A real-time quantitative PCR based on molecular beacon for detecting Brucella infection / PCR quantitativo em tempo real, baseado em guia molecular para detecção de infeção por Brucella

O objetivo deste comunicado é desenvolver um método quantitativo PCR em tempo real, baseado em guia molecular (MB) (MB-qPCR) para detecção de infecção por espécies de Brucella, e avaliar seu potencial de utilização clínica. Os primers e as sondas MB foram desenhados para amplificação específica e determinação de sequência conservada do código do gene para os primeiros 58-aa da proteína de membrana externa OMP-2a, que é compartilhada em cinco espécies de Brucella epidêmicas. A avaliação metodológica foi realizada por análise de sensibilidade, especificidade, coeficiente de variação intra e inter, e a linearidade do qPCR. O potencial diagnóstico foi avaliado comparando-se o método qPCR desenvolvido com ensaios de exames bacteriológicos convencionais, incluindo os testes de soroaglutinação convencionais (SATs) e os testes do Rosa Bengala (RBPTs). O método exibiu alta sensibilidade (tão baixo quanto 50 cópias) e grande faixa de linearidade (102-108 cópias). Nenhuma reação cruzada foi encontrada com bactéria clínica comum. A sensibilidade diagnóstica foi superior ao exame bacteriológico, e a especificidade diagnóstica foi superior ao SAT ou ao RBPT. Um método MB-qPCR altamente sensível e específico para DNA de Brucella foi estabelecido com sucesso, provando ser uma ferramenta útil no diagnóstico molecular de brucelose.(AU)

To optimize dual molecular beacon detection system for rapid identification of Mycobacterium tuberculosis / 国际检验医学杂志

Objective To optimize the experimental system of dual molecular beacon to rapidly detect My-cobacterium tuberculosis and its resistant strains.Methods Fluorescence quantitative PCR was carried out by selecting different magnesium ion concentration,annealing temperature and primer concentration respectively. Finally,the optimum reaction conditions were obtained.Results In order to ensure the efficiency of amplifica-tion and no non-specific amplification,the final selection of the best conditions were as follows,the concentra-tion of Mg2+was 3.0 mmol/L,annealing temperature was 60 ℃,and the concentration of primers was 0.3 mmol/L.Conclusion The optimal condition of dual molecular beacon experiment was established,which en-sured that the detection of Mycobacterium tuberculosis by molecular beacon quantitative PCR had the advanta-ges,such as simple operation,rapid speed,high sensitivity(the minimum detection limit was 1 CFU/mL)and specificity(only Mycobacterium tuberculosis complex including drug-resistant strains could be detected),good reproducibility(coefficient of variation was < 5%)and other advantages.The study provides the necessary conditions for the dual molecular beacon detection of Mycobacterium tuberculosis.

PCR detection of the Nocardia gene SecA1 using molecular beacon probe / 中国人兽共患病学报

Molecular beacon probe was designed based on a specific DNA sequence of Nocardia to PCR detection of thisbacterium.The strains of Nocardia、Gordina and Rhodococcus were inoculated in Brain Heart Infusion Agar medium separately,then the growth condition was observed,DNA was extracted as a template;the molecular beacon probe was designed based on the partial secA 1 gene sequences of Nocardia strains,and the probe was added into the reaction system of real time fluorescence quantitative PCR (RT-PCR),and the fluorescence signal was tested at the end of PCR.Showed that the amplified secA1 gene of Nocardia could produce positive fluorescence signal in RT-PCR,but those of Gordonia and Rhodococcus with control groups showed negative results because of no fluorescence signal.In conclusion as a housekeeping gene,secA1 is an ideal target molecule to identify the actinomycetes strains on the species level in the systematic evolution research,and the technique of fluorescence molecular beacon probe is accurate,rapid and sensitive for detecting the Nocardia strains with secA1 gene.

Detection of Thrombin Using Double Quenching Molecular Beacon / 分析化学

A double quenching molecular beacon ( MB) with simple structure was designed based on organic quencher and G bases, and a simple detection method for thrombin was developed using this MB. In this MB, FAM and BHQ-1 were selected as fluorophore and organic quencher, three continuous nucleotides with G base were connected with BHQ-1, and the loop of MB was designed as a nucleic acid aptamer of thrombin. In the absence of thrombin, the MB was in the stem-loop structure, the fluorophore FAM was close to BHQ-1 and G bases, the fluorescence of FAM was dual quenched by BHQ-1 and G bases, and the fluorescence signal of FAM was very weak. In the presence of thrombin, MB specifically bound thrombin and formed a G-quadruplex structure. The stem-loop structure of the MB was destroyed, and FAM was separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. Under the optimal conditions, the fluorescence intensity of FAM exhibited a good linear relationship with concentration of thrombin in the range of 0. 4-40. 0 nmol/L, and regression equation was △I=24. 63C (nmol/L)+ 13. 06 (R2 =0. 9972) with the detection limit of 0. 18 nmol/L (3σ, n=9). The average recoveries of this method in serum samples were 96. 3%-98. 7%, which indicated that the method had high accuracy.

