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PubMed2000

Frequent detection of HIV-1 RNA but low rates of HIV-1 isolation in cervicovaginal secretions from infected women.

Information regarding the presence of HIV-1 in the female genital tract is necessary to gain insight into the mechanism of HIV-1 heterosexual transmission. Herein, we present the results of a study on virus isolation and HIV-1 RNA detection from cervicovaginal lavage (CVL) samples from 25 HIV-1 seropositive women. Despite detectable levels of HIV-1 RNA in 88% of CVL samples, HIV-1 was isolated in only four (19%) samples. Although HIV-1 shedding in cervicovaginal secretions is a common event at all disease stages, the recovery of infectious virus in cell cultures appears to be rare; this renders viral isolation in studies aimed to evaluate the infectivity of cervicovaginal secretions relatively useless.

Cervix Uteri↗
PubMed2001

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations.

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).

Base Sequence↗
PubMed1997

Effects of formalin fixation and prolonged block storage on detection of hepatitis C virus RNA in liver tissue.

It has been suggested that prolonged formalin fixation and block storage adversely affect hepatitis C virus (HCV) ribonucleic acid (RNA) detection in tissue by reverse transcriptase-polymerase chain reaction (RT-PCR). We attempted to determine whether short-term perfusion fixation (3-5 days) or prolonged formalin storage adversely affects the detection of HCV RNA in paraffin-embedded tissue in comparison with 24-h fixation. Also, we examined the effects of prolonged storage of paraffin blocks on the sensitivity for HCV detection. We performed RT-PCR in formalin-fixed explanted livers from 20 liver allograft recipients known to be HCV positive (10 with specimens stored for 2-4 years and 10 with specimens stored for > 4 years). We compared the results of perioperative needle liver biopsy specimens fixed overnight with liver sections fixed by perfusion for 3-5 days and bulk liver tissue stored in formalin for years (mean, 6.25 years; range, 2-11 years). HCV RNA was detected in 100%, 85%, and 0% of specimens fixed for 24 h, 3-4 days, and years, respectively. We conclude that HCV can be readily detected in tissue fixed by formalin overnight, sensitivity decreases slightly with intermediate-length fixation, and HCV is rendered undetectable by prolonged fixation. In addition, retention of formalin-fixed tissue in paraffin blocks does not affect the sensitivity of HCV detection.

Formaldehyde↗
PubMed1997

In situ hybridization in living cells: detection of RNA molecules.

Fluorescence hybridization is a widely used technique in cell biology and pathology for detecting specific nucleic acid (DNA and RNA) sequences in fixed cells. This technique does not, however, provide dynamic information on the intracellular behavior of the targeted molecules. The aim of this work was to investigate possibilities of labeled DNA probes for RNA detection in cells that are maintained alive. Such techniques will provide useful tools for studying dynamic cellular processes such as RNA distribution and transport from transcription sites to translation sites by means of fluorescence microscopy. First a reversible, nonperturbing cell permeabilization procedure was developed using streptolysin O. This procedure was used to introduce oligodeoxynucleotides and fluorochrome-labeled DNA probes specific for 28S ribosomal RNA (2.1 kb) into living cells, which were then analyzed by fluorescence microscopy. The results showed that: (i) no increased cell death or growth perturbation was observed after permeabilization, (ii) introduction of a 28S RNA-specific probe (plasmid and oligonucleotides) into living cells led to bright nucleoli and a low cytoplasmic signal, and (iii) negative control probes did not lead to any fluorescent staining. These results indicate that specific hybridization of labeled nucleic acid probes takes place while cells are maintained under normal physiological conditions.

Animals↗
PubMed2003

Development and evaluation of the nuclisens basic kit NASBA for the detection of RNA from Candida species frequently resistant to antifungal drugs.

We describe a Nucleic Acid Sequence Based Amplification (NASBA) protocol to detect 6 different Candida species (Candida krusei, Candida glabrata, Candida inconspicua, Candida dubliniensis, Candida norvegensis, Candida lusitaniae) and compare it to a PCR assay. NASBA showed a sensitivity of 1 Colony Forming Unit and detected RNA from all 6 Candida species within 1 working day. All 5 patients with documented candidiasis showed identical results by both methods. This assay offers a sensitive, specific and fast possibility to detect yeast RNA.

Antifungal Agents↗
PubMed2001

Noncoding RNA gene detection using comparative sequence analysis.

