In this experiment, a novel biotin-avidin conjugation probe was synthesized and employed in the detection of reverse-phase protein microarray. Firstly, the proportion of the biotin-avidin conjugation probe was optimized. Then the rat IgG and goat anti-rat IgG system was served as a model to optimize the fabrication conditions of reverse-phase protein microarray, including the non-specific absorption of streptavidin-Cy3 molecules, spotting buffer as well as protein activities. At last, the biotin-avidin conjugation probe was applied to the detection of the reverse-phase protein microarray. The results show that the protein microarray prepared by using BSA spotting buffer could prevent non-specific absorptions of fluorescent molecules and improve the sensitivity, effectively. In addition, compared with traditional biotin-avidin system, the detection limit could be improved four times using the biotin-avidin conjugation probe. In conclusion, the biotin-avidin conjugation probe has its merits of easy synthesis, low price and could be further conjugated with other signal amplification techniques, which is promising to be used in the detection of protein microarray.
Avidin ; chemistry ; Biotin ; chemistry ; DNA Probes ; Immunoglobulin G ; analysis ; immunology ; Protein Array Analysis ; methods

OBJECTIVETo investigate the specificity of the dual-functionalized nanoparticles probes (NPs) self-assembled with colloidal gold.

METHODS13-nm gold nanoparticles were prepared with citrate reduction of HAuCl(4). These gold nanoparticles were sequentially functionalized with the specific single-strand oligonucleotide of HA gene of influenza A virus (H1N1) and disulfide molecules of m/z at 693. The NPs solution showed the red formation. The magnetic microparticles (MPs) were modified with another specific single-strand oligonucleotide in HA gene of H1N1. The sandwich complexes (MP-Target-NPs) were formed by the target DNA with the MPs and the NPs. The color change in the solution was observed and the dehybridization product was detected by MALDI TOF MS. Moreover specificity of the probes was investigated with nano-water (as a blank control) and the different target DNAs including complementary DNA,non-complementary DNA and two DNAs of one base mismatch, respectively.

RESULTThe red formation and the positive signal in MS detection of reporter mass code 693 ([M+Na](+)) were observed,which indicated the formation of sandwich complexes formed only when the completely complementary target DNAs were presented in the solution. No color formation changes and no peak signal detected by MALDI TOF MS were observed,showing that none of target of interest (nano-pure water),non-complementary DNA and two DNAs of one base mismatch existed in the systems,which indicated no sandwich complexes formed between the target DNAs and the two probes.

CONCLUSIONConsidering the simple preparation procedure and high specificity,the dual-functionalized gold nanoparticle probes would be widely and increasingly used in nucleic acid analysis. In particular,it would have broad application prospects in early diagnosis of diseases,single nucleotide polymorphism (SNP) typing and so on.

DNA Probes ; chemistry ; Gold Colloid ; chemistry ; Influenza A Virus, H1N1 Subtype ; genetics ; Metal Nanoparticles ; chemistry ; Oligonucleotides ; genetics ; Sensitivity and Specificity

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.
Animals ; DNA ; genetics ; DNA Probes ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Humans ; Isotope Labeling ; methods ; Metagenomics ; methods ; Molecular Probe Techniques ; Sequence Analysis, DNA ; methods

We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle (AuNP) probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3' end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5' end of target DNA, and two biotin-labeled signal probes (T10 and T40) with different lengths. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a "sandwich" format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.
Colorimetry ; methods ; DNA Probes ; chemistry ; genetics ; DNA, Bacterial ; genetics ; Gold ; chemistry ; Humans ; Metal Nanoparticles ; chemistry ; Mycobacterium tuberculosis ; isolation & purification ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; methods

OBJECTIVETo establish a new nucleic acid hybridization detection technique which may be used in medical genechips.

METHODSThe specific DNA fragment was detected by sequential two hybridization of fluorescence probe with template DNA and fixed DNA probe.

RESULTSFluorescence probe two-hybridization (FPTH) was applied to genechips for the detection of sex-transmitted pathogens from culture strains, and the results showed that the values of fluorescence density of the positive groups decreased remarkably when compared with those of the negative group. Both the sensitivity and specificity for detecting clinical samples are higher than 90%. There is no need of any additional reagent in hybridization procedure, and the hybridization detection can be accomplished in 40 minutes.

CONCLUSIONThe FPTH technique is rapid, simple and reliable, it can also make the clinical detection process completely automatic and integrative.

DNA Probes ; chemistry ; genetics ; DNA, Bacterial ; genetics ; Fluorescent Dyes ; chemistry ; Humans ; Neisseria gonorrhoeae ; genetics ; Nucleic Acid Hybridization ; methods ; Ureaplasma urealyticum ; genetics

OBJECTIVETo develop a simple and reliable method for intensifying the hybridization signals of gene chips.

