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At least 19 recordsLinked to original sources
PubMed1991

Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections.

A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and 35S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

DNA Probes, HPV↗
PubMed1989

Simultaneous detection of Escherichia coli heat-stable and heat-labile enterotoxin genes with a single RNA probe.

A single RNA probe was synthesized and used to detect simultaneously the methanol-soluble heat-stable enterotoxin and heat-labile enterotoxin genes in Escherichia coli strains. The results with the biotinylated or radioactive probe correlated 100% with the biological assay results for both toxins. The RNA probe detected the three known heat-stable enterotoxin A alleles.

Alleles↗
PubMed1989

Immunodetection of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to DNA-RNA hybrids.

A quantitative, nonisotopic hybridization assay which measures specific DNA-RNA hybrids is described. A biotinylated RNA probe is reacted in solution with a DNA target and the labeled hybrids are immobilized onto a solid phase surface with an antibiotin antibody. Bound hybrids are detected with a beta-galactosidase-labeled monoclonal antibody against DNA-RNA hybrids and are quantitated with the addition of a fluorogenic substrate. In a model system using pSP65 or pGEM4 plasmids and transcripts, biotinylated RNA probes allowed detection of 5 pg of DNA in 10(6) pg of exogenous nucleic acids in 1000 min. Signals generated in the system depended on input target length. A nucleic acid target of 25 bases was still detectable in the assay. Human immunodeficiency virus type 1 (HIV-1) DNA was amplified in the polymerase chain reaction with Taq polymerase and a set of primers for the pol gene, one of which contained T7 RNA polymerase promoter sequences. A HIV-RNA probe of 326 bases was transcribed with T7 RNA polymerase using polymerase chain reaction (PCR) amplified DNA as a template. The RNA probe of 326 bases performed as well as a RNA probe of 2588 bases for detection of a DNA segment of 355 bp. For detection of dilutions of HIV-1 with PCR, a set of primers (outer set) was used for amplification of HIV-1 DNA. In a separate reaction a set of primers nested between the first set generated through PCR an amplified DNA fragment with the T7 promoter. This fragment was transcribed for the synthesis of a biotinylated RNA probe. This probe could then be reacted with material amplified with the outer set of primers. Ten copies of HIV-DNA could be detected with this procedure.

Antibodies, Monoclonal↗
PubMed1989

Sex determination of bloodstains by the use of a ribonucleic acid (RNA) probe.

An asymmetric ribonucleic acid (RNA) probe, which represents a Y-chromosome specific nucleotide sequence, was applied to the sex origin of bloodstains by using a nonisotopic Southern hybridization method. The RNA probe was in vitro transcribed from the recombinant of the Y-chromosome specific deoxyribonucleic acid (DNA) sequence and transcription-vector Bluescript SK M13+. The use of this RNA probe can lower the detection limit of the nonisotopic DNA analyzing method to 1/8 of that reached by our previously reported method in which a DNA probe was used.

Blood Stains↗
PubMed1989

Rotavirus gene detection with biotinylated single-stranded RNA probes.

Biotinylated single-stranded RNA probes from two of the eleven genome segments of the simian rotavirus SA11 were synthesized from cloned DNA and used in dot-blot and Northern-blot hybridization assays. Different types of membranes and conditions to prepare and use synthetic non-radioactive transcript probes were evaluated to obtain optimal test results. Nytran membranes showed the highest sensitivity and lowest backgrounds for hybridization with biotinylated RNA probes. When a gene 6 single-stranded biotinylated probe was used in a dot-blot format, test sensitivity was 0.1 ng for detection of homologous RNA and 0.4-1.5 micrograms for detection of RNA from heterologous rotavirus strains. When used in Northern blots, detection with this gene 6 probe required 1 ng of total SA11RNA or 50 ng of heterologous RNA to be applied to the gels for transfer. Simultaneous hybridization with probes from two different genes on one membrane showed a detection level similar to that seen with single probes alone. The advantages of using biotinylated single-stranded RNA probes to detect or characterize the genes of viruses with double-stranded RNA genomes are shown.

Biotin↗
PubMed1988

Development and application of RNA probes for the study of picornaviruses.