BACKGROUND: Noncoding RNA genes produce transcripts that exert their function without ever producing proteins. Noncoding RNA gene sequences do not have strong statistical signals, unlike protein coding genes. A reliable general purpose computational genefinder for noncoding RNA genes has been elusive. RESULTS: We describe a comparative sequence analysis algorithm for detecting novel structural RNA genes. The key idea is to test the pattern of substitutions observed in a pairwise alignment of two homologous sequences. A conserved coding region tends to show a pattern of synonymous substitutions, whereas a conserved structural RNA tends to show a pattern of compensatory mutations consistent with some base-paired secondary structure. We formalize this intuition using three probabilistic "pair-grammars": a pair stochastic context free grammar modeling alignments constrained by structural RNA evolution, a pair hidden Markov model modeling alignments constrained by coding sequence evolution, and a pair hidden Markov model modeling a null hypothesis of position-independent evolution. Given an input pairwise sequence alignment (e.g. from a BLASTN comparison of two related genomes) we classify the alignment into the coding, RNA, or null class according to the posterior probability of each class. CONCLUSIONS: We have implemented this approach as a program, QRNA, which we consider to be a prototype structural noncoding RNA genefinder. Tests suggest that this approach detects noncoding RNA genes with a fair degree of reliability.

Algorithms↗
PubMed1993

Interest of the detection of hepatitis C virus RNA in patients with alcoholic liver disease. Comparison with the HBV status.

HCV-RNA detection was investigated in 66 chronic alcoholic patients divided into 3 groups according to the severity of liver injury: group 1 included 22 chronic alcoholics without cirrhosis, group 2, 20 patients with alcoholic cirrhosis and group 3, 24 patients with alcoholic cirrhosis and hepatocellular carcinoma. The 'nested' polymerase chain reaction (PCR) technique amplifying the 5' non-coding region was used to detect HCV-RNA. For comparison, ELISA1, ELISA2 and RIBA2 tests (Ortho Diagnostics System) were also used to detect anti-HCV antibodies. Finally HBV markers (HBsAg, anti-HBc and anti-HBs antibodies) were detected in all patients as well as HBV-DNA by PCR. In group 1, only 1 patient (4.5%) showed an HCV-RNA-positive PCR, while 3 patients (13.6%) were found to have anti-HCV antibodies detected by RIBA2. In group 2, 3 patients (15%) showed positive PCRs, whereas 4 patients (20%) had anti-HCV antibodies. Finally, in group 3, the PCR was positive in 3 patients (12.5%), while 9 (37.5%) had anti-HCV antibodies. All patients with positive PCRs showed positive anti-HCV antibodies detected by second-generation assays. On the other hand, these patients often had past HBV infection markers but rarely had HBV-DNA detected by PCR. These results suggest that in chronic alcoholic patients, regardless of the severity of liver injury, HCV replication is rarely observed by PCR. Indeed, replication is only observed when anti-HCV antibody detection is positive in second-generation assays, particularly with strong reactivity against C33-C and C22-3 antigens. The relatively high prevalence of anti-HCV antibodies in this population compared to the usual rates could be explained by the age, geographic and perhaps even socioeconomic origin of the patients.

Adult↗
PubMed1989

Use of synthetic oligonucleotide probes to detect rhinovirus RNA.

Current methods of detecting a human rhinovirus (HRV) infection are either based on isolation of virus in appropriate susceptible cell lines, which is time-consuming and requires considerable expertise, or are dependent on knowing the serotype. The existence of over 100 immunologically distinct serotypes makes serotype specific assays, such as ELISA, unsuitable for general diagnostic assays. In this study a general rhinovirus assay is described which utilises synthetic oligonucleotides as probes in a filter hybridization assay. The probes are designed to bind to short but highly conserved regions of the rhinovirus genome. Indeed, the probes successfully detected all 57 rhinovirus serotypes tested. Furthermore, the test was used to demonstrate rhinovirus infection in clinical samples from 57 volunteers, inoculated with HRV, collected on six consecutive days. Clinical samples were taken prior to inoculation and on days 2-7 after inoculation. The filter hybridization assay gave results comparable to virus culture on days 2 and 3 post-inoculation, but was more sensitive on subsequent days.

Base Sequence↗
PubMed2002

Detection of HCV RNA in saliva of patients with hepatitis C virus infection by using a highly sensitive test.

