[Methodology research and preliminary assessment of Mycobacterium tuberculosis detection by immunomagnetic beads combined with functionalized fluorescent quantum dots].

OBJECTIVE: To establish a detection method for Mycobacterium tuberculosis (MTB) by immunomagnetic beads combined with functionalized fluorescent quantum dots technology, and to investigate the optimal test condition and the diagnostic value of this method. METHODS: MTB standard strain H37Rv was used as detection object. Nanobeads and quantum dots were prepared by using wet chemical method, and conjugated separately with MTB binding peptide H8 to obtain immunomagnetic beads and functionalized fluorescent quantum dots, which could react with H37Rv simultaneously and form a ternary complex structure. Based on measurement of the fluorescence value and observation under fluorescence microscopy to determine if MTB existed in the sample, a new detection method of MTB using nanotechnology was established. The optimal detection concentration and reaction time of immunomagnetic beads and quantum dots were investigated, and the detection limit and specificity of this detection method were evaluated by using bacterial suspension and simulation sputum samples. RESULTS: By fluorescence microscopy examination, it was found that conjugated immunomagnetic beads and functionalized fluorescent quantum dots both bound with H37Rv and formed the ternary complex structure. The fluorescent value ratio of the experimental group and the control group could be 4:1. The best detection concentration of immunomagnetic beads and functionalized fluorescent quantum dots was 100 mg/L and the optimal incubation time was 2 h. The detection limit of H37Rv bacterial suspension and simulation sputum sample were both 10(3) CFU/ml. The detection results for 3 non-mycobacteria were all negative, while for the 12 types of NTM, only Mycobacterium parafortuitum, Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium fortuitum were positive, and others were all negative. CONCLUSION: The detection method of immunomagnetic beads combined with fluorescence quantum dots can be a new detection method for MTB, but the clinical value needs to be evaluated further.

[The study of inter-simple sequences repeat genotyping based on one pentanucleotide repeat sequences in mycobacteria].

OBJECTIVE: To establish inter-simple sequences repeat (ISSR) molecular makers based on (CAGCG)n repeat sequence in mycobacteria. METHODS: The distribution of pentanucleotide repeat sequence (CAGCG)n in mycobacterial genomes was analyzed by MICdb 2.0 software in the microsatellite database. ISSR primer MISP6 based on (CAGCG)n sequences was designed and tested in mycobacterial strains, which included 17 mycobacterial strains and 41 Mycobacterium tuberculosis clinical strains. RESULTS: The abundances of pentanucleotide repeat sequences (CAGCG)n were high in most of the mycobacterial genomes and they were mainly located in the coding regions. The results of ISSR analysis in mycobacteria showed that 15 reference strains from mycobacteria were clustered into 2 major clusters. The first cluster contained 2 subtypes and the second cluster contained 4 subtypes. Forty-one clinical strains from Mycobacterium tuberculosis were divided into 2 major clusters by the analysis of MISP6 primer, and each cluster had 2 subtypes. CONCLUSION: ISSR primer MISP6 based on (CAGCG)n sequences can be used as a genetic marker to genotype mycobacterial strains.

[Cross-resistance between rifampin and rifabutin in multidrug resistant Mycobacterium tuberculosis complex strains].

OBJECTIVE: To study the cross-resistance between rifampin and rifabutin in multidrug resistant Mycobacterium tuberculosis complex strains, and therefore to provide laboratory data for using rifabutin in the treatment of multidrug resistant tuberculosis. METHODS: The MIC(90) of rifabutin and rifampin against 99 multidrug resistant Mycobacterium tuberculosis clinical strains were determined by microplate assays. Statistical analysis was performed by using the χ(2) test and the t test. RESULTS: The cross-resistance rate between rifampicin and rifabutin was 85.9% (85/99), but the MIC(90) of rifabutin (≤ 16 mg/L, median 2 mg/L) was significantly lower than that of rifampicin (≥ 2 mg/L, median > 32 mg/L). The cross-resistance rate increased with the resistance level of rifampicin. The cross-resistance strains in the lower and the medium groups were 0/9 and 5/9 respectively, while the strains of the high rifampicin-resistant group were almost all cross-resistant (98.8%, 80/81). CONCLUSION: Rifabutin had activities against rifampin resistant Mycobacterium tuberculosis complex strains in vitro, and therefore may be used as an alternative for the treatment of multidrug resistant tuberculosis.

[Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis complex].

OBJECTIVE: To identify and evaluate a new nucleic acid amplification (NAA) test target for specific detection of Mycobacterium tuberculosis (MTB) complex (MTC). METHODS: MTC-specific fragment was obtained by ISSR genotyping technology. Primer pairs were designed based on the sequences of MTC-specific fragment and tested in 211 mycobacterial strains including 107 MTC strains and 104 nontuberculous mycobacteria (NTM) strains. IS6110 element (specific identification of MTC strains) and 16s rRNA gene (specific identification of Mycobacterium) amplification were used as a control to evaluate the efficacy of the NAA test target in the detection of MTC strains. RESULTS: One MTC-specific fragment with the length of 588 bp, located in 315947 - 316534 of the genome from MTB reference strain H(37) Rv, were obtained, cloned and sequenced. MTC-specific primer pairs MTCF/R were designed based on these sequences. All 211 mycobacterial strains accurately produced the genus-specific 16s rRNA amplicon. All MTC strains were positive in the MTCF/R PCR amplification while 99% MTC strains (106/107) were positive in the amplification of IS6110 sequences. All NTM strains were negative in both IS6110 and MTCF/R PCR amplification. CONCLUSIONS: The MTC-specific fragment developed in this study can be used as a new NAA test target to correctly distinguish MTC from NTM.

[Screening of tuberculosis specific antibody binding peptides].

OBJECTIVE: To screen the specific antibody binding peptides of tuberculosis from phage-displayed random phage display (Ph.D.)-7 peptide libraries with purified IgG from tuberculosis serum, and to provide the basis for the development of serological detection reagent of tuberculosis. METHODS: Purified IgG of tuberculosis serum was used as solid ligand to screen the binding peptide from the Ph.D.-7 peptide library, according to the biopanning process of absorption, elution and amplification. Purified IgG of health people serum was used as the molecule of counter selection during the second and third selection. Phages were enriched after 3 rounds of screening, then 20 single phages separately eluted by IgG of tuberculosis and health people were randomly selected on each direction of the determination plates and amplified. The single chains DNA were extracted as template for sequencing. The combination abilities of selected clones to IgG of tuberculosis and health people were tested by indirect enzyme linked immunosorbent assay (ELISA), and the positive clone was identified. Serum samples, from 47 patients with tuberculosis and 37 healthy people vaccinated with BCG, were verified by positive phage clones using phage-ELISA. The verifying results of 24 serum samples used for panning were separately analyzed statistically. RESULTS: After 3 rounds of panning, remarkable enrichment of phages that could specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 12 sequences were obtained. 12 phage clones with different sequences were amplified and detected with indirect ELISA and single phage H12 showed higher affinity with IgG of tuberculosis (S/N ≥ 2.1) and was identified as the positive clone. It was found that, in indirect ELISA with single Phage H12, the A(450) value of tuberculosis patients (0.105 ± 0.010) was significantly higher than that of healthy individuals (0.070 ± 0.005), and the t value was 2.912 (P < 0.0001). The A(450) value of 12 serum samples of tuberculosis patients and 12 samples of health individuals used for panning were 0.144 ± 0.016 and 0.052 ± 0.004, and the t value was 5.69 (P < 0.0001). CONCLUSION: By using phage-displayed random peptide libraries, we obtained the specific antibody binding peptides of tuberculosis, which showed specific binding activity with IgG of tuberculosis. It can provide a basis for the establishment of a new serological detection method of tuberculosis with peptide.

[Fast identification of mycobacteria in microtiter liquid culture].