Single-stranded RNA probes were developed from cloned cDNA fragments derived from four picornaviruses; poliovirus type 1, coxsackievirus B3, ECHOvirus 9 and Theiler's murine encephalomyelitis virus. In comparative testing with a nick-translated cDNA probe, the RNA probe was found to be 10-100-fold more sensitive. Hybridization conditions were optimized for RNA probes to picornavirus targets. Longer hybridization times gave an improved signal, as did a temperature of 50 degrees C. Formamide concentration had little effect on signal strength. A crude transcription mix performed as well or better as a probe than did more purified preparations of RNA. Finally, a combination of the three probes derived from human picornaviruses detected all 16 serotypes of human enteroviruses tested. Used individually, the four probes detected different spectra of animal and human picornaviruses, shedding more light on the interrelationships among these pathogens.

DNA↗
PubMed1987

Identification of negative strand and positive strand RNA of canine distemper virus in animal tissues using single stranded RNA probes.

In this report, we describe a technique for identifying negative strand (genome) and positive strand (messenger) RNA of canine distemper virus (CDV) in dog tissues by using single stranded RNA probes. Plasmids (pSP64-P and pSP65-P) which contain insert DNA corresponding to the P gene of CDV were transcribed by SP6 polymerase in the presence of radioisotope to produce radiolabeled single stranded RNA probes. RNA transcribed from pSP65-P is complementary to the negative strand (genome) and RNA produced from pSP64-P is complementary to the positive strand (message) of CDV. The binding specificity of the single stranded RNA probes was determined on Northern-blots. The use of these RNA probes in hybridization assays resulted in greater sensitivity and specificity than that obtained from double stranded DNA probes (either whole plasmids or purified insert DNA) which were labeled by the nick translation reaction. We also describe the making of single stranded DNA probes by reverse transcription labeling of complementary RNA. The complementary RNA was produced by the transcription of cloned DNA (pSP64-P and pSP65P). Single stranded RNA probes and single stranded DNA probes were similar in sensitivity. The single stranded RNA and DNA probes were applied to ethanolacetic acid fixed tissue sections from dogs infected with CDV-A75/17. We used 32P-labeled probes in tissue hybridizations and 35S-labeled probes in in situ hybridizations to identify negative and positive stranded CDV RNA. In this report we demonstrate that single stranded RNA and DNA probes can be used successfully in tissue hybridization and in situ hybridization assays to study viral expression in this virus-host system.

Animals↗
PubMed1986

Use of [32P]RNA probes for the dot-hybridization detection of potato spindle tuber viroid.

A dot-hybridization assay using 32P-labelled RNA probes (+RNA and cRNA) transcribed from potato spindle tuber viroid (PSTV) cDNA was described. A complete cDNA copy of PSTV, originally cloned in pBR 322 (pAV 401) was subcloned in the BamHI site of a 'Riboprobe' cloning vectors pSP 64 and pSP 65 in opposite orientations. The reconstructed plasmids were designated pDX 1 and pDX 4, respectively. Transcription of pDX 1 and pDX 4 plasmids by SP6 RNA polymerase resulted in the generation of PSTV-specific RNA (+RNA) and PSTV complementary RNA (cRNA), respectively. The cRNA probe was much more sensitive than the +RNA probe and the nick-translated cDNA probe from the plasmid pAV 401 for the detection of PSTV in clarified plant sap. As little as 1.4 pg of purified PSTV mixed in clarified sap from uninoculated tomato leaves has been detected using cRNA probe. A relatively simple procedure using cetyltrimethyl ammonium bromide (CTAB) as nucleic acid precipitant and an enrichment step for the purification of PSTV was described.

DNA Restriction Enzymes↗
PubMed1990

Analysis of RNA stability and (-) strand content in viral infections using biotinylated RNA probes.

