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At least 253 records · Page 14Linked to original sources
PubMed1997

HIV-1 detection in cervicovaginal secretions during pregnancy.

OBJECTIVE: To assess the reproducibility of and factors associated with HIV detection in cervicovaginal secretions (CVS). DESIGN: Longitudinal study of 43 HIV-1-infected pregnant women in Paris. METHODS: HIV DNA was detected in peripheral blood mononuclear cells (PBMC) by Amplicor and gag nested polymerase chain reaction (PCR) assays. The HIV genotype was determined by heteroduplex mobility assay. Amplicor and gag nested PCR assays were performed on serial CVS samples for HIV DNA detection, and the HIV Monitor test was used for HIV RNA detection in plasma and CVS. RESULTS: A total of 144 CVS samples were collected from the women included in the study. HIV-1 DNA was detected in 36 (25%) of the 144 samples, from 16 (37.2%) of the 43 women. Results of HIV-1 DNA detection were concordant in the first two samples in 27 (84.4%) of the 32 women with at least two CVS samples. The last CVS sample collected in each woman was HIV-1 DNA-positive in 13 (30.2%) of the 43 women. Three factors were found to be independently associated with HIV-1 DNA detection in CVS: HIV-1 subtype B, absence of zidovudine therapy, and microbial cervicovaginal infection. HIV RNA was detected in CVS from 10 (23.3%) out of 43 women and correlated with DNA detection in the same sample and HIV RNA detection in plasma. CONCLUSIONS: DNA and RNA PCR can be used to detect HIV in cells and supernatants of CVS. These techniques may be useful in cohort studies to investigate HIV transmission and to evaluate the efficacy of antiretroviral drugs to reduce HIV excretion.

Adult↗
PubMed1983

A rapid method for detecting specific RNA transcripts by hybridization to DNA probes in solution.

A method is described for detecting specific transcripts in crude mixtures of RNA. The method employs hybridization of single-stranded or double-stranded radioactive DNA probes in solution, followed by electrophoretic separation of the hybrid and probe on agarose and visualization by radioautography. The procedure offers the advantages of decreased preparation time and increased sensitivity over currently used methods.

Autoradiography↗
PubMed2002

Sensitive detection of RNA viruses associated with gastroenteritis by a hanging-drop single-tube nested reverse transcription-PCR method.

The detection of the human RNA viruses, calicivirus and astrovirus, requires high sensitivity and broad reactivity. A novel single-tube nested reverse transcription-PCR (RT-PCR) method is described here, in which all of the required reagents are included in the one tube; however, those required for the nested amplification are separated in a "hanging drop" in the cap to be introduced by centrifugation after the RT and first-round cDNA amplification steps. Broad reactivity was obtained by using primer cocktails covering the published sequence variation in the primer targets. The method was evaluated with clinical fecal samples from outbreak and sporadic cases. Norwalk-like virus types 1 and 2 and rotavirus were the causal agents in 10 of 12 outbreaks. A viral agent was detected in 44% of 197 samples from sporadic infections in patients presenting to community health centers and a children's hospital. Interestingly, whereas rotavirus was more common than astrovirus in patients presenting to the hospital (33 and 7.6%, respectively), the reverse was true for patients presenting to community health centers (4.2 and 34%, respectively).

Adolescent↗
PubMed1994

A sensitive PCR method for detecting HCV RNA in plasma pools, blood products, and single donations.