OBJECTIVE: This research was to establish a method for fast identification of mycobacteria in microtiter liquid culture and to evaluate its clinical value. METHODS: 2-thiophenecarboxylic acid hydrazide (TCH) and paranitrobenzoic acid (PNB) at different concentrations were added into liquid culture in 96-well plate. Different mycobacterium standard strains were incubated in liquid culture with PNB and TCH for 7 to 10 days. According to the growth assay for 15 mycobacterium strains and 30 mycobacterium tuberculosis strains, the best PNB and TCH concentration were determined. A total of 424 clinical mycobacterium isolates were identified by microtiter liquid culture at the best PNB and TCH concentration. The results of microtiter liquid culture were compared with those of PCR and DNA sequencing. RESULTS: The best concentration of PNB was 200 µg/ml in microtiter liquid culture. Compared with the results of PCR, the sensitivity and specificity for identification of mycobacterium tuberculosis complex in microtiter liquid culture were 97.8% (306/313) and 100.0% (107/107) respectively and those for non-tuberculosis mycobacteria in microtiter liquid culture were 100.0% (107/107) and 96.5% (306/317) respectively. The best concentration of TCH was 0.5 µg/ml. Compared with the results of PCR, the sensitivity of mycobacterium tuberculosis in microtiter liquid culture was 100.0% (305/305). The specificity remained under and more studies were needed. CONCLUSION: In microtiter liquid culture with PNB and TCH, mycobacteria can be identified in 7 to 10 days. The results were accurate and the process was simple without expensive equipments. This method meets clinical needs and can be used in all level hospitals in China.

[Evaluation of microscopic observation drug susceptibility for drug susceptibility testing of mycobacterium tuberculosis in smear-positive sputum].

OBJECTIVE: To evaluate microscopic observation drug susceptibility (MODS) for mycobacterium tuberculosis drug susceptibility in smear-positive sputum. METHODS: Drug susceptibility of mycobacterium tuberculosis in 275 smear-positive sputum samples collected from TB patients were detected directly by MODS. The susceptibility of seven antimicrobials including streptomycin, isoniazid, rifampicin, ethambutol, levofloxacin, amikacin and capromycin were detected MODS. At the same time the sputum sample were cultured in MGIT 960 tube and the positive isolates were tested for drug susceptibility by MGIT 960 system. The results of MODS were analyzed and compared with that of MGIT 960. RESULTS: Of 275 smear-positive sputum, MODS detected 235 (85.45%). Results of MODS were obtained in a median time of 18 days (5 - 39 d). For the 235 MODS-positive samples, the compliance rates of MODS to MGIT of 7 drugs were 90.21% (212/235), 88.09% (207/235), 93.62% (220/235), 87.23% (205/235), 92.34% (217/235), 88.51% (208/235) and 86.81% (204/235) respectively. The sensitivity of MODS method were 83.33% (90/108), 85.11% (120/141), 90.74% (98/108), 85.71% (78/91), 86.73% (85/98), 76.92% (40/52) and 77.08% (37/48). The specificities of MODS method were 96.06% (122/127), 92.55% (87/94), 96.06% (122/127), 88.19% (127/144), 96.35% (132/137), 91.80% (168/183) and 89.30% (167/187) respectively. CONCLUSION: MODS is an optimal alternative method for direct and rapid drug susceptibility of sputum with high accuracy in a timely and affordable way in resource-limited settings.

[In vitro activities of clofazimine against different drug-resistant types of Mycobacterium tuberculosis].

OBJECTIVE: To study the in vitro antituberculous activities of clofazimine (CLF) to different drug-resistant types of Mycobacterium tuberculosis. METHODS: The minimal inhibitory concentration (MIC) of CLF and isoniazid (INH), rifampicin (RFP), ofloxacin (OFLX), amikacin (AK), and capreomycin (CPM) against sensitive, single-drug resistant (SDR), poly-drug resistant (PDR), multi-drug resistant (MDR), and extensive-drug resistant (XDR) Mycobacterium tuberculosis strains isolated clinically were determined by microplate assays. RESULTS: The MICs of CLF for sensitive, SDR, PDR, MDR and XDR strains of clinically isolated Mycobacterium tuberculosis were 0.06 - 4.00 mg/L, 0.03 - 4.00 mg/L, 0.06 - 8.00 mg/L, 0.06 - 8.00 mg/L, 0.03 - 8.00 mg/L. For the sensitive group, the MIC of CLF (0.06 - 4.00 mg/L) was higher than that of INH (0.06 - 0.25 mg/L) and RFP (0.06 - 0.25 mg/L), while there was no significant difference among OFLX (0.06 - 2.00 mg/L), AK (0.06 - 4.00 mg/L), and CPM (0.50 - 4.00 mg/L). For the single-drug resistant group, there was no significant difference among CLF (0.03 - 4.00 mg/L), INH (0.06 - 0.25 mg/L), and RFP (0.06 - 0.25 mg/L), but the MIC of CLF was lower than that of OFLX (0.25 - 8.00 mg/L), AK (0.06 - 4.00 mg/L), and CPM (0.50 - 8.00 mg/L). For the MDR group, there was no significant difference between CLF (0.06 - 8.00 mg/L) and AK (0.25 - 8.00 mg/L), but the MIC of CLF was lower than that of OFLX (0.125 - 8.00 mg/L) and CPM (0.50 - 8.00 mg/L). For the XDR group, the MIC of CLF (0.03 - 8.00 mg/L) was lower than that of others. CONCLUSION: CLF showed good in vitro activity against Mycobacterium tuberculosis, especially MDR and XDR strains.

