Mouse nerve growth factor suppresses neuronal apoptosis in valproic acid-induced autism spectrum disorder rats by regulating the phosphoinositide-3-kinase/serine/threonine kinase signaling pathway.

BACKGROUND: Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by deficits in social communication and restrictive behaviors. Mouse nerve growth factor (mNGF), a neurotrophic factor, is critical for neuronal growth and survival, and the mNGF treatment is considered a promising therapy for neurodegeneration. In light of this, we aimed to evaluate the effect of mNGF on neurological function in ASD. METHODS: An ASD rat model was established by intraperitoneal injection of valproic acid (VPA). Social behavior, learning, and memory of the rats were measured. TdT-mediated dUTP Nick-end labeling and Nissl assays were performed to detect neuronal apoptosis and survival in the hippocampus and prefrontal cortex. Apoptosis-related proteins and oxidative stress markers were detected. RESULTS: mNGF improved locomotor activity, exploratory behavior, social interaction, and spatial learning and memory in VPA-induced ASD rats. In the hippocampus and prefrontal cortex, mNGF suppressed neuronal apoptosis, increased the number of neurons, superoxide dismutase, and glutathione levels, and decreased reactive oxygen species, nitric oxide, TNF-α, and IL-1ß levels compared with the VPA group. In addition, mNGF increased the levels of Bcl-2, p-phosphoinositide-3-kinase (PI3K), and p-serine/threonine kinase (Akt), and decreased the levels of Bax and cleaved caspase-3, while the PI3K inhibitor LY294002 reversed these effects. CONCLUSION: These data suggest that mNGF suppressed neuronal apoptosis and ameliorated the abnormal behaviors in VPA-induced ASD rats, in part, by activating the PI3K/Akt signaling pathway.

A Meta-Analysis on the Impact of Prenatal and Early Childhood Antimicrobial Use on Autism Spectrum Disorders.

OBJECTIVE: To investigate the impact of prenatal and early childhood antimicrobial use on autism spectrum disorders (ASD). DATA SOURCES: We searched PubMed and Embase databases for relevant studies from inception to August 2022. STUDY SELECTION AND DATA EXTRACTION: Peer-reviewed, observational studies were all acceptable. Raw data were extracted into a predefined worksheet and quality analysis was performed using the Newcastle-Ottawa Scale. DATA SYNTHESIS: Nineteen studies were identified in the meta-analysis. Prenatal antimicrobial exposure was not associated with ASD (P = 0.06 > 0.05), whereas early childhood antimicrobial exposure was associated with an increased odds ratio of ASD (OR = 1.17, 95% CI = [1.08-1.27], P value < 0.001). The sibling-matched analysis, with a very limited sample size, suggested that neither prenatal (P = 0.47 > 0.05) nor early childhood (P = 0.13 > 0.05) antimicrobial exposure was associated with ASD. Medical professionals may need to take the possible association into consideration when prescribing an antimicrobial in children. CONCLUSIONS: Early childhood antimicrobial exposure could increase the incidence of ASD. In future studies, it would be necessary to control for confounding factors, such as genetic factors, parenteral age at birth, or low birthweight, to further validate the association.

TCHis mitigate oxidative stress and improve abnormal behavior in a prenatal valproic acid-exposed rat model of autism.

Troxerutin is known for its anti-inflammatory and antioxidative effects in nerve impairment. The purpose of this study is to investigate the effect of troxerutin and cerebroprotein hydrolysate injections (TCHis) on prenatal valproic acid (VPA)-exposed rats. The VPA was administered to pregnant rats on gestational day 12.5 to induce a model of autism. The offspring were given the treatment of TCHis on postnatal day (PND) 21-50. On PND 43-50, the behavioral analysis of offspring was performed after the treatment of TCHis for 1 h. On PND 50, the offspring were harvested and the brains were collected. The hippocampus and prefrontal cortex were isolated for relevant biochemical detections. The administration of TCHis increased pain sensitivity and improved abnormal social behaviors in prenatal VPA-exposed rats. Prenatal exposure of VPA induced neuronal loss and apoptosis, enhanced reactive oxygen species (ROS) production, and promoted oxidative stress in hippocampus and prefrontal cortex, whereas these effects were reversed by the postnatal treatment of TCHis. In addition, postnatal administration of TCHis ameliorated mitochondrial function in hippocampus and prefrontal cortex of prenatal VPA-exposed rats. This study concluded that postnatal treatment of TCHis reduced oxidative stress and ameliorated abnormal behavior in a prenatal VPA-induced rat model of autism.