Although current manufacturing processes appear to efficiently inactivate hepatitis C virus (HCV), it is possible that contaminated blood products may result from failure of some stage during manufacture or from virus overload of plasma pools used for preparation of products. While antibody screening probably removes the majority of HCV positive donations, some donations which are antibody-negative but HCV positive may be included in pools. The HCV RNA content of plasma pools from paid and voluntary donors was investigated by polymerase chain reaction (PCR). A sensitive PCR method using a single pair of primers from the 5' non-coding region of the HCV genome and a "hot-start" was established and shown to be as sensitive as the more conventional nested PCR (which uses two pairs of primers). The majority of pools from paid donors (prescreening) were HCV RNA positive, while all pools from voluntary donors were both antibody and RNA negative. Intravenous immunoglobulins prepared from contaminated pools were RNA negative despite having high antibody levels, indicating satisfactory clearance of the virus during manufacture. The virus load of the pools was at least a thousand-fold lower than that of single donations, possibly as a result of treatment during the production of the pools or the presence of factors in pools which reduce the sensitivity of some part of the PCR assay. The HCV content of a plasma donation was determined as 3.6 x 10(6) genomes/ml by an end point dilution method. Thus a simple and sensitive PCR assay was established for detecting HCV RNA in plasma pools and blood products.

Base Sequence↗
PubMed1996

A three-hybrid system to detect RNA-protein interactions in vivo.

RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infection by RNA viruses. However, in spite of the central importance of these interactions, few approaches are available to analyze them rapidly in vivo. We describe a yeast genetic method to detect and analyze RNA-protein interactions in which the binding of a bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in vivo. We demonstrate that this three-hybrid system enables the rapid, phenotypic detection of specific RNA-protein interactions. As examples, we use the binding of the iron regulatory protein 1 (IRP1) to the iron response element (IRE), and of HIV trans-activator protein (Tat) to the HIV trans-activation response element (TAR) RNA sequence. The three-hybrid assay we describe relies only on the physical properties of the RNA and protein, and not on their natural biological activities; as a result, it may have broad application in the identification of RNA-binding proteins and RNAs, as well as in the detailed analysis of their interactions.

Base Sequence↗
PubMed1998

Nucleotide sequence of seed- and pollen-transmitted double-stranded RNA, which encodes a putative RNA-dependent RNA polymerase, detected from Japanese pear.

The nucleotide sequence of the largest double-stranded (ds) RNA (named dsRNA1) of three species of seed- and pollen-transmitted dsRNA species detected from Japanese pear was analyzed, and one strand was found to contain a single long open reading frame (ORF) of 1434 nucleotides that encoded a putative polypeptide containing 477 amino acid residues with a molecular mass of 54.9 kDa. This polypeptide contained amino acid sequence motifs conserved in putative RNA-dependent RNA polymerases of RNA viruses. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Slow sedimentation of the dsRNA fraction suggests that the dsRNAs may be unencapsidated. The concentration of dsRNAs in the host, Japanese pear, was about 16 times higher than that from a cryptic virus, radish yellow edge virus (RYEV). These results suggest that the dsRNAs were not from cryptic viruses. Partial nucleotide sequences of the two smaller dsRNAs (named dsRNAs 2 and 3) and two other dsRNAs (named dsRNAs 4 and 5) detected from only the Japanese pear cultivar (cv.) Akita Tazawa 3 Gou were analyzed, and encoded nearly the same amino acid sequence encoded by dsRNA1.

Amino Acid Sequence↗
PubMed2006

Cerebrospinal fluid is an efficient route for establishing brain infection with feline immunodeficiency virus and transfering infectious virus to the periphery.

Like human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV) invades and infects the central nervous system (CNS) soon after peripheral infection. The appearance of viral RNA is particularly prominent in the cerebrospinal fluid (CSF), suggesting an efficient route of virus transfer across the blood-CSF barrier. This raises the concern whether this route can establish a stable viral reservoir and also be a source of virus capable of reseeding peripheral systems. To examine this possibility, 200 mul of cell-free NCSU1 FIV or FIV-infected choroid plexus macrophages (ChP-Mac) was directly injected into the right lateral ventricle of the brain. Negative controls were sham inoculated with uninfected ChP-Mac or virus-free culture supernatant and positive controls were infected systemically by intraperitoneal (i.p.) injection. Intracerebroventricular (i.c.v.) inoculation with cell-free FIV resulted in high levels of plasma FIV RNA detected as early as 1 to 2 weeks post inoculation in all cats. In each case, the plasma viremia preceded the detection of CSF viral RNA. Compared to i.p. cats, i.c.v. cats had 32-fold higher CSF viral loads, 8-fold higher ratios of CSF to plasma viral load, and a 23-fold greater content of FIV proviral DNA in the brain. No FIV RNA was detected in plasma or CSF from the cats inoculated with FIV-infected ChP-Mac but an acute inflammatory response and a slight suppression of the CD4+:CD8+ ratio were observed. These results indicate that free FIV circulating in the CSF promotes infection of the CNS and provides a highly efficient pathway for the transfer of infectious virus to the periphery.

