Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA detection”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed2003

[Application of one step RT-PCR assay for detection of flavivirus RNA in mosquitoes].

The conditions of one step RT-PCR method for detection of virus RNA in field-collected mosquitoes, and preservation period of infected mosquitoes for one step RT-PCR were examined. We compared several virus RNA extraction methods with artificially contaminated mosquito pools with dengue virus (DV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) with a known amount of plaque forming unit (PFU) to establish the condition of one step RT-PCR. In this study, most effective RNA extraction method was ISOGEN-LS extraction combined with supernatant of centrifuged mosquito homogenates. Detection limit of one step RT-PCR using flavivirus universal primer in ten mosquitoes/tube (pool) was 10 PFU of DV, JEV and YFV, 1 PFU of each viruses using species-specific primer respectively, in one hundred mosquitoes/tube, 100 PFU/tube using universal primer pairs, 10 PFU/tube using species-specific primer pairs respectively. Dengue virus infected single mosquito was mixed with 99 un-infected mosquitoes, and tested by one step RT-PCR. We could detect single infected mosquito in pools containing 99 un-infected mosquitoes. Aedes aegypti and Aedes albopictus were inoculated intrathoracically with a mouse-adapted strain of dengue-1 virus and were kept up to 30 days at different temperature. Then examined by one step RT-PCR to determine the appropriate mosquito handling method and the condition of transportation. Positive result was obtained up to 30 days after the mosquito died naturally. These results suggested that we could detect flavivirus RNA tested not only from live mosquitoes but also dead mosquitoes as well, and could apply one step RT-PCR as a rapid, specific, and highly sensitive tool for flavivirus surveillance.

Animals↗
PubMed2005

Dynamics of viremia in early hepatitis C virus infection.

BACKGROUND: It is important to characterize viral dynamics in early hepatitis C virus (HCV) infection to further our understanding of viral pathogenesis and the potential for secondary transmission in acute infection through blood transfusion or other routes. STUDY DESIGN AND METHODS: Serial units given by 77 source plasma donors who had evolved from HCV RNA-negative to HCV RNA-positive by nucleic acid amplification technology (NAT) screening with 512-unit pool-NAT or were followed from RNA detection to antibody conversion were tested by individual NAT and quantitative RNA assays. RESULTS: During the ramp-up phase when exponential growth occurs, HCV viral load doubled every 10.8 hours (95% confidence interval [CI], 9.9-12.0). Intermittent viremia was observed before the ramp-up phase in 37 of 50 panels with the earliest detectable viremic bleed occurring 63 days before the estimated onset of ramp-up. The plateau phase or high-titer viremic period that occurs between ramp-up and seroconversion was estimated to last 56.3 days (95% CI, 44.8-67.8). CONCLUSIONS: Intermittent low-level HCV viremia can occur as much as 2 months before the periods of exponential increase in viral load and the high-titer plateau-phase viremia that usually precede seroconversion. Animal inoculation studies are in progress to evaluate if transfusion of low-level viremic plasma can transmit HCV infection.

Blood Donors↗
PubMed1988

A rat testis-specific hsp70 gene-related transcript is coded by a novel gene from the hsp70 multigene family.

To establish the identity of testis-specific hsp70 gene-related transcript, the expression pattern of the hsp70 gene family in testis and liver of rats subjected to whole body hyperthermia was investigated. In control liver two hsp70 gene-related transcripts of about 2.2 and 2.5 kb were detected. Increased body temperature resulted in high accumulation of 2.5 kb RNA and in the appearance of abundant amounts of another heat-induced transcript of approx. 2.7 kb. In testis of control rats only one hsp70 gene-related transcript has been identified and hyperthermia did not affect its level. This transcript migrated in 1% agarose-formaldehyde gel slightly faster than the 2.5 kb heat-induced RNA detected in liver and epididymis. The uniqueness of the testicular hsp70 gene-related transcript has been confirmed by its hybridization properties. At stringent conditions this transcript did not hybridize with a human hsp70 gene-related probe in contrast to the 2.5 and 2.7 kb heat-induced RNA species.

Animals↗
PubMed1997

Quantification of HIV in semen: correlation with antiviral treatment and immune status.