Electroacupuncture at Zusanli ameliorates the autistic-like behaviors of rats through activating the Nrf2-mediated antioxidant responses.

OBJECTIVE: Emerging evidence suggests that acupuncture plays a neuroprotective role in autism. This study aimed to explore the effect of electroacupuncture at Zusanli (ST36) on autistic-like behaviors and the underlying mechanism. METHOD: Pregnant rats were administered with valproic acid (VPA) on gestational day 12.5 to induce an autism spectrum disorder (ASD) model. The pups were given electroacupuncture at ST36 daily from postnatal day (PND) 28-48. On PND28, the adenoviral vector containing small interfering RNA Nrf2 (Ad-siRNA-Nrf2) was injected into the prefrontal cortex of rats. The behavioral analysis was performed on PND 44-48. On PND48, the animals were euthanized and the brains were collected for further detection. Nissl staining was performed to detect neuronal viability. The biochemical markers of oxidative stress were subsequently measured. RESULT: Electroacupuncture at ST36 ameliorated the locomotor activity, social behavior, spatial learning and memory and repetitive behavior compared with ASD rats. It was notable that the electroacupuncture decreased oxidative stress markers in the tissues of prefrontal cortex, enhanced translocation of nuclear factor erythroid2-related factor2 (Nrf2) from cytoplasm to nucleus, and up-regulated the levels of NADP(H) quinone oxidoreductase (NQO1) and heme oxygenase (HO-1). However, these effects induced by electroacupuncture at ST36 were abolished after injection of Ad-siRNA-Nrf2. CONCLUSION: These data suggested that electroacupuncture at ST36 protected nerve function in ASD rats through Nrf2 activation and the antioxidant response.

2-aminopurine suppresses the TGF-ß1-induced epithelial-mesenchymal transition and attenuates bleomycin-induced pulmonary fibrosis.

The epithelial-mesenchymal transition (EMT) is a multifunctional cell process involved in the pathogenesis of numerous conditions, including fibrosis and cancer. Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by fibroblast accumulation and collagen deposition in the lungs. The fibroblasts involved in this process partially originate from lung epithelial cells via the EMT. Evidence suggests that the EMT contributes to progression, invasion, and metastasis of various types of cancer. We screened a series of 80 compounds for the ability to interfere with the EMT and potentially be applied as a therapeutic for IPF and/or lung cancer. We identified 2-aminopurine (2-AP), a fluorescent analog of guanosine and adenosine, as a candidate in this screen. Herein, we demonstrate that 2-AP can restore E-cadherin expression and inhibit fibronectin and vimentin expression in TGF-ß1-treated A549 lung cancer cells. Moreover, 2-AP can inhibit TGF-ß1-induced metastasis of A549 cells. This compound significantly attenuated bleomycin (BLM)-induced pulmonary inflammation, the EMT, and fibrosis. In addition, 2-AP treatment significantly decreased mortality in a mouse model of pulmonary fibrosis. Collectively, we determined that 2-AP could inhibit metastasis in vitro by suppressing the TGF-ß1-induced EMT and could attenuate BLM-induced pulmonary fibrosis in vivo. Results of this study suggest that 2-AP may have utility as a treatment for lung cancer and pulmonary fibrosis.

Genetic Polymorphisms of SLCO1B1, CYP2E1 and UGT1A1 and Susceptibility to Anti-Tuberculosis Drug-Induced Hepatotoxicity: A Chinese Population-Based Prospective Case-Control Study.

BACKGROUND: Drug transporters and drug-metabolizing enzymes have been linked to drug-induced hepatotoxicity. Solute carrier organic anion transporter family member 1B1 (SLCO1B1), cytochrome P450 2E1 (CYP2E1), and UDP glucuronosyltransferase 1A1 (UGT1A1) were selected as candidate genes to explore their association with susceptibility to anti-tuberculosis drug-induced hepatotoxicity (ATDH). METHODS: Thirty-four tag single nucleotide polymorphisms (tagSNPs) in SLCO1B1, CYP2E1, and UGT1A1 with 10-kb expansion up- and down-stream were genotyped in 461 patients with ATDH and 466 patients without ATDH in a prospective 1:1 matched case-control study. The frequencies and distributions of genotypes and haplotypes were compared between the groups using three genetic models (dominant, recessive, and additive) to identify associations with susceptibility to ATDH. RESULTS: Patients with the rs4149034 G/A, rs1564370 G/C, and rs2900478 T/A genotypes of SLCO1B1 had a significantly lower risk of ATDH, while those carrying the rs2417957 T/T and rs4149063 T/T genotypes had an increased risk. The rs4148323 A/A genotype of UGT1A1 was found to significantly reduce the risk of ATDH. Haplotype analysis showed the TGTG, TTTC, and GTTC haplotypes of SLCO1B1 were associated with an increased ATDH risk, whereas the GACC haplotype was related to a reduced risk. The ATG haplotype of UGT1A1 reduced the risk of ATDH. Moreover, treatment outcomes in tuberculosis patients were further affected by genetic variants of SLCO1B1. CONCLUSIONS: Genetic polymorphisms of SLCO1B1 and UGT1A1 were found to be associated with susceptibility to ATDH. Molecular identification of susceptibility genes provides a theoretical foundation for predicting the likelihood of ATDH and predicting treatment outcomes in tuberculosis patients.