[Evaluation of the isothermal RNA amplification assay for Mycobacterium tuberculosis in sputum samples].

OBJECTIVE: To evaluate the use of isothermal RNA amplification assay for detection of Mycobacterium tuberculosis (SAT-TB) in sputum samples. METHODS: A total of 244 sputum samples from patients with pulmonary tuberculosis and those with other lung diseases were detected by SAT-TB and Lowenstein-Jensen (L-J) culture. The samples with different results between SAT-TB and L-J culture were tested by Mycobacterium tuberculosis PCR fluorescence diagnostic kits. The sensitivity and specificity of SAT-TB were calculated according to the results of L-J culture. The detection rates of SAT-TB and L-J culture were analyzed according to the clinical diagnosis and the difference of the 2 methods were analyzed by chi-square test. RESULTS: With the result of L-J culture as the reference, the sensitivity and the specificity of SAT-TB were 92% (71/77) and 86% (143/167), respectively. The accordance rate of SAT-TB and L-J culture was 88% (214/244). For tuberculosis patients, the detection rate of L-J culture and SAT-TB was 42% (75/177) and 54% (95/177) respectively. The difference between the detection rates of SAT-TB and L-J was significant by chi-square test (χ² = 4.527, P < 0.05). CONCLUSIONS: SAT-TB is a rapid, sensitive and specific method for detection of Mycobacterium tuberculosis in clinical sputum samples.

[Screening of the binding peptides of Mycobacterium tuberculosis H(37)Rv].

OBJECTIVE: To screen the Mycobacterium tuberculosis H(37)Rv binding peptide using phage-displayed random peptide libraries, and to analyze the binding capacity of the peptide with Mycobacterium tuberculosis. METHODS: Inactive Mycobacterium tuberculosis H(37)Rv was used for screening of the binding peptide from the Ph.D.-7 peptide library, and Mycobacterium smegmatis was used for reverse screening during the 2(nd) to 4(th) rounds of screening. After 4 rounds of screening, single phages were randomly selected for DNA sequencing. The selected clones were tested by indirect enzyme linked immunosorbent assay (ELISA). The peptide of positive clone, which showed the highest affinity, was synthesized in vitro with fluorescent markers. The specific combination of the peptide with 16 mycobacterium standard strains and 3 other microbes (Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans) were observed by fluorescence microscopy. RESULTS: After 4 rounds of biopanning, remarkable enrichment of the phages that specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 5 sequences were obtained. Five phages with different sequences were detected using indirect ELISA and all of them were found to be positive clones. Phage 8 showed the highest affinity with target molecule. The peptide of phage H8 was synthesized in vitro with fluorescent markers, and it was confirmed that the peptide could bind with H(37)Rv and other 15 mycobacterium including Mycobacterium smegmatis, but not with 3 other microbes. CONCLUSIONS: By using phage-displayed random peptide libraries, we obtained the binding peptide of H(37)Rv. It was shown that the peptide could bind with Mycobacterium tuberculosis specifically, which provided a new way for the detection of Mycobacterium tuberculosis in vitro.

Diagnosis of Alzheimer's Disease Based on Deeply-Fused Nets.

AIM AND OBJECTIVE: Fast and accurate diagnosis of Alzheimer's disease is very important for the care and further treatment of patients. Along with the development of deep learning, impressive progress has also been made in the automatic diagnosis of AD. Most existing studies on automatic diagnosis are concerned with a single base network, whose accuracy for disease diagnosis still needs to be improved. This study was undertaken to propose a method to improve the accuracy of the automatic diagnosis of AD. MATERIALS AND METHODS: MRI image data from the Alzheimer's Disease Neuroimaging Initiative were used to train a deep learning model to achieve a computer-aided diagnosis of Alzheimer's disease. The data consisted of 138 with AD, 280 with mild cognitive impairment, and 138 normal controls. Here, a new deeply-fused net is proposed, which combines several deep convolutional neural networks, thereby avoiding the error of a single base network and increasing the classification accuracy and generalization capacity. RESULTS: Experiments show that when differentiating between patients with AD, mild cognitive impairment, and normal controls on a subset of the ADNI database without data leakage, the new architecture improves the accuracy by about 4 percentage points as compared to a single standard based network. CONCLUSION: This new approach exhibits better performance, but there is still much to be done before its clinical application. In the future, greater research effort will be devoted to improving the performance of the deeply-fused net.