OBJECTIVE: This study examined the concentration of HIV in semen and the effects of biological factors on HIV excretion. METHODS: Semen samples from 101 men at different stages of the disease were evaluated by quantitative HIV culture and HIV RNA detection. Blood plasma samples were available from 56 patients. The effects of CD4 and CD8 count, blood plasma RNA levels, treatment status and clinical staging on the shedding of HIV were evaluated. RESULTS: HIV RNA levels in semen correlated with quantitative HIV culture of seminal cells and a strong association of positive seminal cell culture with high RNA levels was observed. CD4 count and antiviral treatment were inversely correlated with the concentration of HIV in semen. Blood plasma HIV RNA values were correlated with HIV RNA levels in semen, although some patients had highly discrepant results. CONCLUSIONS: The strong correlation between seminal cell culture and concentration of HIV RNA in seminal plasma suggested that HIV detected in seminal plasma was released by productively infected cells in the male genital tract. The study showed that the concentration of HIV in semen, which was likely to be correlated with HIV infectivity, was a function of the immune status of the HIV-infected individual. The results suggested that antiviral therapy may have reduced the concentration of HIV in semen.

Adult↗
PubMed1988

Transcripts of the Drosophila blastoderm-specific locus, terminus, are concentrated posteriorly and encode a potential DNA-binding finger.

The commitment of cells to specific fates, as well as the transitions in the cell cycle and transcription that occur at the cellular blastoderm stage of Drosophila embryogenesis, suggest that there are genes with unique functions expressed specifically at this stage. In an attempt to identify such genes, we used molecular screening to isolate several loci which encode blastoderm-specific transcripts (Roark et al., (1985). Dev. Biol. 109, 476-488). We report here the complete nucleotide sequence of one of these genes, terminus (ter), which maps to 75C1,2. The predicted ter protein possesses a transcription factor IIIA (TFIIIA)-like putative Zn-binding, DNA-binding finger. The ter RNA, detected by in situ hybridization, is distributed uniformly in the embryo during the syncytial blastoderm stage, and then becomes more concentrated in the posterior during the late cellular blastoderm stage. During gastrulation, the RNA is most concentrated in the amnioproctodeal invagination; it is also found at a lower concentration in the ventral furrow and in the anterodorsal neurogenic region. By the end of germ band extension, ter RNA is no longer detected.

Amino Acid Sequence↗
PubMed2003

Occult hepatitis C virus infection in type II mixed cryoglobulinaemia.

Mixed cryoglobulinaemia, when not secondary to other well-defined immunological disorders, is commonly associated with hepatitis C virus (HCV) infection. However, a minority of cases lack evidence of HCV infection and are, therefore, defined as 'true essential' mixed cryoglobulinaemias. We thoroughly investigated three such patients to determine the aetiology of this disorder. Antibodies to HCV (anti-HCV) and HCV RNA, detected by sensitive enzyme-linked immunosorbent and polymerase chain reaction assays in serum and in concentrated cryoglobulins, were repeatedly negative in the three patients. Despite the lack of evidence for HCV infection, two of them were still treated with interferon alpha-2a assuming unrecognized viral infection. Both patients demonstrated excellent clinical and laboratory responses, but cryoglobulinaemia relapsed after the withdrawal of therapy. At the time of relapse, HCV RNA genomic sequences were detected for the first time in the cryoprecipitates of both patients. In the third case, HCV RNA was demonstrated for the first time during a flare of cryoglobulinaemia coincident with varicella infection. In all three patients anti-HCV antibodies remained negative throughout follow-up. We conclude that some apparently 'essential' forms of mixed cryoglobulinaemia can be caused by occult HCV infection. Interferon therapy can be taken into consideration in such HCV-negative cases.

Adult↗
PubMed1990

Detection of hepatitis C virus RNA in sera and liver tissues of non-A, non-B hepatitis patients using the polymerase chain reaction.

