Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.     

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization.

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.     

Synthetic oligodeoxyribonucleotide probes for detection of Listeria monocytogenes

A 500-base-pair DNA fragment of a presumptive beta-hemolysin gene of Listeria monocytogenes has been used to identify this organism by a modified colony hybridization technique. We have cloned this DNA fragment into M13 bacteriophage vectors and sequenced it by a dideoxynucleotide sequencing technique. From this sequencing information, several oligodeoxyribonucleotides were synthesized and used as synthetic probes to identify L. monocytogenes. The probes were specific for L. monocytogenes and did not react with any other Listeria strains in a colony hybridization assay. In particular, one of these probes (AD07) was used to detect L. monocytogenes in artificially contaminated raw-milk and soft-cheese samples.     

PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)     

Synthetic oligodeoxyribonucleotide probes for detection of Listeria monocytogenes.

A 500-base-pair DNA fragment of a presumptive beta-hemolysin gene of Listeria monocytogenes has been used to identify this organism by a modified colony hybridization technique. We have cloned this DNA fragment into M13 bacteriophage vectors and sequenced it by a dideoxynucleotide sequencing technique. From this sequencing information, several oligodeoxyribonucleotides were synthesized and used as synthetic probes to identify L. monocytogenes. The probes were specific for L. monocytogenes and did not react with any other Listeria strains in a colony hybridization assay. In particular, one of these probes (AD07) was used to detect L. monocytogenes in artificially contaminated raw-milk and soft-cheese samples.     

Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes

Two general methods which exploit the reactivity of sulfhydryl groups toward maleimides are described for the synthesis of oligonucleotide-enzyme conjugates for use as nonradioisotopic hybridization probes. In the first approach, 6-maleimidohexanoic acid succinimido ester was used to couple 5'-thiolated oligonucleotide to calf intestine alkaline phosphatase to provide a 1:1 conjugate in 80-85% yield. The second strategy employed N,N'-1,2-phenylenedimaleimide to cross-link thiolated horseradish peroxidase or beta-galactosidase with a 5'-thiolated oligonucleotide in 58% and 65% yields, respectively. The oligonucleotide-alkaline phosphatase conjugate was able to detect 6 amol of target DNA in 4 h, while the horseradish peroxidase conjugate was found to be 40-fold lower in its sensitivity of detection by using dye precipitation assays.     

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes

N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.     

Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs

Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.     

Dramatically improved RNA in situ hybridization signals using LNA-modified probes

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.     

Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate

Hybridizations with RNA probes were performed in 3-6 M guanidine thiocyanate concentrations where cells can be solubilized. At these concentrations the melting temperature of hybrids and the optimum temperature of hybrid formation are greatly reduced. The rate of hybridization in GuSCN at room temperature was greater than that in 50% formamide at 42 degrees C. Hybridizations were performed on DNA and RNA in cells dissolved in GuSCN without prior nucleic acid purification.