Biomedical subjects

Xiao-jun Yan

Publications and source records attributed to Xiao-jun Yan.

5 recordsLinked to original sources

PubMed2005

[A rapid and high throughput method for apoE genotyping].

OBJECTIVE: To develop a new method that can determine the apolipoprotein E(apoE) genotypes rapidly in high throughput. METHODS: Genome DNA samples were extracted from the anticoagulated peripheral blood samples of 79 patients with Alzheimer's disease(AD) and 63 healthy individuals, and the 492 bp apoE gene fragments including 112 and 158 codons were amplified by polymerase chain reaction (PCR). With one PCR product, three recombined alleles (epsilon 2, epsilon 3 and epsilon 4) of apoE gene as controls were obtained by cloning and site-directed mutagenesis. The excess primers and dNTPs in all PCR products were removed by treatment with clean up reagents, then template-directed dye-terminator incorporation reaction (TDI) was performed and R110 or TAMRA labeled Acyclo-terminators were added into the mutation sites specifically. Fluorescence polarization value (FP) was measured using victor 2 multilabel counter and the polymorphisms in 112 and 158 condons of apoE gene were investigated. RESULTS: The apoE genotypes in recombined plasmid controls and all serum samples were analyzed using the authors' TDI-FP method, and the reliability and specificity were confirmed by DNA sequencing. The frequency of epsilon 4 allele in patients was significantly higher than that in controls, suggesting that apoE epsilon 4 allele gene is a risk factor for late-onset AD. CONCLUSION: TDI-FP is an easy, reliable and high throughput technology in analyzing polymorphism of apoE gene; it can be used in the prediction of susceptibility to AD in elderly individuals. Furthermore, it is an ideal method for large-scale screening and for studying the relationship between the allelic and genotypic frequencies of apoE and other diseases.

Aged↗

PubMed2005

[Detection of antibody against Helicobacter pylori UreB by fluorescence polarized immunoassay using single epitope synthetic peptide as antigen].

AIM: To develop a new method for antibody detection based on fluorescence polarization (FP) technique. METHODS: BALB/c mice were immunized with single epitope synthetic 8 branches peptide antigen (SVEVGKVADL)8 of Helicobacter pylori (Hp) UreB protein. FP values of the corresponding linear peptide antigen labeled with FITC in different concentration were measured to determine the optimal concentration of the antigen. The antigenicity of the synthetic linear peptide was identified by FP assay. Then, the antibody-positive and negative murine sera were used in FP assay to determine the optimal dilution factor of serum samples. To apply FP technique in Hp antibody detection, 126 human serum samples were detected either by FPIA (fluorescence polarized immunoassay) using the single epitope linear synthetic peptide as antigen or by a commercial ELISA kit. Then, receiver operating characteristic (ROC) curve analysis was performed on the FPIA results by MedCalc software. RESULTS: The UreB single epitope peptide had strong antigenicity. 1.0 nmol/L of the synthetic linear peptide labeled with FITC was the optimal concentration of the antigen and the optimal dilution factor of serum sample was 1:25. In 77 Hp antibody-positive serum samples detected by ELISA, 66 samples were positive by FP detection method. The sensitivity and specificity of the FPIA assay was 85.7% and 98%, respectively. CONCLUSION: The single epitope synthetic peptide antigen can be used in FP method for rapid detection of serum antibody against Hp UreB. The established FP assay for antibody detection may be used in clinical diagnosis in the future.

Animals↗

PubMed2005

[Rapid detection of rifampin resistance of Mycobacterium tuberculosis using high-throughput pyrosequencing technique].

OBJECTIVE: To develop a pyrosequencing approach to rapid detection of rifampin resistance in Mycobacterium tuberculosis based on characterization of all possible mutations in the 81 bp core region. METHODS: Two pyrosequencing sequencing primers and 1 pair of PCR primers were chosen for pyrosequencing analysis. The sensitivity of the pyrosequencing approach was determined by assaying PCR products generated from 10-fold serial dilutions of the DNA from Mycobacterium tuberculosis H(37) Rv strains. The efficacy of the pyrosequencing approach was evaluated by analyzing clinical Mycobacterium tuberculosis isolates with known antibiotic phenotypes. RESULTS: Rifampin resistance could be determined within 2 hours after PCR amplification and single-stranded template preparation by using only two pyrosequencing reactions. About 50 fg DNA/reaction was required in order to get sufficient PCR product for producing a long, clear and accurate pyrosequencing pattern. A total of 41 Mycobacterium tuberculosis isolates were tested and the results were concordant with those based on drug susceptibility testing and conventional DNA sequencing. CONCLUSION: The pyrosequencing technique is simple to perform and can be used as a rapid, high-throughput and efficient method for detecting rifampin resistance in Mycobacterium tuberculosis.

Antitubercular Agents↗

PubMed2004

[Detection of KatG 315 gene mutation associated with isoniazid resistance in Mycobacterium tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection technology].

OBJECTIVE: To study the relationship between KatG 315 mutation and isoniazid (INH) resistance in M. tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP), and to develop a new method that can detect INH-resistant M. tuberculosis precisely and quickly. METHODS: Isolates of M. tuberculosis resistant to INH from 82 tuberculosis patients were cultured on culture medium with different INH concentrations. DNA of all M. tuberculosis samples was extracted and amplification of 271 bp KatG gene fragment was carried out by polymerase chain reaction (PCR). After digestion of the excess primers and dNTPs in PCR products by clean-up reagents, template-directed dye-terminator incorporation reaction was performed, and the Acyclo-terminators labeled Rhodamine 110 (R110) or 6-carboxytetramethylrhodamine (TAMRA) were incorporated into the detection primers specifically. Then fluorescence polarization value was measured using Victor2 multi-label counter and the genotypes of the mutation site in 315 condon of KatG gene of all samples were investigated. RESULTS: There were 15 out of the 29 isolates of M. tuberculosis with high INH-resistance showed G-->C mutation in 315 condon of KatG gene, including 4 isolates with both mutated and non-mutated trains, the mutation frequency being 52%. In 32 isolates of M. tuberculosis with low INH-resistance, 15 were mixed infection, the mutation frequency being 47%. No mutation in 315 condon of KatG gene was found in 21 susceptible isolates of M. tuberculosis. CONCLUSIONS: The G-->C mutation in 315 condon of KatG gene was associated with INH-resistance of M. tuberculosis. TDI-FP technology was a reliable, easy to use and high-through method to detect mutation of KatG gene. It may be a useful technique for the diagnosis of INH-resistance in the future.

Antitubercular Agents↗

PubMed2004

[Detection of HPV 58 and cloning of its E7 gene in cervical cancer].

OBJECTIVE: To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer. METHODS: HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3) cells, and induced to express 58 E7 protein by IPTG. RESULTS: Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M(r) 16 x 10(3) was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins. CONCLUSION: HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.

Adult↗