Sera obtained from patients with non-A, non-B hepatitis were examined for the presence of hepatitis C virus (HCV) genome by using the reverse transcription-polymerase chain reaction assay, as well as for antibody to HCV (anti-HCV) by using an enzyme-linked immunosorbent assay (ELISA). We also examined the presence of HCV RNA in liver tissue obtained by surgical resection of hepatocellular carcinoma. Among 33 patients, HCV RNA was detectable in 21 (64%), and the antibody was also positive in 21 (64%). Eighteen (55%) patients were positive for both assays. The two assays gave inconsistent results in 3 patients who were positive for HCV RNA but negative for anti-HCV, and in 3 other patients who were negative for HCV RNA and positive for anti-HCV. HCV RNA was also detected in 6 out of 10 non-cancerous liver tissue specimens and in 3 out of 7 tumor tissue specimens. Using the polymerase chain reaction, the HCV genome was detected directly in many specimens obtained from patients with non-A, non-B hepatitis, suggesting the presence of replicating virus in patients positive for anti-HCV. In addition, the differing results of the two assay systems suggest that the application of both is important for evaluation of the status of HCV infection.

Adult↗
PubMed1996

Rapidly progressive hepatitis C in a patient with common variable immunodeficiency.

UNLABELLED: A 15-year-old girl with common variable immunodeficiency contracted hepatitis C, which progressed to liver cirrhosis and finally to hepatic failure 5 years later. Since she was agammaglobulinaemic, hepatitis C virus (HCV) infection was diagnosed on the basis of HCV-RNA detection. Quantification of her sera showed high levels of HCV-RNA (more than 10(7) copies of RNA/mL), which implied active viral replication. There were no other hepatotoxic factors except HCV infection. The initial liver biopsy at 16 years of age and the autopsy confirmed a rapid progression in liver histopathological change over 4 years. CONCLUSION: Contrary to the widely held view of a benign short-term prognosis in paediatric hepatitis C, progressive fatal liver disease can develop in some patients with HCV infection. Such a rapid progression of liver injury provides the rationale for antiviral therapy in at least certain high risk groups of these children. Hepatitis C may progress rapidly in an immune deficiency condition.

Adolescent↗
PubMed2002

Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus.

The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognized. We applied several computational methods to detect RNA secondary structure in the coding region of hepatitis C virus (HCV), including thermodynamic prediction, calculation of free energy on folding, and a newly developed method to scan sequences for covariant sites and associated secondary structures using a parsimony-based algorithm. Each of the prediction methods provided evidence for complex RNA folding in the core- and NS5B-encoding regions of the genome. The positioning of covariant sites and associated predicted stem-loop structures coincided with thermodynamic predictions of RNA base pairing, and localized precisely in parts of the genome with marked suppression of variability at synonymous sites. Combined, there was evidence for a total of six evolutionarily conserved stem-loop structures in the NS5B-encoding region and two in the core gene. The virus most closely related to HCV, GB virus-B (GBV-B) also showed evidence for similar internal base pairing in its coding region, although predictions of secondary structures were limited by the absence of comparative sequence data for this virus. While the role(s) of stem-loops in the coding region of HCV and GBV-B are currently unknown, the structure predictions in this study could provide the starting point for functional investigations using recently developed self-replicating clones of HCV.

Base Sequence↗
PubMed2000

Enteroviral infection causing fatal myocarditis and subclinical myopathy.

UNLABELLED: Enteroviral RNA detection in myocarditis and dilated cardiomyopathy is rare. Enteroviral particles and RNA have recently been identified in patient's skeletal muscle, suggesting that skeletal more than heart muscle hosts the virus in chronic infection. Enteroviral RNA and virus-like particles were found in the myocardium and in the skeletal muscle of two patients with fatal myocarditis: a 39 year old man who died five days after the onset of febrile flu; and a 49 year old woman, assisted for 50 days with a left ventricular assist device, who then died from cerebral haemorrhage. Automated sequencing, alignment, and sequence comparison confirmed the enteroviral origin of polymerase chain reaction products and excluded contamination. These findings agree with prior observations of enteroviral localisation in the skeletal muscle of patients with dilated cardiomyopathy, and further support the hypothesis that skeletal rather than heart muscle may host the virus and serve as a reservoir in cardiomyopathies related to chronic infection. KEYWORDS: enterovirus; myocarditis; viral particles; skeletal muscle

Adult↗
PubMed1984

Identification and in vitro capping of a primary transcript of human mitochondrial DNA.