Non-radioactive biotinylated RNA probes specific for plus (+) and minus (-) sense RNAs of brome mosaic virus (BMV) were synthesized in vitro from a plasmid bearing a 200 base pair fragment complementary to the 3' terminus of each of the three genomic RNAs of the virus. Using virion RNAs isolated from BMV infected barley plants, the sensitivity of biotinylated RNAs as hybridization probes was compared with that of 32P-labeled probes in Northern hybridization assays. Although the sensitivity of biotinylated and 32P-labeled probes is similar (approximately 5 pg), the time required to detect the RNA bands was much less than for autoradiography; (-) sense RNAs could be detected in 30 min whereas 48 h or more were required by autoradiography. The value of biotinylated probes for following RNA stability was exemplified by the detection of supplied inocula in protoplasts 24 h post-inoculation. Quantitation of relative accumulation of progeny (+) and (-) sense RNAs by densitometry of the Northern blots probed with biotinylated RNAs paralleled that of radiolabeled probes. The application of these probes was extended to the detection of RNAs in barley protoplasts and BMV infected plant sap by dot hybridization. In these tests, viral RNAs were detected in as few as 250 protoplasts and sap dilutions up to 1:2000. The merits of these non-radioactive probes in monitoring the replication events by the detection and quantitation of mutant progeny RNAs of BMV are discussed.

Biotin↗
PubMed1991

Plasmodium berghei: quantitation of in vitro effects of antimalarial drugs on exoerythrocytic development by a ribosomal RNA probe.

A stage-specific ribosomal RNA probe has been used to quantitate exoerythrocytic development of Plasmodium berghei in primary cultures of mouse hepatocytes. Parasite rRNA could be detected as soon as 6 hr after sporozoite invasion and was increased during schizogony to a maximum at 48 hr, when mature schizonts were identified by microscopy. As few as 10 exoerythrocytic schizonts could be detected by filter blot hybridization, followed by autoradiography and liquid scintillation counting. By hybridizing the culture rRNA samples with either parasite-specific or universal rRNA probes, the in vitro tissue schizonticidal activity and hepatotoxicity of primaquine, two of its analogues, and pyrimethamine, could be assessed. After a 48-hr exposure of the culture to serial dilutions of each drug, a quantitative relationship was demonstrated between the decrease of the parasite rRNA and the increase of the drug concentrations. No significant parasite-specific rRNA could be detected at the concentration achieving complete inhibition of schizont formation but causing no cytotoxic effects on host hepatocytes. In contrast to microscopic-based assays, this molecular approach provides an objective and quantitative in vitro method for rapid screening and evaluation of tissue schizonticidal antimalarials.

Aminoquinolines↗
PubMed1990

RNA probes detect nucleotide sequence homology between members of two different nairovirus serogroups.

Cloned cDNA derived from the small (S) and medium (M) genomic RNA segments of Dugbe (DUG) virus, isolate ArD44313, a member of the Nairobi sheep disease (NSD) serogroup of nairoviruses (family, Bunyaviridae) was used to prepare 32P-labelled DNA and RNA probes. The S and M segments of six isolates of DUG virus all hybridised to both DNA and RNA probes, although the M segment of isolate KT281/75 reacted only weakly. Of nine other nairoviruses tested, representing all the six other serogroups within the Nairovirus genus, none hybridised to the DNA probes. However, under conditions of low stringency, the DUG S and M RNA probes hybridised to the respective S and M segments of Ganjam (GAN) virus (another member of the NSD serogroup). The DUG S RNA probe also hybridised to the S segments of Crimean-Congo haemorrhagic fever (CCHF) virus and Hazara (HAZ) virus (members of the CCHF serogroup). The indicated sequence relationships between DUG, GAN, CCHF and HAZ viruses show that the NSD serogroup is more closely related to members of the CCHF serogroup than it is to nairoviruses of the other five serogroups.

Animals↗
PubMedDate not supplied

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes.

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.

Animals↗
PubMed1990

In situ hybridization for light and electron microscopy: a comparison of methods for the localization of viral RNA using biotinylated DNA and RNA probes.

