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At least 91 records · Page 5Linked to original sources
PubMed2005

Collaborative study to evaluate a working reagent for West Nile virus RNA detection by nucleic acid testing.

BACKGROUND: A nucleic acid test (NAT) assay reference reagent for West Nile virus (WNV) RNA, consisting of heat-inactivated WNV grown in tissue culture and diluted in pooled, negative human plasma, was evaluated and quantitated in a collaborative study in which 14 laboratories participated. STUDY DESIGN AND METHODS: Participants were requested to assay serial half-log and log dilutions of the reagent to determine the RNA endpoint. A single endpoint for each such dilution series was calculated with the maximum likelihood method, which assumes that the probability of a positive result at a given dilution follows a Poisson distribution. The calculated endpoint was used to give an estimated "NAT-detectable units per mL" (not necessarily equivalent to genome equivalents/mL or copies/mL). The assays used by participants included qualitative and quantitative NAT assays and both the commercial WNV assays (Chiron and Roche). RESULTS: The estimated number of detectable units per mL for the 14 laboratories varied from log 2.0 to 3.0 with the exception of two outliers. The overall mean titer for all the assays was log 2.52 detectable units per mL (330 detectable units/mL). Multiple testing of individual vials by two laboratories indicated that there was no evidence of vial-to-vial variation in WNV content of the reference reagent. CONCLUSION: A reference reagent for WNV NAT assays has been established. The mean titer of the reagent, with the results from 14 laboratories, was 330 detectable units per mL.

Humans↗
PubMed1998

Diagnostic implications of albumin messenger RNA detection and cytokeratin pattern in benign hepatic lesions and biliary cystadenocarcinoma.

Cytokeratin (CK) patterns and albumin messenger RNA (mRNA) are investigated in 24 patients with benign hepatic lesions (7 patients with focal nodular hyperplasia [FNH], 10 with hepatocellular adenomas [HA], 1 with biliary hamartoma, 4 with biliary cysts, 2 with cystadenomas) and in 8 patients with cystadenocarcinoma, a rare liver malignancy. The lesions and surrounding tissue of the hepatocytic components expressed CK 8 and 18 at immunohistochemistry, whereas the biliary elements evidenced CK 8 and 18 and CK 7 and 19. The albumin mRNA, as detected by in situ hybridization (ISH), revealed different distributions in the hepatocytes of FNH and HA. In the benign biliary lesions, the normal hepatocytes surrounding the tumors expressed albumin mRNA, whereas the biliary structures did not. Interestingly, in the cystadenocarcinomas, albumin mRNA was observed not only in the hepatocytes of residual parenchyma, but also in neoplastic bile duct cells lining the carcinomatous cysts; no signal was identified in the nonneoplastic biliary elements. This indicates that cystadenocarcinomas have a mixed biological phenotype and suggests they could arise either from pluripotent cells or from neoplastic cells that reacquire epigenetic features. Our results suggest two possible diagnostic applications for albumin ISH: on routine sections, it could represent an important tool for distinguishing between cystadenoma and cystadenocarcinoma; and on fine needle biopsy specimens, it could reduce uncertainty between FNH and HA.

Adenoma↗
PubMed1984

Use of a 2-5A analogue probe for detecting RNA ligase and RNA ligase substrates in mammalian cell extracts.

The compound ppp(A2'p)3A3'[32P]pCp is a commercially available radioactive analogue of the 2',5' oligoadenylate series ppp(A2'p)nA, n greater than or equal to 2, commonly referred to as 2-5A. It is used as a probe for measuring concentrations in competition radiobinding and radioimmune assays. We have found that incubation of the probe with extracts from HeLa, CV1, or neuroblastoma cells results in its covalent attachment to two size classes of RNA: the first includes a major species with a molecular weight of approximately 350,000, the second is much smaller (40 +/- 5 nucleotides in length) and could represent tRNA half-molecules. Ligation is to the 3' end of the probe molecule with formation of a 3',5'-phosphodiester bond. Thus, probe ligation provides a sensitive and convenient assay for the detection not only of RNA ligase(s) but also of ligatable RNAs (such as the putative tRNA half-molecules) in mammalian cell extracts.

