Detecting Micro-luminescence of 330 Codon in emb gene of Mycobacterium tuberculosis by Molecular Beacon Hybridization / 中华医院感染学杂志

OBJECTIVE To design molecular beacon detecting embB330 codon of ethambutol-resistant Mycobacterium tuberculosis(MTB),meanwhile,and try to detect fluorescence of mutation site of embB330 codon in liquid by fluorescence microscope by compareing the mutation strains and standard strains.METHODS The software,Beacon designer,was used to design molecular beacon detecting embB330codon and detecting fluorescence signal from hybridization between the amplified product and probe by fluorescence microscope,and to confer to the sequencing results.RESULTS The difference between PCR products from standard strain and ethambutol-resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope.We detected fluorescent light signal between the 33 ethambutol-resistant strains and 10 H37RV standard strains.The rate of ethambutol-resistant strains was about 3%,and the rate of sequencing was about 3%.CONCLUSIONS The technology of molecular beacon effectually can detect mutation single base site of embB330codon.Fluorescence microscope owns characteristics such as high sensitiveness to detect the fluorescent light.

Quick Detecting Mutation Site of 315 Codon in katG Gene of INH resistant MTB by Stem-ring Molecular Probe in Liquid / 中华医院感染学杂志

OBJECTIVE To detect 315 codon of mutation site in katG of isoniazid(INH)-resistant Mycobacterium tyberculosis(MTB) by stem-ring molecular probe quickly and detect out the fluorescence sign of hybridization between amplified products of katG 315 codon and probe in liquid by fluorescence spectrophotometer.The results were confirmed by sequencing.METHODS The software,Beacon designer,was used to design the katG 315 codon stem-ring molecular probe and the amplification system,and the relationship between the way and sequencing of the amplification products were compared.RESULTS The difference between PCR products from standard strain and INH-resistant one was significant in detecting the fluorescent light by use of fluorescence spectrophotometer.We detected fluorescent light signal between the 16 INH resistant strains and 10 H37RV standard strains.The resistant rate to INH detected was about 44%,and the rate of coincidence was about 97.5%.CONCLUSIONS The stem-ring molecular probe technology show high sensitivety in detecting mutation site of nucleic acid.The rate of coincidence is good between fluorescence spectrophotometer way and sequencing.

Detection of Mycobacterium tuberculosis by Fluorescence Chip of Molecular Beacon Probe / 中华医院感染学杂志

OBJECTIVE To have a research on the detection of Mycobacterium tuberculosis by fluorescence chip of molecular beacon probe.METHODS Fluorescence chip for the detection of M.tuberculosis was designed,based on the better bonding force and specificity of molecular beacon and target molecule.Hybridization efficiency was optimized,which included the concentration and purity of molecular beacon probe,hybridization temperature,hybridization time,the prescription of hybridization solution and the comparing of molecular beacon fluorescence signals of 4 molecules.RESULTS Molecular beacon fluorescence signal approached the highest when the final concentration of Mg was 5 mmol/L and the amplification efficiency of PCR approached the highest.Half extended molecular beacon probe could bind nice with chip when it was middle labeled with quench group.The results of 3 hybridization solutions were as follows: 0.15% SDS better than 0.2% SDS,10?SSC better than 5?SSC.Strong signal of hybridization reaction could be detected when the concentration of Cy3-BDH molecular beacon probe was 15 ?mol/L,the concentration of FAM-DABCLYE probe was 9 ?mol/L and the reaction time was 7 h.The efficiency of fluorescence quenching was higher when the molecule was consisted of Cy3-BDH,other than Cy3-DABCLYE.CONCLUSIONS The design of half extended molecular beacon probe tactfully makes use of the advantages of the high bonding force and specificity of molecular beacon probe on the chip technology.Fluorescence chip of molecular beacon for M.tuberculosis has satisfactory detecting rate,specificity and sensibility.

Detecting 43Codon of High Mutation Site in rpsL of STR Resistant MTB by Molecular Beacons Technology:An Experimental Study / 中华医院感染学杂志

OBJECTIVE To design molecular beacon probe of 43 Codon of high mutation site in rpsL of STR resistant MTB and its amplification system, meanwhile, to find the way of detecting the fluorescent light and making a qualitative judgment by use of fluorescence microscope and image analysis software. METHODS The software, Beacon designer, was used to design the 43 Codon molecular beacon probe and the amplification system, and the fluorescence microscope and image analysis software were used to detect the fluorescent light and make a qualitative judgment. RESULTS The strap of amplification product was shown clearly. The difference between PCR products from standard strain and STR resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope. The rate of resistant STR detected was about 80% (P

Piezoelectric Immunoglobulin Microarray Immunosensor / 中华医院感染学杂志

OBJECTIVE To develop new 2?5 type of piezoelectric immunoglobulin microarray immunosensor for determination of immunoglobulin.The energy converters are 10MHz AT-cut quartz crystals with gold-coated(electrodes).The monoclonal antibodies of immunoglobulin are immobilized onto the surfaces of crystals gold(electrodes) by staphylococcal protein A(SPA).METHODS The standard substance and serum were detected to find the detection time,temperature and specificity of the immunosensor.RESULTS The piezoelectric quartz(crystal) immunoglobulin microarray immunosensor could complete the detection in 15 min without nonspecific(response).Under the optimized conditions,the experimental results showed that the piezoelectric immunosensor had good(response) to immunoglobulin whose frequency shifts were linearly dependent on immunoglobulin (concentration) in different range.The piezoelectric microarray immunosensor had been used to detect(immunoglobulin) in serum,the analytical results given by this method were in satisfactory agreement with those given by traditional (immunoassay),with correlation coefficient of 0.94,0.94,0.94,0.92 and 0.91.(CONCLUSIONS) Piezoelectric microarray immunosensor for the determination of immunoglobulin is of high(sensitivity),high specificity,high analysis speed,unnecessary labelling,simple operation,real-time detection,(repeated) use,etc.It is suitable for (detecting) of immunoglobulin and should be used for clinical detection.

Immobilization of Antibody on Piezoelectric Immunoglobulin Immunosensor / 中华医院感染学杂志

OBJECTIVE To develop a new type of piezoelectric immunoglobulin microarray immunosensor for(determination) of immunoglobulin.METHODS The energy converters were 10MHz AT-cut quartz crystals with gold-coated electrodes.The monoclonal antibodies of immunoglobulin were immobilized onto the surfaces of(crystals) gold(electrodes) by SPA to study the best immobilization yield of the antibodies at different condition.Gold-(protein) A complex was highly stable and was believed to be Van der Waals in nature.We did experiments to find the best temperature-time condition about the binding of SPA and the gold surface and to explore the influence(about) the immobilization of antibody by the concentration and the pH of the antibody solution.RESULTS At the same antibody concentration, it had the highest immobilization yield of SPA at a low-temperature-long-time(combination).The most immobilization yield was obtained at the 5mg/ml concentration of antibody solution.The optimum pH for antibody immobilization was 7.2.CONCLUSIONS SPA for the immobilization of antibody is of(simple) operation,and time-saving.It is a good way to immobilize antibody on the gold electric surface of the quartz(crystal).