A discrete primary transcript of HeLa mitochondrial DNA containing 12 S rRNA, tRNAPhe, and 19 +/- 2 nucleotides upstream has been identified. The 5' ends of heavy strand encoded RNAs adjacent to the D-loop region of mitochondrial DNA were mapped by primer extension and S1 nuclease protection to a site 19 +/- 2 nucleotides upstream from the start of tRNAPhe. Hybridization of a tRNAPhe-specific probe to a blot of mitochondrial RNA detected a discrete species with a molecular weight consistent with an RNA containing 12 S rRNA, tRNAPhe, and a short upstream leader sequence. The di- or triphosphate nature of the 5' end of this polycistronic RNA was demonstrated by in vitro capping with [alpha-32P]GTP and guanylyltransferase. S1 nuclease protection of the purified in vitro capped RNA established that the 5' end, 19 +/- 2 nucleotides upstream from tRNAPhe, is an initiation site for heavy strand transcription.

Base Sequence↗
PubMed2006

Multicenter quality control of the detection of HIV-1 genome in semen before medically assisted procreation.

Couples in whom the man is HIV-1-positive may use medically assisted procreation in order to conceive a child without contaminating the female partner. But, before medically assisted procreation, the semen has to be processed to exclude HIV and tested for HIV nucleic acid before and after processing. The performance was evaluated of the technical protocols used to detect and quantify HIV-1 in 11 centers providing medically assisted procreation for couples with HIV-1 infected men by testing panels of seminal plasma and cells containing HIV-1 RNA and/or DNA. The performance of these tests varied due to the different assays used. False positive results were obtained in 14-19% of cases. The sensitivity for RNA detection in seminal plasma was 500-1,000 RNA copies/ml, over 500 RNA copies/10(6) cells in semen cells, and for DNA detection in semen cells 50-500 DNA copies/10(6) cells. The use of silica-based extraction seemed to increase the assay performance, whereas the use of internal controls to detect PCR inhibitor did not. This first quality control highlights the need for technical improvements of the assays to detect and quantify HIV in semen fractions and for regular evaluation of their performance.

DNA, Viral↗
PubMed1994

A technique for the sequential isolation of RNA and DNA from embryos developed for screening for viruses.

A technique has been developed to detect RNA or DNA viruses in single embryos. This technique is based on sequential RNA and DNA isolation from the embryo and analysis of this nucleic acid by polymerase chain reaction. A constitutively expressed embryo gene (alpha subunit ATPase) is also analysed as an internal standard. Extraction of RNA was more efficient than that of DNA, however, positive results were obtained for the internal standard gene for 84% (RNA) and 30% (DNA) of embryos tested.

Animals↗
PubMed1996

Failure to detect measles virus RNA, by reverse transcription-polymerase chain reaction, in peripheral blood leucocytes of patients with multiple sclerosis.

We have examined peripheral blood leucocytes (PBLs) from 17 multiple sclerosis patients, two patients with rheumatoid arthritis, one case of acute childhood measles and one case of subacute sclerosing panencephalitis, as well as 19 healthy adult controls for measles virus (MV) RNA, by the technique of reverse transcription-polymerase chain reaction. MV nucleocapsid gene specific primers were used to amplify all PBL-derived cDNA samples. These proved to be negative with the exception of the sample derived from the acute measles case. Selected cases were examined further, using fusion gene and matrix gene specific primers. MV RNA could not be detected.

Adult↗
PubMed2005

Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells.