Animals↗
PubMed2003

Binding affinity and inhibitory potency of neomycin and streptomycin on the Tat peptide interaction with HIV-1 TAR RNA detected by on-line acoustic wave sensor.

The binding of two aminoglycoside antibiotics, neomycin and streptomycin, to a segment of the transactivation responsive region (TAR) RNA of the human immunodeficiency virus, and their inhibitory potency to disrupt the interaction of the RNA with a regulatory Tat protein-derived peptide, have been studied using a flow-through acoustic wave detector system. Binding affinity is directly correlated with the inhibitory potency of these molecules and the acoustic wave detection system shows that neomycin exhibits at least a ten-fold greater affinity for TAR RNA and that it is also a more potent inhibitor than streptomycin. These results are in agreement with previous studies. However, unlike the time-consuming batch-based assays, use of the flow-through format offers considerable potential for the rapid screening of the chemistry of relatively small-molecule-nucleic acid binding events.

Acoustics↗
PubMed1997

[Comparison of anti-hepatitis C virus (HCV) IgM and HCV-RNA detection in serum samples of patients with HCV infection].

Anti-HCV IgG is the routine assay for the diagnosis hepatitis C. But the method is ineffective in early detection of hepatitis C. We have detected anti-HCV IgM in hospital hepatitis C patients by Enzyme-Linked Immunosorbent Assay (ELISA) and compared the results with those of HCV-RNA and total antibody. Our studies indicate that anti-HCV IgM is detected in all hepatitis C marker positive sera. But the amount of anti-HCV IgM in HCV-RNA positive sera is significantly higher than that of in the corresponding negative sera. This subjects that anti-HCV IgM and HCV-RNA are somewhat correlated and they have different clinical interests. Anti-HCV IgM can be expected and hepatitis activities and its actual clinical interests are to be further studied.

Antibodies, Anti-Idiotypic↗
PubMed2006

HCV RNA detection by TMA during the hepatitis C antiviral long-term treatment against cirrhosis (Halt-C) trial.

For making treatment decisions related to chronic hepatitis C, the utility of HCV RNA tests with increased sensitivity has not been defined. Prior interferon nonresponders with advanced fibrosis (n = 1,145) were retreated with peginterferon alpha-2a and ribavirin. Patients who were HCV RNA-negative by a polymerase chain reaction (PCR)-based assay (Roche COBAS Amplicor HCV Test, v. 2.0; lower limit of detection [LOD] 100 IU/mL) at week 20 (W20) received treatment for 48 weeks. Stored specimens were tested using the Bayer VERSANT HCV RNA Qualitative (TMA) Assay (LOD 9.6 IU/mL) and compared to PCR results for the ability to predict sustained virological response (SVR; defined as undetectable HCV RNA by PCR at W72). Nearly all PCR-positive samples (1006/1007, 99.9%) were positive as assessed by TMA. Among 1,294 PCR-negative samples, 22% were TMA-positive. Negative TMA results were more predictive of SVR than were negative PCR results at W12 (82% vs. 64%, P < .001) and at W20 (66% vs. 52%, P = 0.001). SVR was more likely the earlier TMA had become negative during treatment (82% at W12, 44% at W20, 20% at W24). Among 45 patients who were TMA-positive but were PCR-negative at W20 and W24, none achieved SVR (95% CI: 0%-8%). Approximately 10% of patients with a single positive TMA result at the end of treatment still achieved SVR. In conclusion, negative TMA results at or after W12 were superior to negative PCR results for predicting SVR. In patients with negative PCR results during treatment, a single positive TMA test did not exclude SVR, although persistently positive tests did.

Antiviral Agents↗
PubMed2001

Screening of active lyssavirus infection in wild bat populations by viral RNA detection on oropharyngeal swabs.