Hot electron-induced cathodic electrochemiluminescence at oil film-covered carbon paste electrode and application to nano-molar determination of catechol.

Hot electron-induced cathodic electrochemiluminescence of the Ru(bpy)(3)(2+)/S(2)O(8)(2-) system was investigated at an oil film-covered carbon paste electrode (CPE) under cathodic pulse polarization for the first time. Compared with other electrodes, the CPE is of lower background, better stability and reproducibility. The method is also applied to the determination of catechol. Under the optimum conditions, the linear correlation between the quenched ECL intensity (ΔI) and the logarithm of catechol concentration (logC(catechol)) was observed over the range of 2.0×10(-10) mol/L-4.0×10(-9) mol/L and 4.0×10(-9) mol/L-4.0×10(-7) mol/L with the limit of detection (LOD) of 2.0×10(-10) mol/L, which is lower than the other reported methods. The proposed method is applied to determine catechol in reservoir water. The mean recoveries of 83.3%-99.0% and the relative standard deviations (RSDs) of 0.8%-2.2% were obtained.

[Study on the correlation between polymorphisms of genes with susceptibility to tuberculosis and drug-resistant tuberculosis in Chinese Han population].

OBJECTIVE: To investigate the distribution of polymorphisms of SLC11A1 gene, VDR gene, MBL gene and IFNG gene with susceptibility to tuberculosis (TB) in Chinese Han population suffering from drug-sensitive TB and drug-resistant TB so as to identify the correlation between gene polymorphisms and the development of drug-resistant TB. METHODS: Single nucleotide polymorphisms (SNP) of VDR gene, SLC11A1 gene, MBL gene, IFNG gene were typed and analyzed by pyrosequencing, Real-time Probe and SNaPshot among 229 patients with drug-sensitive TB and 230 patients with drug-resistant TB. RESULTS: The polymorphic foci of VDR gene from the drug-sensitive TB group and the drug-resistant TB group showed no significant difference (P > 0.05). The genotype of INT4 site and allelic frequency of SLC11A1 gene for drug-sensitive TB group were significantly different from those for drug-resistant TB group (P = 0.031, 0.046). If recessive inheritance was assumed, the genotypes of INT4 site from the two groups were significantly different (OR = 5.756, 95%CI: 1.261 - 26.269, P = 0.011). Considering the relationship between OR values under various combination, our findings confirmed that the genetic mode of INT4 site was in accordance with recessive inheritance. The genotypes of Q/P site and allelic frequencies of MBL gene from drug-sensitive and drug-resistant groups were significantly different (P = 0.029, 0.033). The difference still existed under the hypothesis of recessive inheritance (OR = 9.290, 95%CI: 1.167 - 73.949, P = 0.011). The polymorphic foci of IFNG gene from the two groups showed no significant difference. CONCLUSION: INT4 sites on SLC11A1 gene and Q/P site on MBL gene were probably associated with the development of drug-resistant TB in Chinese Han population. Further study on this issue would be helpful in locating the population at high risk of drug-resistant TB and exploring the effective intervention to decrease the incidence of this disease.

Selection and application of peptide mimotopes of MPT64 protein in Mycobacterium tuberculosis.