[The effects of gene mutation related to drug resistance and drug efflux pump in extensively drug-resistant tuberculosis clinical isolates].

OBJECTIVE: To explore the effects of 2 major drug-resistant mechanisms in clinically isolated strains of extensively drug-resistant tuberculosis (XDR-MTB). METHODS: Genomic DNA of 10 XDR-MTB strains isolated from Shanghai Pulmonary Hospital were extracted. The main gene mutations related to drug resistance and 15 SNPs unique to XDR-MTB clinical isolate KZN605 reported by the Broad Institute in USA were detected by sequencing. The changes of minimal inhibition concentration (MIC) of XDR-MTB isolates were detected before and after the addition of efflux pump inhibitors verapamil, CCCP and reserpine in liquid cultures. RESULTS: The mutation of rpoB, katG and rpsL occurred in all XDR-MTB strains. The mutation of gyrA, gyrB and rrs occurred in 9 strains, 2 strains and 6 strains respectively. There was no mutation of tlyA in all the strains. Most of the SNPs in KZN 605 strains were not detected in the clinical strains. The clinical strains showed no significant changes of MICs, except 1 strain for which the MIC of ofloxacin decreased by 16 times after addition of the efflux pump inhibitors. CONCLUSIONS: The gene mutations related to drug resistance are the key mechanism for the clinical XDR-MTB strains, while the efflux pumps partly play a role in the drug resistance to fluoroquinolones. The detailed mechanism of efflux pump mediated drug resistance to other anti-TB drugs needs further study.

[Associated factors for the infection of Beijing genotype Mycobacterium tuberculosis].

OBJECTIVE: to analyze the risk factors for the infection of Beijing genotype Mycobacterium tuberculosis (MTB) and the relationship to drug resistance and clinical symptoms. METHODS: sputum samples were collected from patients with pulmonary tuberculosis who were admitted to the hospital during July, 2007 to March, 2008. The sputum was cultured with L-J method, and then the bacterial DNA was isolated and genotyped with VNTR-7 (variable-number tandem repeats, VNTR) and RD105 deletion method respectively. The association between different genotypes and risk factors was analyzed. RESULTS: a hundred and sixteen clinical sputum isolates were obtained from 172 positive sputum cases. There were 112 isolates of MTB, and isolates of non-tuberculosis mycobacterium (NTM). Among the 97 isolates from Shanghai, Zhejiang and Jiangsu areas, Beijing genotype accounted for 86.6% (84/97), and non-Beijing genotype for 13.4% (13/97). The rates of MDR (multi-drug resistance), PDR (poly-drug resistance) and single drug resistance in Beijing genotype were not significantly higher than those in non-Beijing genotype. Among the risk factors, female gender, and CD(4)/CD(8)< 1 in patients with newly-treated tuberculosis, were associated with higher rate of Beijing genotype, chi(2) = 4.436, 4.494 and all P < 0.05, respectively. The Beijing genotype isolates were subdivided into 31 VNTR-7 types, and the distribution of quantity and resistance among different VNTR-7 genotypes was not even. A large number of MTB isolates (47.6%, 40/84) and drug resistant isolates (43.6%, 17/39) were among four VNTR-7 genotypes. CONCLUSION: Beijing genotype is the most prevalent MTB in Shanghai, Zhejian and Jiangsu areas. Female gender and low CD(4)/CD(8) ratio in patients with newly-treated TB are risk factors for infecting Beijing genotype MTB, which may have no relationship with drug resistance and clinical symptoms.

Epileptic Seizure Detection in EEG Signals Using a Unified Temporal-Spectral Squeeze-and-Excitation Network.