Brain analysis cannot be used for the investigation of active lyssavirus infection in healthy bats because most bat species are protected by conservation directives. Consequently, serology remains the only tool for performing virological studies on natural bat populations; however, the presence of antibodies merely reflects past exposure to the virus and is not a valid marker of active infection. This work describes a new nested reverse transcription (RT)-PCR technique specifically designed for the detection of the European bat virus 1 on oropharyngeal swabs obtained from bats but also able to amplify RNA from the remaining rabies-related lyssaviruses in brain samples. The technique was successfully used for surveillance of a serotine bat (Eptesicus serotinus) colony involved in a case of human exposure, in which 15 out of 71 oropharyngeal swabs were positive. Lyssavirus infection was detected on 13 oropharyngeal swabs but in only 5 brains out of the 34 animals from which simultaneous brain and oropharyngeal samples had been taken. The lyssavirus involved could be rapidly identified by automatic sequencing of the RT-PCR products obtained from 14 brains and three bat oropharyngeal swabs. In conclusion, RT-PCR using oropharyngeal swabs will permit screening of wild bat populations for active lyssavirus infection, for research or epidemiological purposes, in line not only with conservation policies but also in a more efficient manner than classical detection techniques used on the brain.

Animals↗
PubMed1992

Unexpected heterogeneity in E2A/PBX1 fusion messenger RNA detected by the polymerase chain reaction in pediatric patients with acute lymphoblastic leukemia.

The t(1;19)(q23;p13) is the most common recurring chromosomal translocation in childhood acute lymphoblastic leukemia (ALL) and has been associated with adverse prognosis. It involves the rearrangement of two genes, PBX1 and E2A, resulting in the production of transforming chimeric DNA-binding proteins. In all previous reports in which the presence of a chimeric transcript was described, the fusion point between the coding sequences of E2A and PBX1 was found to be constant at the RNA level. We have used RNA-based polymerase chain reaction (PCR) for the detection of E2A/PBX1 messenger RNAs (mRNAs) in children with ALL at the time of diagnosis. Of 21 patients exhibiting this rearrangement, 3 (14%) expressed a variant E2A/PBX1 transcript in addition to the expected one. The relative amounts of the two chimeric mRNAs varied between the patients, but remained constant in the same patient during different stages of the disease. Sequence analysis showed an identical insertion of 27 bp at the E2A/PBX1 junction of the variant RNA species, the translation of which would result in the replacement of Val478 by 10 amino acids. The inserted sequence has not been detected in any other human transcript besides the variant E2A/PBX1 RNA species and probably represents a splicing variant of the chimeric RNA. We conclude that a subset of pediatric patients with ALL that carry the E2A/PBX1 rearrangement express two types of the chimeric mRNA. The biologic significance of this additional E2A/PBX1 transcript is discussed.

Adolescent↗
PubMed2004

[Clinical significance of detecting RNA and anti HEV antibody in convalesent sera in patients with acute HEV hepatitis].

OBJECTIVE: To investigate the anti hepatitis E virus (HEV) and HEV RNA in acute and convalescent sera of patients with NonA-E acute hepatitis. METHODS: The serum samples were taken from 95 patients who were diagnosed as acute NonA-E hepatitis. Enzyme immunoassay (EIA) was used for detecting anti-HEV Immunoglobulin G (IgG, Genolable and Wantai EIA anti-HEV kits). RT-PCR amplification of HEV RNA was based on the open reading frame 2 region of HEV and the PCR products were sequenced. RESULTS: Sera from 95 patients who were negative for anti-HEV in acute phase were followed up for 11-35 days to detect the anti-HEV antibody in recovery phase, 16/95 (16.84%) were positive for anti-HEV (wantai EIA anti-HEV kits). Ten (62.50%) were positive for HEV RNA in acute phase. Sequence analysis showed that 4 were HEV genotype. 6 were HEV genotype; 12/95 (12.50%) were positive for anti-HEV (Genolable EIA anti-HEV kits). Seven were positive for HEV RNA; 4 belonged to HEV genotype, 3 were HEV genotype. CONCLUSION: It is significant and necessary to detect anti HEV antibody and HEV RNA in patients with HEV infection during acute phase and convalesent phase.

Acute Disease↗
PubMed1973

Maternal histone messenger RNA: detection by molecular hybridization.