Hepatitis C virus (HCV) is transmitted primarily by direct percutaneous exposures to blood. Since HCV RNA has been found in saliva, it has been suggested that saliva might also be a source of infection. HCV RNA in saliva from plasma HCV RNA positive patients was tested by a highly sensitive PCR method. HCV RNA was detected in 32 out of 61 saliva specimens (52.4%). No correlation was found between the presence of HCV in saliva and age, sex, identified risk factors for HCV infection, time lapsed since the diagnosis, transaminases and alkaline phosphatase values and stimulated salivary flow. A statistically significant relation between plasma HCV RNA viral load and saliva HCV RNA detection was observed (P<0.001). In conclusion, HCV RNA is often present in saliva of HCV infected patients, with plasma viral load being the only known predictable factor. Further studies on salivary HCV RNA are needed.

Adult↗
PubMed2002

Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato.

AIMS: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. METHODS AND RESULTS: It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine-killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead. The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. CONCLUSIONS: NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.

Base Sequence↗
PubMed1995

Single-step reverse transcription-polymerase chain reaction for hepatitis C virus RNA with DNA enzyme immunoassay hybridization.

A single reverse transcription-polymerase chain reaction (SRT-PCR) for HCV RNA detection, confirmed by hybridization of amplified products with biotinylated probes using DNA enzyme immunoassay (DEIA), was compared to nested-PCR (N-PCR) on 20 sera from patients with chronic (n = 18) or acute (n = 2) hepatitis. Results obtained by SRT-PCR confirmed by DEIA and by N-PCR identical. All but one of the patients with chronic hepatitis and positive HCV serology as well as the patients with acute hepatitis had detectable HCV RNA in serum; all patients with chronic hepatitis and indeterminate HCV serology and all controls (n = 5) were negative by the two PCR methods. Both SRT-PCR and N-PCR remained positive after 7 x 10(-2) and 5 x 10(-4) dilutions of two HCV RNA-positive sera. The threshold of detection for SRT-PCR was 15 RNA copies per assay, as assessed by testing serial dilutions of an in vitro synthesized 5'-NCR HCV RNA transcript. SRT-PCR confirmed by DEIA for HCV RNA appears to be as sensitive and specific as N-PCR. Furthermore, it is easier to perform, with less of contamination, is less time-consuming, requires fewer enzymes, and it permits semi-quantification of PCR products.

Acute Disease↗
PubMed2005

Specific detection of RNA on ultra-thin sections.

In this paper, we describe a specific method for ultrastructural detection of RNA. Our method is based in the bromination "in situ" of uridine residues in the RNA, which allows the detection of brominated RNA with specific antibodies against bromo-deoxyuridine. With this method we can achieve high specificity and resolution, and it can be applied to Epon or acrylic resin embedded material.

Antibodies↗
PubMed1998

Hepatitis C--role of perinatal transmission.

To evaluate the role of perinatal transmission in the spread of hepatitis C virus (HCV), we screened a cohort of pregnant intravenous drug using (IVDU) women for HCV antibody detection; where seropositive HCV RNA detection by polymerase chain reaction (PCR) was found we followed the infants longitudinally for HCV antibody and HCV RNA. Serum prevalence for HCV for this population was 80% with HCV RNA detected in 50%. Recruitment and follow-up over a 3-year period of a cohort of 83 seropositive women, their 91 newborns and 16 siblings of newborns, showed that there had been a 3% perinatal transmission rate with 1 sibling also infected. These positive cases were defined as transient in 1 case (HCV RNA positive by PCR at 1 month, but seronegative and HCV RNA negative at 10 months of age), 2 unevaluable (HCV RNA positive at 2 months of age, but patients lost to follow-up), and 1 chronic infection in a child at 34 months (positive HCV RNA and seropositive 34-month sibling). Maternal HCV RNA levels for those with infected infants was a mean 40-fold greater than those whose babies were uninfected, although this did not reach statistical significance. Of the remaining infants, the majority (93%) had lost passively acquired maternal antibodies by 9 months of age and all by 12 months. Of 18 women who were HCV seropositive and breast feeding (66% of whom were HCV RNA positive in their sera), none had detectable HCV RNA in breast milk. Hence we conclude that transmission of HCV from mother to infant appears to be of low frequency and positivity appears to correlate with maternal circulating viral load.

Adult↗
PubMed1990

Flow injection analysis and real-time detection of RNA bases by surface-enhanced Raman spectroscopy.

Surface-enhanced Raman scattering (SERS) spectroscopy has been successfully interfaced with a flow injection analysis system to detect RNA bases in real time. Four of the major base components of RNA, uracil, cytosine, adenine, and guanine, were introduced into the flow injection system and were mixed with a Ag sol prior to SERS measurements. Several experimental parameters including pH, temperature, flow rate, and tubing materials were examined, and their impact on the SERS spectra is presented here. The feasibility of interfacing flow injection based SERS detection methods with liquid or high-performance liquid chromatography for the detection of individual components in a complex mixture is also assessed.