Animals↗
PubMed2005

Detection of human immunodeficiency virus type 1 RNA by in situ hybridization in oral mucosa epithelial cells from anti-HIV-1 positive patients.

Several in vitro studies have shown that HIV-1 can infect CD4 negative epithelial cells of different origin including normal human oral keratinocytes, but whether this infection of mucosal epithelial cells occurs in vivo is still unclear. In this report, the presence and cell types infected by HIV-1 in paraffin embedded oral mucosa biopsies from 17 anti-HIV-1 positive patients have been examined by in situ hybridization and immunohistochemistry. As controls, oral mucosa biopsies from eight patients without HIV-1 infection markers were also analyzed. The results showed that 8 out of the 17 anti-HIV-1 positive patients had HIV-1 RNA detectable in plasma. Positive hybridization signals were observed in the mucosa biopsies from 14 of the 17 anti-HIV-1 patients (82.3%). The mean percentage of cells showing HIV-1 RNA was 2.64% +/- 1.77% (range: 1% to 5.5%). No differences in the mean percentage of HIV-1 infected cells were found between patients with and without HIV-1 RNA in plasma (3.01% +/- 1.57% vs. 3.4% +/- 1.27% respectively), or between untreated patients and patients under antiretroviral therapy (2.83% +/- 1.63% vs. 3.42% +/- 1.29% respectively). Immunohistochemical detection of S-100 antigen, cytokeratin and CD4 showed that hybridization signals appeared in cytokeratin positive cells and CD4 positive cells but not in S-100 positive cells. In conclusion, this study has demonstrated that HIV-1 infects and replicates in oral mucosa epithelial cells in vivo and that these cells could represent a reservoir of the virus that may escape to the currently used antiretroviral therapy.

Adult↗
PubMed2004

[Detection of RNA of SARS coronavirus in hospital sewage].

OBJECTIVE: In order to explore the existence of SARS coronavirus (Co-V) and/or its RNA in sewage of hospitals administered SARS patients. METHODS: A novel electropositive filter was used to concentrate the SARS-CoV from the sewage of two hospitals administered SARS patients in Beijing, including twelve 2,500 ml sewage samples from the hospitals before disinfection, and ten 25,000 ml samples after disinfection; as well as cell culture, RT-PCR and sequencing of gene to detect and identify the viruses from sewage. RESULTS: There was no live SARS-CoV detected in the sewage in this study. The nucleic acid of SARS-CoV had been found in the 12 sewage samples before disinfection from both hospitals by semi-nested PCR. After disinfection, SARS-CoV RNA could only be detected from the samples from the 309th Hospital, and the others were negative. CONCLUSION: It provides evidence that there is no live SARS-Cov in the sewage from hospitals with SARS patients though SARS-CoV RNA can be detected.

Hospitals↗
PubMed2004

Multiplex detection of RNA expression in Drosophila embryos.

We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven differ-ent transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent transcript multiplex fluorescent in situ hybridization. These multiplex methods fill a current technological gap between high-resolution in situ hybridization with one or two fluorescently labeled probes and low-resolution but genome-wide microarray RNA profiling and should be of great utility in establishing gene networks.

Animals↗
PubMed1996

Specific detection of RNA molecules by fluorescent in situ hybridization in living cells.

