BACKGROUND: A peptide nucleic acid (PNA) probe-based real-time PCR (PNAqPCR(TM) TB/NTM detection kit; PANAGENE, Korea) assay has been recently developed for the simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) in clinical specimens. The study was aimed at evaluation of the performance of PNA probe-based real-time PCR in respiratory specimens. METHODS: To evaluate potential cross-reactivity, the extracted DNA specimens from Mycobacterium species and non-mycobacterial species were tested using PNA probe-based real-time PCR assay. A total of 531 respiratory specimens (482 sputum specimens and 49 bronchoalveolar washing fluid specimens) were collected from 230 patients in July and August, 2011. All specimens were analyzed for the detection of mycobacteria by direct smear examination, mycobacterial culture, and PNA probe-based real-time PCR assay. RESULTS: In cross-reactivity tests, no false-positive or false-negative results were evident. When the culture method was used as the gold standard test for comparison, PNA probe-based real-time PCR assay for detection of MTBC had a sensitivity and specificity of 96.7% (58/60) and 99.6% (469/471), respectively. Assuming the combination of culture and clinical diagnosis as the standard, the sensitivity and specificity of the new real-time PCR assay for detection of MTBC were 90.6% (58/64) and 99.6% (465/467), respectively. The new real-time PCR for the detection of NTM had a sensitivity and specificity of 69.0% (29/42) and 100% (489/489), respectively. CONCLUSIONS: The new real-time PCR assay may be useful for the detection of MTBC in respiratory specimens and for discrimination of NTM from MTBC.
Bronchoalveolar Lavage Fluid/microbiology ; DNA Probes/chemistry/metabolism ; DNA, Bacterial/*analysis ; Humans ; Molecular Typing/*methods ; Mycobacterium tuberculosis/*genetics/isolation & purification ; Nontuberculous Mycobacteria/*genetics/isolation & purification ; Nucleic Acid Hybridization ; Peptide Nucleic Acids/chemistry/*metabolism ; *Real-Time Polymerase Chain Reaction ; Respiratory System/*microbiology ; Sputum/microbiology

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

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Female ; Ferric Compounds ; chemistry ; Genes, erbB-2 ; genetics ; Humans ; Magnetics ; Microscopy, Atomic Force ; methods ; Molecular Probe Techniques ; Nucleic Acid Probes ; chemistry ; genetics ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; ultrastructure ; RNA, Messenger ; genetics ; metabolism

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

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

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.
Bacteremia ; diagnosis ; genetics ; Bacterial Typing Techniques ; Bacteriological Techniques ; DNA Probes ; Genetic Techniques ; Listeria monocytogenes ; metabolism ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotides ; chemistry ; RNA, Ribosomal ; chemistry ; RNA, Ribosomal, 23S ; genetics ; Stem Cells

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

Two non-radioactive probes using digoxigenin or biotin were developed for detecting canine herpesvirus (CHV) and compared for their sensitivities by in situ hybridization (ISH) in formalin fixed, paraffin embedded sections, which has been used routinely in veterinary fields. Sections of the CHV-infected cell preparation were subjected to several different ISH protocols using digoxigenin- or biotin-labeled probe respectively. Results were compared for the hybridization and background signal intensities. The best result was obtained by the optimized ISH protocol using digoxigenin-labeled probe for detection of CHV DNA. The optimized ISH assay, which developed in this study, may be a valid tool for the study of pathogenesis and diagnosis of CHV infection.
Animals ; Biotin/diagnostic use ; Cell Line ; DNA Probes/chemistry/genetics ; DNA, Viral/chemistry/genetics ; Digoxigenin/diagnostic use ; Dog Diseases/diagnosis/*virology ; Dogs ; Herpesviridae Infections/diagnosis/*veterinary/virology ; Herpesvirus 1, Canid/genetics/*isolation&purification ; In Situ Hybridization/methods/*veterinary ; Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity

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

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