A competition-hybridization assay has been developed for the majority of sequences present in embryonically synthesized histone messenger RNA. The assay permits the first direct demonstration of specific "maternal" messengers in unfertilized sea-urchin eggs. The molecular size of histone messenger RNA stored in the egg appears to be the same as that of histone messenger RNA synthesized by the embryo. Maternal histone messenger RNA is found in the soluble phase of egg homogenates, in the form of ribonucleoprotein particles, unassociated with ribosomes.

Animals↗
PubMed1984

A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation.

Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks. Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8. A [P32] DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis. A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed.

Animals↗
PubMed1987

Preprotachykinin messenger RNA detected by in situ hybridization in striatal neurons of the human brain.

Sections from postmortem human brain were processed for in situ hybridization histochemistry using a 35S-labelled RNA probe transcribed from a cDNA coding for the human preprotachykinin which contains both substance P and K. Labelled neurons were observed in the caudate nucleus and the putamen but not the cerebellum. The labelled cells were of medium size and their distribution and morphology were compatible with previous data on substance P-like immunoreactivity in the human brain. The results confirm the presence of preprotachykinin mRNA in a subpopulation of striatal neurons in the human and show that in situ hybridization can be used to detect specific neurotransmitter-related mRNAs in postmortem tissue from normal and diseased humans.

Aged↗
PubMed2001

[Analysis of the primary structure of hantavirus RNA, detected in organs of patients that died from HFKS in the Republic of Bashkortostan].

Fragments of the minor S and middle M segments of Hantavirus genome RNA were isolated by reverse transcription-polymerase chain reaction (RT-PCR) from the organs of patients who died from hemorrhagic fever with the renal syndrome (HFRS) during an epidemic outbreak of this infection in the Bashkortostan Republic in 1997-1998. The sizes of the resultant PCR fragments were 317 p. n. for S segment and 416 p. n. for M segment. Their primary structures (GENBANK registration numbers AJ133581 and AJ133582) were determined and they were compared with the nucleotide sequences of the respective genome sites of Hantavirus strain CG1820/Ufa-83 isolated from Clethrionomys in Bashkortostan in 1983. The homologies of nucleotide sequences of the corresponding sites of S and M segments of RNA isolate of 1997 and CG1820/Ufa-83 were 87.7 and 96.6%, respectively. The authors discuss the probability of circulation of hantaviruses differing by individual features of primary structure of genome RNA in Bashkortostan.

Amino Acid Sequence↗
PubMed2002

Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection.

Multiplexed detection of oligonucleotide targets has been performed with gold nanoparticle probes labeled with oligonucleotides and Raman-active dyes. The gold nanoparticles facilitate the formation of a silver coating that acts as a surface-enhanced Raman scattering promoter for the dye-labeled particles that have been captured by target molecules and an underlying chip in microarray format. The strategy provides the high-sensitivity and high-selectivity attributes of gray-scale scanometric detection but adds multiplexing and ratioing capabilities because a very large number of probes can be designed based on the concept of using a Raman tag as a narrow-band spectroscopic fingerprint. Six dissimilar DNA targets with six Raman-labeled nanoparticle probes were distinguished, as well as two RNA targets with single nucleotide polymorphisms. The current unoptimized detection limit of this method is 20 femtomolar.

Coloring Agents↗
PubMed1983

RNA splicing in neurospora mitochondria: structure of the unspliced 35S precursor ribosomal RNA detected by psoralen cross-linking.

The structure of the unspliced 35S precursor rRNA of Neurospora mitochondria was studied by psoralen photochemical cross-linking. The results show that when the 35S RNA is cross-linked in ribonucleoprotein particles (RNPs) under appropriate conditions, the predominant configuration is a 2.2 kb intron loop which brings opposite splice sites into proximity; that the predominant secondary structural feature in the free RNA is a relatively large hairpin (length = 0.105 kb) in the center of the molecule at or near the 5' splice site; that the intron loop and the central hairpin are different configurations of sequences at or near the 5' splice site; and that the intron loop is stabilized by protein components of RNPs. Based on the structures detected by psoralen photochemical cross-linking, we propose a mechanism for the splicing of the Neurospora mitochondrial precursor rRNA. We propose further that certain features of this mechanism may be relevant to the splicing of other RNAs, including eucaryotic mRNAs.

Base Sequence↗