The antisense therapeutic strategy makes the assumption that sequence-specific hybridization of an oligonucleotide to its target can take place in living cells. The present work provides a new method for the detection of intracellular RNA molecules using in situ hybridization on living cells. The first step consisted in designing nonperturbant conditions for cell permeabilization using streptolysin O. In a second step, intracellular hybridization specificity was evaluated by incorporating various types of fluorescently labeled nucleic acid probes (plasmids, oligonucleotides). Due to its high expression level, the 28S ribosomal RNA was retained as a model. Results showed that: (1) no significant cell death was observed after permeabilization; (2) on living cells, 28S RNA specific probes provided bright nucleoli and low cytoplasmic signal; (3) control probes did not lead to significant fluorescent staining; and (4) comparison of signals obtained on living and fixed cells showed a colocalization of observed fluorescence. These results indicate the feasibility of specific hybridization of labeled nucleic acid probes under living conditions, after a simple and efficient permeabilization step. This new detection method is of interest for investigating the dynamics of distribution of various gene products in living cells, under normal or pathological conditions.

Animals↗
PubMedDate not supplied

[Detection of RNA in vaccine " EnceVir" against tick-borne encephalitis virus].

Fractionation of RNA isolated from the inactivated purified concentrated vaccine "EnceVir" ("Virion", Tomsk, Russia) allowed the authors to reveal predominantly double-stranded RNA and trace quantities of single-stranded RNA sizing mainly less than 200 n. The fragments of tick-borne encephalitis virus (TBEV) were detected among the total RNA by the reverse transcription test, followed by nested polymerase chain reaction with the primers specific to the E gene of TBEV. The findings may suggest that immunity may be induced through activation of Th1 and Th2 after immunization with such an inactivated vaccine for effective elimination of intracellular infection and intracellular virions, respectively.

Base Sequence↗
PubMed1996

A one-hybrid system for detecting RNA-protein interactions.

BACKGROUND: mRNA translation, stability, and localization are controlled by regulatory proteins that bind to specific RNA motifs. Since biochemical isolation of such proteins has often proven to be difficult, a genetic system for studying RNA-protein interactions would be of great utility in the identification of novel RNA binding proteins and in understanding how these proteins recognize particular RNA sequences. The bacteriophage lambda gene product N protein is a sequence specific RNA binding protein that when bound to its target sequence allows RNA polymerase to ignore transcription termination signals. The fact that the binding of N protein to RNA is directly coupled to gene expression suggests that N protein could be used to develop a general system for studying RNA-protein interactions. RESULTS: Our results show that fusion of the RNA binding protein, R17, to N causes antitermination in a beta-galactosidase reporter construct that has the R17 binding site substituted for the normal N target sequence. This system can both detect low affinity interactions as well as discriminate between binding events with equilibrium dissociation constants from 10(-5) to 10(-8)M. The differences in antitermination activity with various mutant binding sites can be reliably detected by colony colour on X-Gal plates as well as by liquid culture assay. CONCLUSIONS: We have demonstrated that N protein can cause antitermination through a heterologous RNA-protein interaction and that the system is capable of detecting RNA-protein interactions of differing affinities. This approach may also be useful in screening libraries for proteins that bind to novel RNA regulatory elements. Our results are also consistent with a model of N protein function in which binding to the nascent transcript increases the effective concentration of N in the vicinity of RNA polymerase leading to antitermination.

Capsid↗
PubMed1985

Polyadenylation of a human mitochondrial ribosomal RNA transcript detected by molecular cloning.

We have identified by molecular cloning a polyadenylated RNA transcript in the human leukemia cell line, K562, which is complementary to a portion of the gene-encoding mitochondrial 16S ribosomal RNA (mt 16S rRNA). The cloned portion of the transcript corresponds to positions 2191-2395 of the human mt genome. The clone represents a cDNA copy of an RNA transcript from the H strand and carries an additional poly(A) tail 21 residues long at its 3'-end. Our data provide direct evidence for polyadenylation of some mt 16S rRNA transcripts.