Considering growing evidence indicating that hepatitis C virus (HCV) replicates in lymphoid cells, establishment of a reliable and sensitive method for detection of HCV in these cells may provide means for monitoring the infection and the efficacy of sterilizing antiviral therapy. In this study, conditions for ex vivo augmentation and detection of the HCV genome in peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C (CHC) or after a sustained virological response (SVR) to antiviral treatment were assessed. Following stimulation with combinations of mitogens and/or cytokines, PBMCs and, in certain cases, affinity-purified T and B cells were examined for HCV positive- and negative-strand RNA by using RT-PCR followed by nucleic acid hybridization, while the presence of viral NS3 protein was determined by flow cytometry. HCV RNA augmentation was assessed by quantification of Southern and dot-blot hybridization signals. The results showed that treatment of peripheral lymphoid cells with mitogens stimulating T- and B-cell proliferation and with cytokines supporting their growth significantly increased HCV RNA detection in patients with both CHC and SVR. This enhancement was up to 100-fold for the HCV genome and fivefold for the NS3 protein compared with untreated cells. In conclusion, HCV RNA can be readily detected in circulating lymphoid cells in progressing hepatitis C and following SVR after ex vivo cell stimulation. As such, this method offers a new investigative tool to study HCV lymphotropism and to monitor virus presence during the course of HCV infection.

Adult↗
PubMedDate not supplied

Use of magnetic beads versus guanidium thiocyanate-phenol-chloroform RNA extraction followed by polymerase chain reaction for the rapid, sensitive detection of enterovirus RNA.

The current study compares the sensitivity of RNA extraction using magnetic beads versus that of a standard extraction method. Streptavadin-coated magnetic beads were labelled with a biotinylated, enterovirus-specific oligonucleotide. RNA was extracted using labelled beads or guanidium thiocyanate-phenol-chloroform from 1, 0.1 and 0.01 TCID50/100 microliters of stock coxsackievirus types A9 and B3, echovirus type 11, enterovirus type 70 and poliovirus type 1. Each strain was tested three times. RNA extraction using magnetic beads was > 50% faster than the standard method. The RNA was amplified using RT-PCR, and the products were detected using agarose gel electrophoresis; 6/15 and 7/15 samples at an initial concentration of 0.01 TCID50/100 microliters were detected using magnetic beads or standard extraction, respectively. Negative-stain electron microscopy was used to determine that 0.01 TCID50/100 microliters of coxsackievirus B3 contained approximately 3 genomes. Thus, use of magnetic beads labelled with an enterovirus-specific oligonucleotide was less toxic, more rapid and as sensitive as the current standard RNA extraction method.

Chloroform↗
PubMed1975

Electron spin resonance for detecting polyadenylate tracts in RNA's.

Electron spin resonance is used to detect RNA's that contain polyadenylate tracts. The method depends on the ability of RNA's that contain polyadenylate sequences to associate with poly(2'-deoxy-2'-fluoro)uridylic acid, which has been spin-labeled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl. The formation of the hybridization product can be detected by monitoring the decrease in mobility of the spin probe.

Adenine Nucleotides↗
PubMed1982

Purification of RNA associated and unassociated forms of 1,4-alpha-glucan branching enzyme from rat liver.

Two molecular forms (BE-I and -II) of 1,4-alpha-glucan branching enzyme were purified from rat liver by affinity chromatography on glycogen-adipoyldihydrazide-Sepharose 4B and glycogen-ethylenediamine-Sepharose 4B, respectively, after hydrophobic chromatography on hexylamine-Sepharose 4B, as single proteins with the same molecular weight of 82,000 on SDS-polyacrylamide gel electrophoresis. By comparing their UV spectra and by RNA detection on polyacrylamide gels after electrophoresis, BE-I and -II were identified to be RNA associated and unassociated forms, respectively. The molecular weights of BE-I and -II estimated by Sephadex G-200 gel filtration were 91,000 and 98,000, respectively, indicating that both forms consist of a monomer. As Be-II had about half the specific activity of BE-I, the RNA component was not essential for the activity of the rat liver branching enzyme, and it was also dissociable from the protein component (BE-II) on polyacrylamide gel electrophoresis at pH 7.3, whereby BE-II showed a heterogeneity of at least three microspecies as revealed by protein staining as well as by activity staining of the gels. Using the antibody against BE-I, BE-I and -II were indistinguishable on immunodiffusion, and the activity inhibition rate of BE-II by the antibody was almost the same as that of BE-I if their specific activities are considered, indicating that the RNA component may have no effect on the enzyme-antibody reaction. Immunochemically no isoenzyme was detected for this enzyme in rat tissues.

1,4-alpha-Glucan Branching Enzyme↗