Chromatography, High Pressure Liquid↗
PubMed1998

GBV-C/HGV coinfection in HIV-1-positive men: frequent detection of viral RNA in blood plasma but absence from seminal fluid plasma.

Sequential paired samples of blood and seminal fluid were obtained from a cohort of 54 HIV-1-infected homosexual males. The prevalence of GBV-C/HGV RNA in the cell-free fractions of some of these patients was determined using reverse-transcription polymerase chain reaction (RT-PCR). To assess the effects of HIV-1 and HCV infection upon GBV-C/HGV RNA status, blood CD4 cell counts, HCV RNA status, and HIV-1 proviral DNA and viral RNA titres were also determined. GBV-C/HGV RNA was detected in 8/30 (27%) of the blood plasma samples obtained at the start of the study, and was present at a frequency of 14/64 (22%) in all the blood plasma samples tested. By contrast, GBV-C/HGV RNA was not detected in the 26 seminal fluid samples obtained at the start of the study, including 8 samples obtained from patients for which GBV-C/HGV RNA was detected in the corresponding blood sample. Of the samples tested for the presence of both GBV-C/HGV and HCV RNA, there was no evidence of coinfection. Although GBV-C/HGV RNA detection rates were significantly higher in individuals with blood CD4 cell counts greater than 200 cells per microlitre, there were no significant differences in the median blood CD4 cell counts or HIV-1 proviral DNA or viral RNA titres observed between the GBV-C/HGV-positive and -negative individuals. The failure to detect GBV-C/HGV RNA in seminal fluid samples obtained from this cohort would suggest that further studies need to be carried out to determine the roles of sexual transmission and of seminal fluid in GBV-C/HGV infection.

CD4 Lymphocyte Count↗
PubMed1998

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.

Cells, Cultured↗
PubMed1990

Detection of RNA on northern blots by negative staining with aurintricarboxylic acid.

Aurintricarboxylic acid (ATA) is a well-known inhibitor of RNA and DNA modifying enzymes and was suggested as a potent RNase inhibitor for preparation of RNA (Hallick et al., 1977, Nucleic Acids Res. 4, 3055-3064). We show that ATA is a very useful stain for detecting RNA on Northern blots and slot blots although it did not fully protect purified RNA in concentrated solution against RNase A.

Animals↗
PubMed2002

Investigation of HGV and TTV infection in sera and saliva from non-hepatitis patients with oral diseases.

AIM: To determine the frequencies of HGV and TTV infections in serum and saliva samples of non-hepatitis patients with oral diseases in Hangzhou area, and to understand the correlation between detected results of HGV RNA and/or TTV DNA in sera and in saliva from the same patients. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection were performed in the serum and saliva samples from 226 non-hepatitis patients with oral diseases, and nucleotide sequence analysis. RESULTS: Twenty-seven (11.9 %) and 21 (9.3 %) of the 226 serum samples were only positive for HGV RNA and TTV DNA, respectively. 10 (4.4 %) and 9 (3.9 %) of the 226 saliva samples were only positive for HGV RNA and TTV DNA, respectively. And 7 (3.1 %) of the serum samples and 2 (0.9 %) of the saliva samples showed the positive amplification results for both HGV RNA and TTV DNA. 12 saliva samples from the 34 patients (35.3 %) with HGV or HGV/TTV viremia and 11 saliva samples from the 28 patients (39.3 %) with TTV or HGV/TTV viremia were HGV RNA detectable, respectively, including two patients positive for both HGV RNA and TTV DNA in serum and saliva samples. No saliva samples from the 226 patients were found to be HGV RNA or TTV DNA detectable while their serum samples were negative for HGV or TTV. Homologies of the nucleotide sequences of HGV and TTV amplification products from the serum and saliva samples of the two patients compared with the reported sequences were 88.65-91.49 % and 65.32-66.67 %, respectively. In comparison with the nucleotide sequences of amplification products between serum and from saliva sample from any one of the two patients, the homologies were 98.58 % and 99.29 % for HGV, and were 98.65 % and 98.20 % for TTV, respectively. CONCLUSION: Relatively high carrying rates of HGV and/or TTV in the sera of non-hepatitis patients with oral diseases in Hangzhou area are demonstrated. Parts of the carriers are HGV and/or TTV positive in their saliva. The results of this study indicate that dentists may be one of the populations with high risk for HGV and/or TTV infection, and by way of saliva HGV and TTV may be transmitted among individuals.

DNA Virus Infections↗