCLUSION: VNTRs genotyping has high relation to serogrouping A and C. The nucleotide sequence variations in the galactosyl-transferase gene related to the serogrouping A and C.

[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.