The intelligent recognition of epileptic electro-encephalogram (EEG) signals is a valuable tool for the epileptic seizure detection. Recent deep learning models fail to fully consider both spectral and temporal domain representations simultaneously, which may lead to omitting the nonstationary or nonlinear property in epileptic EEGs and further produce a suboptimal recognition performance consequently. In this paper, an end-to-end EEG seizure detection framework is proposed by using a novel channel-embedding spectral-temporal squeeze-and-excitation network (CE-stSENet) with a maximum mean discrepancy-based information maximizing loss. Specifically, the CE-stSENet firstly integrates both multi-level spectral and multi-scale temporal analysis simultaneously. Hierarchical multi-domain representations are then captured in a unified manner with a variant of squeeze-and-excitation block. The classification net is finally implemented for epileptic EEG recognition based on features extracted in previous subnetworks. Particularly, to address the fact that the scarcity of seizure events results in finite data distribution and the severe overfitting problem in seizure detection, the CE-stSENet is coordinated with a maximum mean discrepancy-based information maximizing loss for mitigating the overfitting problem. Competitive experimental results on three EEG datasets against the state-of-the-art methods demonstrate the effectiveness of the proposed framework in recognizing epileptic EEGs, indicating its powerful capability in the automatic seizure detection.

[Detection on drug resistance of Mycobacterium tuberculosis by microscopic observation drug susceptibility].

OBJECTIVE: To evaluate the effects of microscopic observation drug susceptibility (MODS) in detecting susceptibility of Mycobacterium tuberculosis (MTB) onto four first line anti-tuberculosis drugs. METHOD: The 24-hole cell culture plates were used to test drug susceptibility of MTB on liquid medium, and the best detecting condition of MODS assay was probed; 66 clinical isolates susceptibility to streptomycin (S), isoniazid (H), rifampin (R) and ethambutal (E) were evaluated by using MODS assay and Lowenstein-Jensen (L-J), thereafter, all the inconcordance of isolates between MODS and L-J were tested for the minimal inhibitory concentrations (MIC). RESULTS: Concordance rate of the susceptibility to S, H, R and E in 66 clinical isolates detected by MODS and L-J was 97.0%, 90.9%, 95.5% and 86.4% respectively. If the results obtained by L-J were taken as a golden standard, the sensitivity, specificity, positive and negative predictive value (PPV and NPV) as well as accuracy of susceptibility test to S detected by MODS was 96.0%, 97.6%, 96.0%, 97.6% and 97.0%; 100%, 85.4%, 81.0%, 100% and 90.9% to H; 96.2%, 95%, 92.6%, 97.4% and 95.5% to R; 73.7%, 91.5%, 77.8%, 89.6% and 86.4% to E. There were 20 inconsistent results of 16 isolates by comparing MODS with L-J, and MIC yielded 16 results of those 14 isolates showing identical results with those of the MODS, while 4 results of other 4 isolates identical with L-J. CONCLUSION: MODS method simultaneously provides drug susceptibility to S, H, R and E. MODS might be one of the rapid tools to diagnosing multidrug-resistant tuberculosis as it is rapid, simple, inexpensive and has high concordance with L-J drug susceptibility test.

[Identification of the Mycobacterium tuberculosis complex by detecting recombinant secretory protein MPT64].

OBJECTIVE: To identify the Mycobacterium tuberculosis complex by detecting the secretory protein MPT64. METHODS: The gene mpt64 was amplified by polymerase chain reaction (PCR) from the genome of Mycobacterium tuberculosis H(37)Rv strain and cloned into expression vector. Immune sera from rabbits by recombinant proteins MPT64, were used to make enzyme-labeled antibodies and coated antibodies. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the secretory protein MPT64 of the culture supernatants in Mycobacterium strains. Results of ELISA were compared to those of the gene mpt64 amplified by PCR. RESULTS: A recombinant vector was constructed. The minimum detectable concentration of MPT64 was 0.01 mg/L. A total of 27 reference strains and 170 clinical isolate strains were evaluated. PCR for Mycobacterium tuberculosis reference strain, Mycobacterium bovis reference strain and Mycobacterium africanum reference strain was positive, but that for other reference stains was negative, consistent with the results of ELISA. In the 170 clinical isolate stains, the positive result of PCR and ELISA was 98.2% (111/113) and 97.3% (110/113) respectively, while the specificity of PCR and ELISA was both 100%, no positive result in non-tuberculosis mycobacterium strains. CONCLUSION: Identification of the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 is rapid, sensitive, and specific, which can be used routinely in clinical laboratories.