Detection of Thrombin Using Double Quenching Molecular Beacon / 分析化学

A double quenching molecular beacon ( MB) with simple structure was designed based on organic quencher and G bases, and a simple detection method for thrombin was developed using this MB. In this MB, FAM and BHQ-1 were selected as fluorophore and organic quencher, three continuous nucleotides with G base were connected with BHQ-1, and the loop of MB was designed as a nucleic acid aptamer of thrombin. In the absence of thrombin, the MB was in the stem-loop structure, the fluorophore FAM was close to BHQ-1 and G bases, the fluorescence of FAM was dual quenched by BHQ-1 and G bases, and the fluorescence signal of FAM was very weak. In the presence of thrombin, MB specifically bound thrombin and formed a G-quadruplex structure. The stem-loop structure of the MB was destroyed, and FAM was separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. Under the optimal conditions, the fluorescence intensity of FAM exhibited a good linear relationship with concentration of thrombin in the range of 0. 4-40. 0 nmol/L, and regression equation was △I=24. 63C (nmol/L)+ 13. 06 (R2 =0. 9972) with the detection limit of 0. 18 nmol/L (3σ, n=9). The average recoveries of this method in serum samples were 96. 3%-98. 7%, which indicated that the method had high accuracy.

Imaging of lung cancer cells with molecular beacons delivered by Chitosan nanoparticles / 解放军医学杂志

Objective: To investigate the effect of a method for imaging lung cancer cells using nanotechnology and molecular beacon (MB) that identifies miR-155 and is delivered by chitosan nanoparticles (CS). Methods: The miR-155 MB modified by locked nucleic acids (LNAs) was designed and synthesized. The CS-MB complex was synthesized by self-assembly method and tested for its physicochemical properties including anti-DNase I features, particle size, zeta potential and so on. The miR-155 MB was transfected with CS as vectors. The abilities of miR-155 MB to identify miR-155 and thus to image lung cancer cells were determined by confocal microscopy. Furthermore, the miR-155 expression levels were detected by qRT-PCR to validated the effect of miR-155 MB. The random sequence molecular beacon (RS MB) was set as a negative control. Results: The CS-MB complex at the weight ratio of 7:1 was best suited for transfection due to its high encapsulation rate, resistance to the degradation by DNase I, small particle size and positive charge. Relatively strong red fluorescence could be detected in the lung cancer cells after transfection of miR155 MB, while that could not be detected in the RS MB group (P<0.05). Moreover, the changing trend in the fluorescence intensity was consistent with that in the miR-155 expression levels. Conclusion: CS nanoparticles can be used as vectors to deliver miR-155 MB for miR-155 identification and lung cancer cell imaging, thus providing new ideas and novel technique for lung cancer diagnosis.

Fluorescent Detection of Specific DNA Sequences Related to Toxoplasma gondii Based on Magnetic Fluorescent Nanoparticles Fe3O4/CdTe Biosensor.

Magnetic fluorescent nanoparticles (Fe3O4/CdTe) were prepared in this work and applied for Toxoplasma gondii DNA detection. First, CdTe quantum dots were synthesized with 3- mercaptopropionic (MPA) capped. Fe3O4 magnetic particles were prepared by hydrothermal method with NaOH as precipitator, and they were surfacely modified with silane coupling agent (KH550). After then, the MPA-capped CdTe QDs were immobilized on the Fe3O4 particles surface via electrostatic interaction, and the Fe3O4/CdTe particles were prepared with the average size of 10 nm. The DNA sensing probe was fabricated through labeling a stem-loop Toxoplasma gondii DNA oligonucleotides with Fe3O4/CdTe (donor) at the 5′ end and BHQ2 (acceptor) at 3′ end, respectively. The assembly prosess was verified by UV-Vis, TEM, IR, XRD etc. The sensitivity characterization of the molecular beacon probe was performed by fluorescence spectrum (FS) with a detection limit of 8.339x10-9M. This chemical strategy can be further applied to prepare the magnetic nanoparticles for DNA detection.

