Consensus PCR and microarray for diagnosis of the genus Staphylococcus, species, and methicillin resistance.

We propose the use of DNA microarray for the discrimination of homologous products after a single PCR amplification with consensus primers. The method was applied to Staphylococcus identification. The femA nucleotide sequences, which are phylogenetically conserved among the staphylococci, were first amplified using a consensus primer pair together with the mecA sequence, a molecular marker for methicillin resistance. Products were then identified on a glass array. The microarray contained five selective DNA capture probes for the simultaneous and differential identification of the five most clinically relevant staphylococcal species (S. aureus, S. epidermidis, S. haemolyticus, S. hominis, and S. saprophyticus), while a consensus capture probe could detect all femA sequences, allowing the identification of the genus Staphylococcus. The mecA sequence hybridized to a specific capture probe. The identification was univocal because only a single capture probe had to be present for each sequence to be identified. The hybridization and identification processes were completed in less than 2 h. Current results demonstrate that low-density microarrays are powerful multigenotypic post-PCR analyzers and could compete with conventional bacteria identification.     

Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.     

Array-based nano-amplification technique was applied in detection of hepatitis E virus

A rapid method for the detection of Hepatitis E Virus (HEV) was developed by utilizing nano-gold labeled oligonucleotide probes, silver stain enhancement and the microarray technique. The 5'-end -NH(2) modified oligonucleotide probes were immobilized on the surface of the chip base as the capture probe. The detection probe was made of the 3'-end -SH modified oligonucleotide probe and nano-gold colloid. The optimal concentrations of these two probes were determined. To test the detection sensitivity and specificity of this technique, a conservative fragment of the virus RNA was amplified by the RT-PCR/PCR one step amplification. The cDNA was hybridized with the capture probes and the detection probes on microarray. The detection signal was amplified by silver stain enhancement and could be identified by naked eyes.100 fM of amplicon could be detected out on the microarray. As the results, preparation of nano-gold was improved and faster. Development time also was shortened to 2 min. Thus, considering high efficiency, low cost, good specificity and high sensitivity, this technique is alternative for the detection of HEV.     

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.     

Focal extraction of surface-bound DNA from a microchip using photo-thermal denaturation

High-throughput, selective extraction of a particular DNA fragment from a mixture of DNA before PCR amplification is becoming increasingly important in the DNA analysis field. Although the latest microchip technology has enabled real-time DNA expression analysis using hybridization between surface-bound probe DNA and sample DNA, the potential of this technology in purification of a small amount of DNA has not been demonstrated. We report here a method for area-selective release and collection of specific DNA, in which an IR laser beam is focused onto surface-bound sample DNA at the target-spotted area to denature hybridized DNA. First, sample DNA labeled with a fluorescent dye was hybridized to a probe DNA immobilized on a chromium-coated chip. A 1053-nm IR laser beam with an intensity of 10-100 mW was then focused on the target area with a spatial resolution of 10 microns, causing the release of the fluorophore-labeled sample DNA as a result of photo-thermal denaturation. Confirmation of the amount of eluted DNA by PCR amplification after collection indicated that more than 10(-20) mol DNA/micron 2 area was eluted from the microchip, representing more than 70% of the chip-bound sample DNA. These results indicate that this method can be applied to the highly sensitive purification of DNA in microchip technology.     

Fabrication of DNA microarrays using unmodified oligonucleotide probes

Microarrays printed on glass slides are often constructed by covalently linking oligonucleotide probes to a derivatized surface. These procedures typically require relatively expensive amine- or thiol-modified oligonucleotide probes that add considerable expense to larger arrays. We describe a system by which unmodified oligonucleotide probes are bound to either nonderivatized or epoxy-silane-derivatized glass slides. Biotinylated PCR products are heat denatured, hybridized to the arrays, and detected using an enzymatic amplification system. Unmodified probes appear to detach from the slide surface at high pH (> 10.0), suggesting that hydrogen bonding plays a significant role in probe attachment. Regardless of surface preparation, high temperature (up to 65 degrees C) and low ionic strength (deionized water) do not disturb probe attachment; hence, the fabrication method described here is suitable for a wide range of hybridization stringencies and conditions. We illustrate kinetics of room temperature hybridizations for probes attached to nonderivatized slides, and we demonstrate that unmodified probes produce hybridization signals equal to amine-modified, covalently bound probes. Our method provides a cost-effective alternative to conventional attachment strategies that is particularly suitable for genotyping PCR products with nucleic acid microarrays.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.     

Highly sensitive and selective colorimetric genotyping of single-nucleotide polymorphisms based on enzyme-amplified ligation on magnetic beads

Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

Hen vitellogenin genes: a study of the distribution of phosvitin phosphoprotein coding sequences using mRNA hybridization with synthetic complementary oligonucleotide probes

Pentadecamer DNA probes were synthesized, having complementary codons for selected unique pentapeptide sequences of low codon degeneracy present in hen phosvitin minor phosphoprotein, hen phosvitin major phosphoprotein, both phosvitin phosphoproteins. These probes were 5'-32P-labelled. Vitellogenin mRNA was isolated from estrogenized chick liver, fractionated by electrophoresis using formaldehyde/agarose gels and blot transferred to nitrocellulose paper. Relative yields of the two vitellogenin mRNAs differed with the extraction method used. The minor phosphoprotein DNA probe formed a hybrid with a 1.6 megadalton component. The remaining two probes hybridized to a 2.3 megadalton component, this being the expected size of a full-length message. The smallest polyadenylated fragment to which the major phosphoprotein DNA probe hybridized was 1.0 megadalton. The remaining two probes hybridized to fragments of 0.7 megadalton and possibly smaller. Phosvitin major phosphoprotein is concluded to be coded for by part of the larger vitellogenin mRNA, while the minor phosphoprotein is coded for by part of the smaller vitellogenin mRNA. Estimates of the distances of the hybridization sites from polyadenylated tails are also given.     

Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.     

一种标记cDNA芯片探针的新方法

探讨mRNA长片段反转录PCR技术(RT-LDPCR)在cDNA芯片微量探针标记和信号放大中的应用.首先提取BEP2D细胞的总RNA,然后用两种不同的方法进行标记,一种为RT-LDPCR,用荧光素Cy3-dCTP进行标记;另一种为传统的RNA反转录,用荧光素Cy5-dCTP进行标记.将两种方法标记好的探针等量混合后与含有440个点(44个基因)的cDNA芯片同时杂交,发现二者具有很高的一致性(0.5＜Cy3/Cy5＞2.0).由于RNA反转录法为cDNA芯片探针标记的传统方法,从而验证了RT-LDPCR用于cDNA芯片探针标记的可行性.RT-LDPCR具有对样品总RNA的需要量少和可对样品中信号进行放大的优点,特别适合于对材料来源受到限制的RNA进行标记.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择