[Expression and purification of CFP32 of Mycobacterium tuberculosis and its serodiagnostic analysis].

OBJECTIVE: To establish a recombinant plasmid of CFP32 of Mycobacterium tuberculosis in E. coli, and to analyze its antigenicity. METHODS: Rv0577 gene was amplified by polymerase chain reaction from genome of Mycobacterium tuberculosis, and then cloned into vector pMD18-T followed by the subclone into the expression vector pET21a. Recombinant CFP32 was expressed and purified. The antigenicity of the recombinant protein was analyzed by using Western-blot. The purified recombinant CFP32 protein was used as an antigen to screen the sera of 7 pulmonary TB patients (n = 97), as well as the other pulmonary disease patients (n = 25), and the clinically healthy controls (n = 38) by ELISA. RESULTS: Recombinant plasmid of CFP32 was established, and be expressed efficiently in E. coli BL21 (DE3). The relative molecular mass of the protein was about 300,000 by SDS-PAGE analysis. The protein purified by Ni-NTA was in a purity over 90%, which was confirmed by Western-blot analysis. ELISA analysis showed its sensitivity and specificity were 63.9% (62/97) and 96.8% (2/63) respectively. CONCLUSION: The recombinant expression plasmid pET21a CFP32 has been constructed and CFP32 proteins has been successfully expressed and be purified in E. coli and, ELISA analysis has identified the recombinant CFP32 as a candidate antigen for TB serodiagnosis.

[Evaluation of pyrosequencing for the detection of rpoB gene mutation in Mycobacterium tuberculosis].

OBJECTIVE: To detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates. METHODS: Using the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC). RESULTS: Among the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively. CONCLUSION: Not only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.

[Selecting and affinity analysis of DNA aptamers to MPT64 protein from Mycobacterium tuberculosis].

OBJECTIVE: To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX technology. METHODS: An in vitro synthesized 78 per random DNA library was subjected to 12 rounds of selection by SELEX (Systematic evolution of ligands by exponential enrichment) method against MPT64 protein. Binding of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. DNAMAN package was employed to analyze the sequences and the second structures of the aptamers. Moreover, target protein was bound to one aptamer and another aptamer modified with biotin together forming a sandwich-like complex, which was captured in microwell, to be tested in negative group including BCG and reference strains from nontuberculous mycobacteria, and positive group including H37Rv, Mycobacterium bovis reference strain, and clinical strains from Mycobacterium tuberculosis. RESULTS: After 12 rounds of selection, high-affinity aptamers to MPT64 was obtained. The OD value at 450 nm of affinity of aptamers to MPT64 protein was from 0. 492 to 1.243, in which 73.3% was over 1.0. Pocket and stem-loops was the basis of aptamers binding to MPT64 protein by the analysis of structures,with several GC pairs among bridges between pocket and stem-loops. The analysis of the sandwich-like complex system based on two aptamers and protein showed that the positive percentage was 87. 9% in the positive group while the negative percentage was 85.7% in the negative group, with positive H37Rv and Mycobacterium bovis, and negative BCG, when the cut-off value for a positive response was 0.61 OD. CONCLUSION: A set of aptamers with considerable binding affinity to MPT64 protein were successfully selected from the initial random DNA library.

[Construction of a human phage display ScFv library against Mycobacterium tuberculosis].

OBJECTIVE: To construct a human phage display single-chain Fv (ScFv) antibody library against Mycobacterium tuberculosis (MTB), for specific ScFv antibody cloning. METHODS: Total RNA was isolated from the lymphocytes of patients with positive serum antibody against MTB and reverse transcribed into cDNA. The heavy chain and light chain variable region gene of human immunoglobulin were amplified individually by PCR and then assembled into ScFv genes. The ScFv genes were ligated into phagemid pCANTAB5S. The human phage display ScFv library against MTB was constructed by transforming the recombinant phagemid into E. coli TG1 with the presence of helper phage M13K07. RESULTS: The human phage display ScFv library containing 10(7) different clones was constructed successfully. CONCLUSIONS: A phage display ScFv library against MTB has been constructed based on the variable region gene of immunoglobulin of the lymphocytes of TB patients and phagemid pCANTAB5S. The specific ScFv antibodies can be screened from this library.