METHODSThe authors added EDTA and another FAM-labeled probe to the normal PCR products, denatured the mixture by heat, and then let the mixture hybridize with the fastened probes on the chip.

RESULTSWith the use of EDTA and another FAM-labeled probe, the hybridization signals increased by 6 times or greater.

CONCLUSIONAdding EDTA and another probe to the normal PCR products is a simple and efficient method to intensify the hybridization signal of chips.

Base Sequence ; DNA Probes ; chemistry ; genetics ; Edetic Acid ; chemistry ; Fluorescent Dyes ; chemistry ; Microscopy, Fluorescence ; Molecular Sequence Data ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

OBJECTIVEEstablish the fluorescent quantitative RT-PCR detection method for rabies virus (RV) and construct RNase-resistant virus-like particles as positive controls.

METHODSAnalyze the database in GenBank, the probe and the primers were designed in the conservative region of N gene of rabies virus and the method of real-time fluorescent quantitative PCR was obtained; On the basis of MS2 phage, with the technology of gene recombination, prepare the RNase-resistant virus-like particles for RV positive controls;

RESULTSRNase-resistant virus-like particles were obtained after prokaryotic expression in E. coli. The designed primers and probe were confirmed to be very specific and conservative, and be sensitive to-concentration of 15 copies/microl.

CONCLUSIONEstablished the method of detecting rabies virus by reverse transcription real-time quantitative PCR,obtained the RNase-resistant and no infectivity virus-like particles as positive controls of rabies virus.

Animals ; DNA Probes ; DNA, Viral ; Dogs ; Humans ; RNA, Viral ; analysis ; Rabies ; virology ; Rabies virus ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Ribonuclease, Pancreatic ; metabolism ; Viral Load

OBJECTIVETo detect the methylenetetrahydrofolate reductase(MTHFR) gene C677T mutation with molecular beacon technique and assess the revant applicability.

METHODSA total of 228 samples were analyzed using molecular beacons which are oligonucleotide probes to become fluorescent upon hybridization. Wild-type molecular beacon and mutant beacon were designed to detect the genotypes of MTHFR gene.

RESULTSAnalysis of the 228 samples indicated that there were three genotypes including 41 homozygous mutants, 113 heterozygous individuals and 74 wild-type individuals. Every sample was identified clearly.

CONCLUSIONThe present method, a closed-tube PCR/hybridization assay, is a simple, high-throughput and fast procedure that is fully automated for detecting gene mutation.

DNA Mutational Analysis ; methods ; Fluorescent Dyes ; chemistry ; Genotype ; Humans ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Oligonucleotide Probes ; chemistry ; genetics ; Point Mutation ; Sensitivity and Specificity

One step TaqMan reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probe was developed for detection of Japanese encephalitis virus (JEV). Real-time RT-PCR was optimized to quantify JEV using the detection system (Rotor Gene 2000 detector) and dual-labeled fluorogenic probes. The gene specific labeled fluorogenic probe for the 3' non-translated region (3' NTR) was used to detect JEV. When the specificity of the assay using specific JEV primers was evaluated by testing three different JEV strains, other swine viruses and bovine viral diarrhea virus, no cross-reactions were detected with non-JE reference viruses. A single tube TaqMan assay was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method. Detection limits of two step and real-time RT-PCR for JEV were 112 TCID50 /ml and 11.2 TCID50 /ml, respectively. Quantification of JEV was accomplished by a standard curve plotting cycle threshold values (Ct ) versus infectivity titer. Real-time RT-PCR assay using single tube method could be used as a sensitive diagnostic test, and supplied the results in real time for detection and quantification of JEV. We could detect JEV RNA genome in plasma samples of pigs inoculated with KV1899 strain at 2 days post inoculation, but couldn't in 41 fetus samples. This assay was sensitive, specific, rapid and quantitative for the detection of JEV from laboratory and field samples.
Animals ; DNA Primers/chemistry ; DNA Probes/chemistry ; Encephalitis Virus, Japanese/genetics/*isolation&purification ; Encephalitis, Japanese/diagnosis/*veterinary/virology ; RNA, Viral/analysis ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction/methods/*veterinary ; Sensitivity and Specificity ; Swine ; Swine Diseases/*diagnosis/virology ; *Taq Polymerase

OBJECTIVETo establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).

METHODSThe probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed.

RESULTSThe sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01).

CONCLUSIONSThe method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase ; chemistry ; metabolism ; Animals ; DNA Probes ; chemistry ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; blood ; diagnosis ; virology ; Hepatitis B virus ; genetics ; Humans ; Molecular Diagnostic Techniques ; methods ; standards ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity