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PubMed1997

Quantitative detection of hepatitis C virus RNA in the serum of patients with chronic hepatitis C treated with interferon: a pilot study.

BACKGROUND: It is not known whether the measurement of serum hepatitis C virus (HCV) RNA by reverse transcription polymerase chain reaction (RT-PCR) could improve the management of patients with chronic hepatitis C being treated with interferon. OBJECTIVES: We analysed, in a pilot study, the relations between the variations of HCV-RNA and alanine aminotransferase (ALT) serum levels in 18 anti-HCV positive patients treated with interferon. STUDY DESIGN: Serum HCV-RNA was measured, using a non competitive coamplification assay (Amplicor HCV Monitor), before (at 3, 2 and 1 months and baseline), during (first, third and sixth month) and after treatment for at least 8 months (range 8-17 months). HCV-RNA levels fluctuations were correlated with those of ALT and treatment outcome. According to the ALT pattern, four patients were non responders, seven partial responders, four relapsers and two long term responders. RESULTS: The median and mean baseline HCV-RNA levels were significantly different in patients infected by HCV type 1, 2 and 3, being 248,449, 235,506; 4170, 17,866 and 22,315, 79,273 molecules per ml, respectively (P < 0.0001). We did not find any significant difference between median and mean baseline viremia of responders and non responders. After 1 month of treatment viremia was below the sensitivity levels of the assay in 77.7% (14/18) of the patients who normalized ALT, at least temporarily. On the contrary, HCV-RNA remained detectable in non responders. CONCLUSIONS: Our data suggest that HCV-RNA detection using Amplicor Monitor at the first month of treatment can be useful to identify non responders, avoiding three additional months of treatment as would be required by ALT monitoring alone. During the post-treatment follow-up, persistence of undetectable HCV-RNA and normal ALT levels helps to identify long term responders from patients with the risk of relapse in spite of biochemical remission.

Adolescent↗
PubMed1999

Detection of dengue virus RNA in patients after primary or secondary dengue infection by using the TaqMan automated amplification system.

In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR. For this amplification technique new primers and special fluorochrome-labeled probes had to be synthesized. During amplification the increasing amount of viral DNA could simultaneously be measured in the tightly sealed tubes. Dengue virus RNA was found in almost all patients (17 of 18), if the samples had been taken soon after the onset of symptoms and before anti-dengue virus antibody had been produced. RNA was detectable in only one of five persons who had anti-dengue virus immunoglobulin M (IgM) antibodies but not yet IgG antibodies. In 30 late samples with both IgG and IgM antibodies viral RNA was no longer demonstrable. In two early samples from two frequent travelers obtained 1 and 2 days after the onset of symptoms significant IgG antibody titers were present but there were no anti-dengue virus IgM antibodies. In these samples a viral load of >5 x 10(6) dengue virus RNA copies (dengue types 1 and 2) was detectable. These findings of a high viral load in the presence of anti-dengue virus IgG antibody are suggestive of a secondary dengue virus infection. In the 20 tourists (17 plus 1 plus 2) in whom viral RNA was found, the dengue virus serotype could be related to the area where the infection had taken place. Most of our patients came from southeast Asia and most frequently had dengue virus type 1 infections (8 of 20).

Base Sequence↗
PubMed1997

Therapy of hepatitis C: patients with normal aminotransferase levels.

An important group of patients with chronic hepatitis C have normal serum alanine aminotransferase (ALT) levels despite having hepatitis C virus (HCV) RNA detectable in serum. These patients are typically identified after donating blood and being found to be positive for antibody to HCV (anti-HCV). A strict definition of this patient population is needed, which should include presence of anti-HCV, HCV RNA detectable by polymerase chain reaction (PCR), and persistently normal ALT levels. These patients are usually asymptomatic, but almost all have histological evidence of chronic hepatitis on liver biopsy. The liver histological lesions are generally mild, and cirrhosis is rare. The long-term outcome of this group of patients with chronic HCV infection is not known, but the prognosis is probably good. In small, uncontrolled trials of alpha interferon in this group of patients, end-of-treatment virological responses occurred in approximately 50% and sustained responses 6 to 12 months afterwards in 20%. These rates of response are not very different from those reported in patients with abnormal ALT levels. Importantly, in most studies, serum ALT levels became abnormal during therapy in approximately half of patients, and levels remained abnormal in a proportion after therapy. These findings suggest that alpha interferon therapy is not usually beneficial and may be harmful in patients with chronic hepatitis C who have normal ALT levels, perhaps as a result of the immunomodulatory actions of alpha interferon that may alter the balance of host immune reactions and viral replication, which seem to be responsible for the liver injury in chronic hepatitis C.

Alanine Transaminase↗
PubMed1996

Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA.

Toward the development of a universal, sensitive and convenient method of DNA (or RNA) detection, electrochemically active oligonucleotides were prepared by covalent linkage of a ferrocenyl group to the 5'-aminohexyl-terminated synthetic oligonucleotides. Using these electrochemically active probes, we have been able to demonstrate the detection of DNA and RNA at femtomole levels by HPLC equipped with an ordinary electrochemical detector (ECD) [Takenaka,S., Uto,Y., Kondo,H., Ihara,T. and Takagi,M. (1994) Anal. Biochem., 218, 436-443]. Thermodynamic and electrochemical studies of the interaction between the probes and the targets are presented here. The thermodynamics obtained revealed that the conjugation stabilizes the triple-helix complexes by 2-3 kcal mol-1 (1-2 orders increment in binding constant) at 298 K, which corresponds to the effect of elongation of additional several base triplets. The main cause of this thermodynamic stabilization by the conjugation is likely to be the overall conformational change of whole structure of the conjugate rather than the additional local interaction. The redox potential of the probe was independent of the target structure, which is either single- or double stranded. However, the potential is slightly dependent (with a 10-30 mV negative shift on complexation) on the extra sequence in the target, probably because the individual sequence is capable of contacting or interacting with the ferrocenyl group in a slightly different way from each other. This small potential shift itself, however, does not cause any inconvenience on practical applications in detecting the probes by using ECD. These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acids due to the stability of the complexes, high detection sensitivity and wide applicability to the target structures (DNA and RNA; single- and double strands) and the sequences.

DNA↗
PubMed1992

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.

Animals↗
PubMed2001

TaqMan 5'-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening.

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5'-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5'-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5'-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a >or=95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.

Blood Donors↗
PubMed1995

A fibroblast cell line cultured from a hypertrophic scar displays selective downregulation of collagen gene expression: barely detectable messenger RNA levels of the pro alpha 1(III) chain of type III collagen.

The present study was designed to investigate the expression of type I, III and VI collagens by a fibroblast cell line initiated from a hypertrophic scar. The same tissue has previously been demonstrated to display markedly elevated expression of type I and III collagen mRNAs in vivo. Unexpectedly, slot-blot and Northern hybridizations revealed a barely detectable steady-state level of pro alpha 1(III) collagen chain mRNA in cultured hypertrophic scar fibroblasts. The levels of pro alpha 1(I) and alpha 2(VI) collagen chain mRNAs were essentially the same in fibroblasts cultured from hypertrophic scar and in fibroblasts cultured from normal skin. However, Northern blot analyses indicated that the ratio of 5.8 kb to 4.8 kb species of pro alpha 1(I) collagen mRNA was slightly reduced in fibroblasts originating from the hypertrophic scar compared to that in normal fibroblasts. When normal fibroblasts were incubated in conditioned medium from hypertrophic scar cultures, the expression of pro alpha 1(III) collagen chain mRNA decreased to a markedly lower level. Our studies suggest that collagen synthesis by fibroblasts in hypertrophic scars is stimulated by humoral factors which are active only in vivo. Furthermore, the results suggest that fibroblasts cultured from hypertrophic scar display a selective downregulation of different collagen genes and that this downregulation is exerted through an autocrine mechanism.

Adult↗
PubMed1990

A herpes simplex virus type 1 variant, deleted in the promoter region of the latency-associated transcripts, does not produce any detectable minor RNA species during latency in the mouse trigeminal ganglion.

In peripheral sensory ganglia latently infected with herpes simplex virus type 1 (HSV-1) transcription is restricted. A set of viral latency-associated transcripts, the LATs, have been characterized by Northern blotting and in situ hybridization. These transcripts have previously been mapped to a 3 kb region of the viral genome within the repeat long region. However, transcription from adjacent regions of the genome can be detected by in situ hybridization, which cannot be detected by Northern blotting. These RNAs are termed minor LATs or m-LAT. In this study we show that in ganglia latently infected with the HSV-1 variant 1704, which is deleted in one complete copy of the LAT gene and in the promoter and 5' portion of the other copy, m-LATs are not detected by in situ hybridization. Furthermore, the levels of DNA in nervous system tissue latently infected with the parental and the 1704 variant virus are similar. Thus we propose that the sequence elements necessary for initiating transcription or stabilizing m-LATs are within the region deleted in variant 1704 that codes for the promoter and the 5' end of the LATs.

Animals↗
PubMedDate not supplied

[Demonstration of the influenza virus A RNA by nucleic acid molecular hybridization using biotin-treated probes].

A probe containing full-size DNA copy of influenza A/USSR/90/70 virus protein gene M labeled with biotin on 32P was used for influenza A virus RNA detection by dot hybridization method. For labeling with biotin, a new method of its administration by chemical modification of nucleic acid was employed. In homologous DNA:DNA hybridization the sensitivity of determinations was less than 1 pg in the biotin-treatment of the probe and 1.25 pg in its radioactive labeling. Hybridization of DNA probe with cytoplasmic RNA isolated from influenza A virus-infected (strains A/USSR/90/77 and A/Texas/77) MDCK cells revealed RNA in the dot corresponding to 4.5-5.5 1g ID50 of virus present in 2 x 10(4) cells. The probe did not bind with negative controls in any dot in all the tests. The results of the study indicate that DNA probes labeled with biotin and 32P and used in dot hybridization for influenza A virus RNA detection in infected cells show the similar sensitivity and specificity.

Biotin↗
PubMed2005

Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription.

A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(-) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.

Animals↗
PubMed1989

Monoclonal antibody assay for detection of double-stranded RNA and application for detection of group A and non-group A rotaviruses.

Fastidious viruses are generally detected in human body fluids by means of immunoassay or nucleic acid hybridization systems. These approaches can be difficult to apply to the detection of viruses which display variations in antigenic or genetic composition. Rotaviruses are examples of viruses which can display such variations. Recently identified antigenic variants, designated as non-group A rotaviruses, cannot be detected by immunoassays or nucleic acid hybridization assays which utilize reagents directed at group A rotavirus strains. The incomplete understanding of the extent of antigenic and genetic variation has inhibited the development of assay systems for all of the non-group A rotaviruses and has limited the study of their role in human disease. While rotaviruses display genetic variation, they all contain a genome which consists of double-stranded RNA. We utilized a monoclonal antibody to devise a sensitive assay for the measurement of double-stranded RNA and applied it to the detection of a wide range of rotaviruses. We found that the assay could detect double-stranded RNA from as few as 10 PFU of standard strains of group A rotaviruses. The assay system was also capable of detecting double-stranded RNA from several strains of group B rotaviruses isolated from calves, rats, and pigs at levels below those at which viral RNA could be visualized by means of polyacrylamide gel electrophoresis. When applied to the detection of double-stranded RNA in serial stools shed by rotavirus-infected children, the assay system was capable of detecting double-stranded RNA in samples in which antigen could not be detected by immunoassay. The specific nature of the double-stranded RNA detected by this assay system could be determined by the elution of the nucleic acids from the monoclonal antibody and the reaction of the RNA with specific nucleotide probes. The measurement of double-stranded RNA offers a potential method for the sensitive detection of a wide range of rotaviruses and other members of the family Reoviridae.

Animals↗
PubMed2006

[Early detection of hepatitis C virus infection using a new combined antigen-antibody detection assay: potential use in HIV co-infected individuals].

BACKGROUND: The aim of this study was to determine the clinical benefit of a new combined antigen-antibody immunoenzymatic assay (Monolisa HCV Ag-Ab Ultra, Biorad) in the setting of acute HCV infection in HIV infected patients. PATIENTS AND METHODS: The performance of this assay was first evaluated in 160 HIV positive samples from uninfected and chronically HCV infected patients. To assess the performance of the Ag-Ab assay in the context of acute hepatitis C, 94 stored frozen serums from 20 recently diagnosed cases were retrospectively tested for HCV-RNA and presence of anti-HCV antibodies, in parallel with the new assay. RESULTS: In HIV infected patients, the sensitivity and specificity of the Ultra assay was 100% with a strong discrimination between positive and negative samples. In HCV acutely infected patients, the Ag-Ab assay significantly reduced the seronegative period, allowing an earlier diagnosis compared to a 3rd generation ELISA in 65% of the cases. The combined assay became positive on the same bleed as the first HCV-RNA detection for 13 patients out of 20. Nevertheless, in one case, characterized by an absence of seroconversion over one year but a continuous viral replication above 1 million IU/ml, the combined assay did not improve HCV infection diagnosis. CONCLUSION: Use of this new assay as a first line screening would significantly reduce the long seronegative window period seen in HCV infection allowing earlier HCV diagnosis and rapid clinical management. However, in case of clinical acute hepatitis C, sensitive HCV-RNA detection should remain the gold standard.

Antigen-Antibody Complex↗
PubMed1999

Isolation of DNA after extraction of RNA To detect the presence of Borrelia burgdorferi and expression of host cellular genes from the same tissue sample.

We are investigating the neuropathogenesis of Lyme disease caused by Borrelia burgdorferi in a nonhuman primate model. In the past, two separate pieces of tissue had to be used when both analyzing for the presence of the spirochete and examining the host response to infection. We have modified a procedure to purify DNA from the same sample after the extraction of RNA. The remaining material containing the DNA was precipitated, and residual organic reagent was removed prior to deproteinization and extraction of the DNA. This procedure now allows us to both assay for the presence of the Lyme microorganism and analyze the host response in the same tissue preparation.

Animals↗
PubMed1994

Detection of HCV RNA by the asymmetric gap ligase chain reaction.

The ligase chain reaction (LCR) and the gap ligase chain reaction (gLCR) are exponential amplification techniques for the detection of DNA sequences in a sample. Both techniques depend on the enzyme, DNA ligase, to join adjacent probes annealed to a DNA molecule. However, DNA ligase joins DNA inefficiency on an RNA target. Consequently, LCR and gLCR cannot amplify RNA efficiency. RNA detection methods using LCR or gLCR require a cDNA synthesis step. The carryover of four dNTPs from the cDNA reaction inhibits gLCR. Although LCR can use cDNA reaction products directly, background generated by blunt-end ligation does not allow the high sensitivity typically needed for HIV or HCV detection. The asymmetric gap ligase chain reaction (AGLCR) is a modification of gLCR that allows for the detection of RNA by using < or = 3 of the 4 nucleotides in the cDNA step and the gLCR step. Fewer than 50 copies of synthetic RNA transcript can be reproducibly detected. HCV, an RNA virus with no DNA intermediate, was chosen as the initial RNA model system. HCV antibody-positive and normal samples were analyzed, and the results were found to correlate with the results obtained using nested RNA-PCR. AGLCR provides a new nucleic acid amplification technique that can aid in the diagnosis of disease when the detection of RNA is critical.

Base Sequence↗
PubMed1990

Serological and genetic characterization of bovine rotaviruses in Thailand by ELISA and RNA-RNA hybridization: detection of numerous non-serotype 6 strains.

A total of 62 fecal specimens positive for rotavirus were collected from diarrheic cows in Thailand in 1988 and 1989. The antigenic properties of rotaviruses in stool were examined by enzyme-liked immunosorbent assays using specific monoclonal antibodies directed at VP4, VP6 or VP7: all the bovine rotavirus strains were determined as subgroup I; none of the strains were reactive with serotype 6-specific monoclonal antibody; and different reactivities of the bovine strains with two anti-VP4 monoclonal antibodies were observed. Polyacrylamide gel electrophoresis of viral RNA exhibited three different RNA electropherotypes. In RNA-RNA hybridization experiments using cell culture-adapted three strains as well as a reference bovine strain (NCDV), RNA from the Thai bovine strains showed very low homology to that from NCDV; only three or four RNA segments were hybridized between the RNAs from Thai samples and NCDV. These results suggested that bovine rotaviruses isolated in Thailand are serologically and genetically distinct from a reference serotype 6 bovine strain, NCDV.

Animals↗
PubMed2002

Identification of OCT6 as a novel organic cation transporter preferentially expressed in hematopoietic cells and leukemias.

OBJECTIVE: Human organic cation transporters (OCTs) play a critical role in the cellular uptake and efflux of endogenous cationic substrates and hydrophilic exogenous xenobiotics. We sought to identify OCT genes preferentially expressed in hematopoietic cells. MATERIALS AND METHODS: We isolated a novel OCT, named OCT6, by data-mining human expressed sequence tag databases for sequences homologous to known OCT genes. We developed a quantitative reverse transcriptase polymerase chain reaction assay to determine the relative expression of this gene in 50 cancer cell lines and in tissues. RESULTS: The two highest expressing cell lines were the leukemia cell lines HL-60 and MOLT4. Quantitative reverse transcriptase polymerase chain reaction analysis using a normal tissue cDNA panel demonstrated that this transport gene is highly expressed in testis and fetal liver, with detectable RNA levels in bone marrow and peripheral blood leukocytes. Unlike other OCT genes, RNA levels were not detectable in placenta, liver, or kidney. To further define the expression of OCT6 in hematopoietic tissues, we measured OCT6 RNA levels in sorted peripheral blood cell populations and found a clear enrichment of OCT6-expressing cells in purified CD34(+) cells. To determine if OCT6 was highly expressed in leukemias, we examined circulating leukemia cells from 25 patients and found high levels of OCT6 RNA in all specimens in comparison with liver, kidney, and placenta. CONCLUSIONS: The results demonstrate the existence of a novel OCT preferentially expressed in human hematopoietic tissues, including CD34(+) cells and leukemia cells. Its narrow tissue distribution, potential for substrate specificity, and close homology to other cell membrane transporters make OCT6 an attractive target for the treatment of leukemia.

Amino Acid Sequence↗
PubMed1993

Differential location of nucleic acids within interchromatin granule clusters.

We have examined in great detail the distribution of nucleic acids within interchromatin granule clusters in different cell types by means of various immunocytochemical approaches. Using the in situ polyadenylate nucleotidyl transferase-immunogold technique for RNA detection or anti-RNA antibodies, we decisively demonstrate the presence of appreciable amount of RNA in clusters of interchromatin granules of untreated cells. Neither the in situ terminal deoxynucleotidyl transferase-immunogold technique nor anti-DNA antibodies, nor the in situ nick-translation technique for DNA detection have revealed any DNA in the interchromatin granule clusters. However, dispersed chromatin sensitive to DNase I are found at the borders and in the close vicinity of interchromatin granule clusters. The results indicate that interchromatin granule clusters should not be nuclear structures directly involved in RNA transcription but rather in some other steps of RNA metabolism.

Animals↗