Highly Sensitive Fluorescent Aptasensor for Thrombin Detection Based on Competition Triggered Rolling Circle Amplification / 分析化学

Based on the competition reaction of target protein, aptamer probe, padlock probe and complementary sequence, a highly sensitive fluorescent aptasensor was developed in this study in combination with rolling circle amplification. In the absence of target protein, the ligation-rolling circle amplification reaction was repressed because the complementary sequence hybridized with aptamer probe to form double-stranded duplex. While in the presence of target protein, the target molecules bound specifically with aptamer probe, inducing displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of E. coli DNA ligase, and the rolling circle amplification process could be accomplished by Phi 29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacon ( the detection probes) , resulting in a significant fluorescence signal. The effects of length of complementary DNA ( CDNA ) sequence and concentration of padlock probe were investigated. Under the optimized experimental conditions, the model target protein thrombin could be highly sensitively detected by the proposed aptasensing system in a linear range of 0 . 067-32 . 4 nmol/L with a detection limit of 0 . 03 nmol/L ( approximately 90 amol target molecules). Moreover, the presented sensing method was universal for other target analysis by skillfully design of the sequence of aptamer probe and related oligonucleotides.

Detection of Specific DNA Sequence with a Non-labeled Molecular Beacon and a Nucleic Acid Dye Hoechst 33258 / 分析化学

A highly sensitive and selective method for specific DNA sequence detection is developed using a non-labeled molecular beacon (MB) and a nucleic acid dye Hoechst 33258. It is demonstrated by a specific DNA sequence of wild-type HBV as a model system. In this strategy, the stem of MB is completely designed as C/G base pairs. In the absence of target DNA, the interaction between Hoechst 33258 and the MBs is very weak,and the fluorescence signals of Hoechst 33258 is very low. In the presence of target DNA, the MBs hybridize with the target DNA and form double-stranded structure. Hoechst 33258 binds to dsDNA, and the fluorescence intensity is significantly enhanced. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibits good linear dependence on target DNA concentration in the range of 2 × 10-10-2 × 10-8 mol/L. The fitted regression equation is △I=3. 3439C(10-10 mol/L) ﹢18. 6949(R2=0. 9982) with a correlation coefficient of 0. 9982 (R2), and the detection limit is 9 × 10-11 mol/L (3σ). The proposed method has good precision, simple operation, fast detection speed, low detection limit, high accuracy and high sensitivity.

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.

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

Progress in Aptazymes / 生物化学与生物物理进展

Aptazymes are a new artificial synzyme, selected from the random oligonucleotide sequence libraries against various of effector molecules. They own the advantages of an aptamer(the receptor site) and the ribozyme (the catalytic active site). Moreover, aptazymes as catalytic molecular beacon provide a new orientation for the quantitative analysis of effector molecules. Aptazymes not only have the application in genomics and proteomics, but also have potential applications in biosensor and DNA AND gate.

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.

REAL-TIME DETECTION OF SURVIVIN mRNA EXPRESSION IN CERVICAL CANCER CELL LINES USING MOLECULAR BEACON IMAGING / 药物分析学报

Objective To detect the expression of survivin mRNA in cervical cancer cell lines using molecular beacon imaging technology. Methods Human cervical cancer cells (HeLa and SiHa) and human fetal lung fibroblast HFL-I were cultured in vitro. After adding 100 nmol/L survivin mRNA molecular beacon, the fluorescent signals were observed under fluorescent microscope. The expressions of survivin in cervical cancer cells and HFL-I cell were examined by immunocytochemical streptravidin-biothin peroxidase (SP) assay at the same time. Results Two kinds of survivin mRNA molecular beacon, with different color fluorescence, had strong fluorescent signal in cervical cancer cell lines, and the signal in SiHa cell line was stronger, but these signals were not found in HFL-I ; Immunocytochemical staining of positive survivin was located in the cytoplasm of cervical cancer cell lines HeLa and SiHa, whereas, no expression of survivin was detected in HFL-I cell line. Conclusion The technology of molecular beacon imaging can be used to detect the expression of survivin mRNA in viable cells successfully, and may provide a new approach to the diagnosis of early stage cervical cancer and the following-up in the clinic.

Establishment of Real-Time PCR and Molecular Beacon Detection Method for Bifidobacteria / 微生物学通报

0.97).The method of Real-time PCR and molecular beacon detection for bifodobacteria has many advantages,such as being sensitive,specific,simple and fast,and this method can be used in situ detection of bifidobacteria quantitatively.