We report procedures for in situ hybridization at the light and electron microscopic level for localization of viral RNA in poliovirus-infected, Lowicryl-embedded cells. We compare specificity and signal intensity of biotinylated, double-stranded DNA and single-stranded, strand-specific RNA probes, both corresponding to the same region of the poliovirus genome. The hybrids were detected with antibiotin antibodies or streptavidin with colloidal gold as a marker. Hybridization with the RNA probe was more sensitive and gave lower background than with DNA. Detection with immunogold proved to be by far more sensitive than with streptavidin. The hybridization and detection protocols for the DNA and the RNA probes could be applied without modification to light microscopic semi-thick sections as well as to electron microscopic ultrathin sections.

Biotin↗
PubMed1989

Detection of DNA targets with biotinylated and fluoresceinated RNA probes. Effects of the extent of derivitization on detection sensitivity.

The substituted nucleotide aminohexyl-ATP (AH-ATP) was used for synthesis of RNA probes from a plasmid template using the T7 phage promoter. Following synthesis, RNA probes were modified by reaction with N-hydroxysuccinimide (NHS) esters of biotin or fluorescein. Nearest-neighbor analysis was used to quantitate both the incorporation of the substituted nucleotide into RNA and the subsequent modification of the incorporated nucleotide by the NHS esters. The results indicate that AH-ATP is efficiently incorporated into RNA and that modification of the amine group is also efficient. The T7 polymerase shows a bias for ATP over AH-ATP and truncated transcripts are produced if 100% AH-ATP is used for synthesis. However, the use of 50% AH-ATP in the synthesis reaction yields full-length RNA probes that contain on average one amine-labeled nucleotide every 12 bases. This RNA is readily modified by the respective NHS esters to obtain one biotin group per 15-18 total RNA bases or one fluorescein group per 25-35 bases. Probes modified with biotin or fluorescein were used to detect picogram levels of target DNA in a dot blot hybridization format.

Adenosine Monophosphate↗
PubMed1989

Detection of the bunyavirus Germiston in VERO and Aedes albopictus C6/36 cells by in situ hybridization using cDNA and asymmetric RNA probes.

Using Germiston virus infected vertebrate (VERO) and invertebrate (Aedes albopictus C6/36) cells, paraformaldehyde-glutaraldehyde fixative allowed the best preservation of cellular morphology and the highest hybridization signals with cDNA and asymmetric RNA probes against the viral S segment. Asymmetric RNA probes always gave higher sensitivity and better specificity of in situ hybridization than the nick-translated symmetric DNA probe in both vertebrate and invertebrate cells. The study of Aedes albopictus C6/36 cells persistently infected with Germiston virus showed that only a small number of cells contained the S segment, and that the replication and transcription of the S segment took place in the cytoplasm of acutely and persistently infected cells.

Aedes↗
PubMed1991

Comparison of biotinylated DNA and RNA probes for rapid detection of varicella-zoster virus genome by in situ hybridization.

We describe a general method for the production of nonisotopic DNA and RNA probes for the detection of the varicella-zoster virus (VZV) genome by in situ hybridization. VZV DNA was extracted from purified viral nucleocapsids, cleaved with restriction enzyme (RE) BamHI, and cloned into plasmid pBR322 by the standard vector insert procedure. We cloned over 85% of the VZV genome and obtained 18 recombinants. Plasmids containing the B, F, G, H, and J fragments of VZV DNA were labeled by the nick translation method with biotin-11-dUTP as the dTTP analog. Additionally, the B fragment was cleaved with RE AvaI, subcloned into the plasmid pGEM-4 transcription vector, and subsequently linearized with REs PstI and EcoRI. RNA was transcribed with T7 or SP6 polymerase, with a substitution of allylamine-UTP as the UTP analog, and labeled with epsilon-caproylamidobiotin-N-hydroxysuccinimide ester. The DNA and RNA probes were used under full-stringency conditions for in situ hybridization with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate-Nitro Blue Tetrazolium as the substrate. When tested under comparable conditions, the RNA probe was slightly more sensitive than was the DNA probe: both probes showed homology only with VZV-infected cells and clinical tissues and not with the other herpesviruses. Probes prepared from variable regions of the genome (fragments F and J) performed as well as did those from conserved regions (fragments B. G. and H). Biotinylated probes have distinct advantages over isotopic probes and retain their full potency for more than 2 years when stored properly.

Biotin↗