Antibody responses can be useful markers of tuberculosis (TB) infection, especially in the screening of extra-pulmonary TB. MPT64 is an important antigen in Mycobacterium tuberculosis (MTB) infection and is used in serological diagnosis. However, large variability in the diagnostic accuracy of MPT64 as a serological tool has limited its application. Phage-displayed random peptide libraries have emerged as a powerful technique to select peptides (epitopes) or mimotopes that may serve as surrogate diagnostic markers in serological tests. In the present study, this method was employed to identify mimotopes of the MPT64 protein of MTB by screening a linear heptapeptide library with rabbit antibodies raised against MPT64 protein. Two antigenic mimotopes (M2 and M6) resembling B-cell epitopes of MPT64 were identified that bound the affinity purified anti-MPT64 polyclonal antibodies and competed with MPT64 for antibody binding. From the results of sequence alignment and a structure modelling figure of MPT64, the sequence of the 2nd to 5th amino acids (DSML) of M2 was totally consistent with the sequence of the 224th to 227th amino acids of MPT64 and the peptide is located on the surface of the space structure of MPT64, suggesting that it might be a linear epitope of MPT64. The recognition of both phage-displayed and synthetic peptides of M2 by the anti-MPT64 polyclonal antibodies also supported this. Although no recurring sequence and no analogue to MPT64 of M6 were found for sequence alignment, the recognition of both phage-displayed and synthetic peptides of M6 by the anti-MPT64 polyclonal antibodies indicated that it might be a mimotope of a conformational epitope of MPT64. According to the results of the reactivity of human sera with synthetic M2 and M6 peptides and MPT64, M2 showed a significantly higher AUC and sensitivity than M6 and MPT64, especially for the sera from sputum-negative TB patients, suggesting that the M2 mimotope may be useful in serological diagnostic testing for TB.

[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.

[The correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of tuberculosis in 459 Han patients].

OBJECTIVE: To study the correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of tuberculosis in Han population. METHODS: Four hundred and fifty-nine tuberculosis inpatients of Han population in Shanghai Pulmonary Hospital from Jan 2007 to Dec 2008 were recruited. The clinical characteristics of tuberculosis (gender, fever, extent of lesions, cavity formation, hemoptysis, initial treatment and retreatment) were observed. The polymorphisms of VDR gene (variants in FokI and TaqI), NRAMP1 gene (variants in INT4, D543N and 3 UTR), MBL gene (variants in HL, YX and QP) and IFNG gene (variants in 874AT) were genotyped by a variety of SNP genotyping techniques. The correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of the disease was analyzed by ANOVAs. RESULTS: The frequency of CC, CT and TT variants of FokI in VDR gene in cases with fever were 54.7% (29/53), 13.2% (7/53) and 32.1% (17/53), respectively, compared to 40.6% (52/128), 30.5% (39/128) and 28.9% (37/128) in cases without fever, the difference being significant (χ² = 6.183, P < 0.05). In patients with CT variants, 15.2% (7/46) had fever, while in patients with non-CT variants, 34.1% (46/135) had fever (χ² = 5.891, P < 0.05), suggesting that patients with CT variants were less likely to have fever. The frequencies of TT + TC and CC variants of QP in the MBL gene in initial treatment cases were 28.3% (60/212) and 71.7% (152/212), respectively, compared to 19.1% (41/215) and 80.9% (174/215) in retreatment cases, the difference being significant (χ² = 5.038, P < 0.05). No significant correlation was observed between the other variants and the clinical characteristics of tuberculosis (χ² = 0.001 - 2.732, P > 0.05). CONCLUSIONS: The polymorphisms of FokI in VDR gene was associated with fever among the clinical characteristics of tuberculosis, and patients with CT variants might be protected from fever. The polymorphisms of QP in MBL gene might be associated with recurrence of tuberculosis.

A reagentless DNA biosensor based on cathodic electrochemiluminescence at a C/C(x)O(1-x) electrode.

A reagentless signal-on electrochemiluminescence (ECL) biosensor for DNA hybridization detection was developed based on the quenching effect of ferrocene (Fc) on intrinsic cathodic ECL at thin oxide covered glassy carbon (C/C(x)O(1-x)) electrodes. To construct the DNA biosensor, molecular beacon (MB) modified with ferrocene (3'-Fc) was attached to a C/C(x)O(1-x) electrode via the covalent bound between labeled amino (5'-NH(2)) and surface functional groups. It was found that the immobilization of the probe on the electrode surface mainly depended on the fraction of surface carbonyl moiety. When a complementary target DNA (cDNA) was present, the stem-loop of MB on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the moving away of Fc from the electrode surface, and the restoring of the cathodic ECL signal. The restoration of the ECL intensity was linearly changed with the logarithm of cDNA concentration in the range of 1.0x10(-11) to 7.0x10(-8)M, and the detection limit was ca. 5.0pM (S/N=3). Additionally, single-base mismatched DNA can be effectively discriminated from the cDNA. The great advantage of the biosensor lies in its simplicity and cost-effective with ECL generated from the electrode itself, and no adscititious luminophore is required.

[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.

[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.

[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.

[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.

[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.