Base Sequence↗
PubMed1995

Real-time fluorescence detection of RNA amplified by Q beta replicase.

Amplification of RNA probes by Q beta replicase can be used to detect a wide range of analytes with a potential sensitivity of a single molecule. A system has been developed in which Q beta amplification of midivariant-(MDV)-based RNA is measured in real time by fluorescence. This was accomplished by including a fluorescent intercalating dye, propidium iodide, in the reactions and monitoring the fluorescence change using a custom fluorometer. The time at which fluorescence is detectable above background is referred to as the "response time" and is calculated using curve-fitting algorithms. A response time is inversely and linearly proportional to the logarithm of the number of template RNA molecules which initiated the reaction. Therefore, this system permits an unknown amount of input RNA probe to be quantified through 11 orders of magnitude when compared to a standard curve. Under the described conditions with MDV RNA, the response time occurs when about 3 x 10(11) RNA molecules are synthesized and occurs within the exponential phase of the reaction, before the number of active enzyme molecules are saturated with RNA templates. This system has been used to determine the replication properties of MDV RNA reporter molecules bearing specific probe sequences and to develop hybridization assays for the clinical diagnostic field.

Base Sequence↗
PubMed1982

Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA.

RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.

Animals↗
PubMed1995

Determination of hepatitis delta virus RNA by polymerase chain reaction in acute and chronic delta infection.

The objective of this study was to evaluate the usefulness of hepatitis delta virus (HDV) RNA detection by polymerase chain reaction (PCR) in acute and chronic D hepatitis and to correlate with HDV-RNA detection by dot blot and hepatic delta antigen. Serum samples from 33 patients with acute hepatitis B surface antigen (HBsAg)-positive hepatitis (15 with hepatitis B and D coinfection, 8 with HDV superinfection, and 10 with acute hepatitis B), 85 patients with chronic HBsAg-positive hepatitis (73 with chronic D hepatitis and 12 with chronic B hepatitis), and consecutive serum samples from nine patients with chronic D hepatitis treated with interferon alfa-2b were studied. HDV-RNA was detected by PCR in 93% of the patients with hepatitis B and D coinfection, in 100% of the patients with hepatitis D superinfection, and in 1 of the 10 patients with acute hepatitis B who subsequently seroconverted to total antibody to hepatitis delta antigen (HDAg), whereas HDV-RNA was found by dot blot technique in 60% of the hepatitis B and D coinfection cases, in 62.5% of the patients with hepatitis D superinfection, and in none of the acute hepatitis B cases. In chronic D hepatitis, HDV-RNA tested positive by PCR assay in 97% of patients with intrahepatic HDAg, in one patient with undetectable hepatic HDAg, and in none of the patients with chronic hepatitis B. In the treated patients, HDV-RNA was observed to become negative by PCR only in the three patients who had a persistent response to interferon.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1988

Direct detection of HIV RNA expression in seropositive subjects.

The polymerase chain reaction (PCR) and reverse transcription were used to assess human immunodeficiency virus type 1 (HIV1) RNA expression in peripheral blood mononuclear cell samples from seropositive subjects. HIV RNA was detected from seropositive subjects who had no symptoms, lymphadenopathy syndrome, and acquired immunodeficiency syndrome. DNA PCR of the samples used for RNA extraction showed that seventeen of eighteen (94%) contained HIV proviral DNA. Eleven (65%) of the seventeen DNA-positive samples were also positive for HIV RNA, including samples from four patients undergoing antiviral drug treatment. Serum HIV antigen assays detected only six (32%) of the nineteen PCR-positive samples. Owing to the speed and high sensitivity of PCR for HIV detection, this technique will be suitable for monitoring antiviral therapy and the virus load of people with HIV infections.

Cells, Cultured↗