The aim of this study was to build a gene chip system with surface plasmon resonance (SPR) technique, for which Gamma-peptide nucleic acid (Gamma-PNA) functioned as a probe, in order to improve sensitivity and its specificity. With the use of self-assembled monolayer (SAM) technology, surface chemistry of two-dimensional structure was used. Gamma-PNA was designed according to the bioinformatics, and was plated on the SPR chip modified by SAM. Subsequently, relevant parameters of the experiment were ensured and optimized. The results showed that the performances of Gamma-PNA probe was little affected by the ion concentration of buffer, and it had a strong light signal in a stable state. As the ion concentration was 0, there were still good hybrid reactions; pH value had less influence upon Gamma-PNA probe, and acid environment of buffer could be better. Gamma-PNA probe combined with sensor technologies achieved made the probe with dispensable labels and real-time detection. It also improved the efficiency of the hybridization and the stability, providing the foundation for clinical application.
Nucleic Acid Hybridization ; Nucleic Acid Probes ; Oligonucleotide Array Sequence Analysis ; methods ; Peptide Nucleic Acids ; genetics ; Sensitivity and Specificity ; Surface Plasmon Resonance

AIMTo explore a method to replace the isotope probe in the PCR-Select differential screening Kit with DIG labeled probe.

METHODSDifferential expressed sequences isolated from Suppression Subtractive Hybridization (SSH) was translocated onto nitric fibrin film. Probes were prepared with DIG-11-dUTP mixed in by PCR. Hybridization and coloration followed routine operation procedures of DIG labeled probe. Positive hybridization results were verified with reverse Northern blot.

RESULTSThe positive results from the PCR-Select differential screening Kit improved with DIG labeled probe achieved 90% correspondence verified by reverse Northern blot.

CONCLUSIONThe PCR-Select differential screening Kit improved with DIG labeled probe maintained high specificity while avoiding isotope pollution. It can replace isotope probe completely.

DNA Probes ; Digoxin ; Nucleic Acid Hybridization ; Polymerase Chain Reaction ; instrumentation ; methods ; Reagent Kits, Diagnostic

The oligonucleotide microarray, a high-throughput polymorphism detection technology, holds great promise for the characterization of complex genetic variance. To achieve greater sensitivity and specificity for it to be an effective platform technology we present results and discuss some of the factors influencing signal intensities and single-mismatch discrimination in array-based mutation/SNP detection. Probes with a series of concentrations were spotted onto the slide in order to find the optimal concentration with the identifiable satisfying signals and the stable ratios between matched and mismatched probes. It was found that under our experimental conditions, when the initial probe concentration is higher than the maximum immobilization capability of the slide (7.5 micrometer), the hybridization signal will be saturated and the ratio between matched and mismatched probes will be more stable than at a lower probe concentration. Considering the cost of probes and the systematic stability, a constant spotting concentration of 10 micrometer was selected. The stability of different types of mismatched oligo-DNA duplexes on the glass surface was also confirmed. The results show that the order of stability of mismatched oligo-DNA duplexes on a glass surface is in general agreement with previous reports conducted using liquid and polyacrylamide gel pads. This suggests that the influence of the mismatched base pair on the stability of the duplex in a solid hybridization system is similar to that in the solution hybridization environment.
Nucleic Acid Heteroduplexes/chemistry ; *Nucleic Acid Hybridization ; *Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes/chemistry ; *Polymorphism, Single Nucleotide ; Research Support, Non-U.S. Gov't

Multiplex ligation-dependent probe amplification (MLPA) is a semiquantitative analysis based on polymerase chain reaction (PCR). It possesses many advantages such as high efficiency, simple operation, low cost and has been wildly applied in researches of diseases associated with copy number variation, point mutation and methylation. Recently, MLPA is combined with DNA chip to become a real high-throughput method and get great improvement in reliability. Here, the progresses of methods and application of MLPA, as well as its limitations are reviewed.
DNA Methylation ; DNA Probes ; analysis ; genetics ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods ; Oligonucleotide Array Sequence Analysis ; Polymorphism, Single Nucleotide

BACKGROUND: Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes. METHODS: PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain. RESULTS: The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676). CONCLUSIONS: Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.
Cell Wall ; Centers for Disease Control and Prevention (U.S.) ; DNA ; Fluorescence* ; In Situ Hybridization* ; Mycobacterium tuberculosis* ; Nucleic Acid Probes* ; Peptide Nucleic Acids ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sputum

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

OBJECTIVETo develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir.

METHODSTwenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples.

RESULTSThis study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method.

CONCLUSIONThe assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.

Amino Acid Substitution ; Drug Resistance, Viral ; Influenza A Virus, H3N2 Subtype ; drug effects ; enzymology ; genetics ; Mutation ; Neuraminidase ; genetics ; Nucleic Acid Probes ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Communicable Diseases ; diagnosis ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza, Human ; diagnosis ; virology ; Microspheres ; Nucleic Acid Amplification Techniques ; methods ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Probes ; genetics ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

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