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Results for “Self-Sustained Sequence Replication”

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PubMed1998

In situ hybridization detection of low copy nucleic acid sequences using catalyzed reporter deposition and its usefulness in clinical human papillomavirus typing.

In situ hybridization (ISH) detection of low copy DNA and RNA sequences using nonisotopic probes has been difficult in the past because of a lack of sensitivity. Several techniques, such as ISH with radioisotopic-labeled probes, in situ polymerase chain reaction, in situ reverse transcription polymerase chain reaction, self-sustained sequence replication, and chemiluminescence, have allowed increased sensitivity but have required specialized and often expensive equipment, lengthy protocols, and in the case of radioactive probes, there has been an associated increased health risk. Catalyzed reporter deposition (CARD) combined with ISH (CARD-ISH) increases the signal-generating potential of labeled hybridized probes and allows the detection of low copy sequences of nucleic acids in formalin-fixed, paraffin-embedded tissue sections. To determine the sensitivity of CARD-ISH to detect nucleic acids in routinely processed specimens, we analyzed the detection of HPV 16 and 18 infection in formalin-fixed, paraffin-embedded sections of cultured cell lines, including CaSki cells with 400-600 copies of HPV 16, HeLa 229 cells with 10-50 copies of HPV 18, and SiHa cells with 1-2 copies of HPV 16 using a conventional ISH method and by CARD-ISH. In addition, 20 cases of clinical specimens previously analyzed for HPV 6, 11, 16, 18, 31, 33, and 51 with the Enzo PathoGene kit (Enzo Diagnostics, Inc., Farmingdale, NY, U.S.A.) were reexamined with the CARD-ISH method. The CARD-ISH system detected one to two copies of HPV 16 in the SiHa cells whereas the conventional ISH method did not. Both methods detected HPV 16 and 18 in CaSki and HeLa 229 cells, respectively. Three clinical cases that were previously negative and two weakly positive cases of HPV infection were all strongly positive with the CARD-ISH system, a 25% increase in the detection of positive cases by CARD-ISH. We also showed for the first time that a cocktail of six biotinylated oligonucleotide probes was capable of detecting one to two copies of HPV 16 in SiHa cells. These results show that the CARD-ISH method increases the sensitivity of nonisotopic ISH to the level of detecting one to two copies of HPV DNA in formalin-fixed, paraffin-embedded tissue sections using biotinylated cDNA or oligonucleotide probes.

3,3'-Diaminobenzidine↗

PubMed1993

Photochemical sterilization of 3SR reactions.

The self-sustained sequence replication (3SR) reaction is an extremely efficient method for amplifying target DNA and RNA sequences that may be present in minute quantities. A serious problem often encountered in its practice is carryover contamination from products of previous 3SR reactions. A postamplification treatment of 3SR reaction products with the photoactive agent 4'-aminomethyl-4,5-dimethylisopsoralen (IP-10) was investigated as an approach for preventing carryover contamination by 3SR amplicons. Initially, inhibition of the amplification reaction by high concentrations of the reagent was observed. This problem was circumvented by developing a gel-based delivery of IP-10, and the method was found to provide highly efficient sterilization (approximately 10(6)-fold) of 3SR amplicons. Evaluation of this strategy on a number of 3SR targets has indicated that the degree of sterilization is dependent on the length of the amplified region and on the concentration of IP-10. It appears that the sterilization effect is caused by covalent modification of the pyrimidine bases of RNA and DNA, which renders them unusable as templates for the 3SR reaction. Modification of a purified RNA transcript with IP-10 was shown to prevent effectively reverse transcription by avian myeloblastosis virus reverse transcriptase (AMV RT). Similarly, treatment of a T7 RNA polymerase promoter-containing DNA template with IP-10 eliminated full-length transcription by T7 RNA polymerase. This isopsoralen method may be used to sterilize multiple 3SR reactions in a clinical assay with a convenient UV irradiation step.

Base Sequence↗

PubMed1997

Cooperative amplification of templates by cross-hybridization (CATCH).

In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model systems for the investigation of fundamental issues in evolutionary optimization. In this work we present a coupled amplification system based on the self-sustained sequence replication (3SR), also known as nucleic acid sequence-based amplification (NASBA), which allows the experimental investigation of evolving molecular cooperation. The 3SR reaction is an isothermal method of nucleic acid amplification and an alternative to PCR. A target nucleic acid sequence can be amplified exponentially in vitro using two enzymes: reverse transcriptase (RT) and a DNA-dependent RNA polymerase (RNAP). A system has been constructed in which amplification of two molecular species is cooperatively coupled. These species are single-stranded (ss)DNA templates (D1 and D2) of lengths 58 and 68 nucleotides, respectively. Coupling occurs when D1 and D2 anneal to each other via a complementary region (DB and DB') situated at the 3' end of each template. RT elongates the hybridized templates producing a double-stranded (ds)DNA of 106 base pairs (bp). This double strand contains two promoters, which are situated on either side of, and directly adjacent to DB, and which are oriented towards each other. These promoters specify two RNA transcripts encompassing, respectively, the D1 and D2 portion of the dsDNA. After hybridization of two primers (P1 and P2) to the transcripts (R1 and R2) and reverse transcription, the ss templates D1 and D2 are regenerated. Amplification cycles of D1 and D2 are coupled cooperatively via the common dsDNA intermediate. Under optimized batch conditions the system shows the expected growth phases: exponential, linear and saturation phase. The enzymes of the 3SR cycle tend to misincorporate nucleotides and to produce abortive products. In future experiments, we intend to use the system for studies of evolutionary processes in spatially distributed systems where new strategies for optimization at the molecular level are possible.

DNA-Directed RNA Polymerases↗

PubMed1998

Detection of dopamine receptor D1A subtype-specific mRNA in rat kidney by in situ amplification.

In recent years, both molecular biological and immunohistochemical techniques, utilizing receptor subtype-specific probes and antibodies to cloned central nervous system dopamine receptors, have revealed their presence in a number of peripheral organs and tissues. Molecular techniques have been hindered by the low abundance of receptor mRNA in these sites, and reverse transcription-polymerase chain reaction (RT-PCR) has been utilized to address this problem. However, RT-PCR is most often employed on either isolated mRNA or microdissected tissue samples, thereby limiting interpretation of whole tissue distribution. The present paper describes the use of a novel self-sustained sequence replication system (3SR) to amplify a target mRNA sequence in situ within the tissue or cell of interest that is then detected with the use of an internal labeled probe, using standard nonisotopic in situ hybridization. Specifically, D1A receptor mRNA was amplified and detected in kidney sections of Wistar-Kyoto rats (WKY). The amplified D1A receptor mRNA was localized to renal arterioles, juxtaglomerular apparatus, and both proximal and distal tubules. mRNA was colocalized to regions shown also to contain D1A receptor protein. D1A receptor mRNA was predominantly localized in the cortex. Specificity of D1A receptor mRNA detection was confirmed by appropriate localization in rat brain sections known to express D1A receptor mRNA. In addition, we confirmed the presence of renal D1A receptor mRNA by RT-PCR. We conclude that D1A receptor mRNA is expressed in a site-specific manner in the WKY kidney. The use of 3SR in situ permits elucidation of site specific mRNA localization in a manner not reported previously.

Animals↗

PubMed1999

Application of nucleic acid amplification in clinical microbiology.

The use of nucleic acid amplification methods in routine clinical microbiology laboratories is becoming increasingly widespread. The theory of polymerase chain reaction is described, including discussion of suitable microbal targets, extraction of nucleic acid from clinical samples, choice of primers, optimization of the process, laboratory design, contamination, and other problems as well as quality control. Other nucleic acid amplification methods such as ligase chain reaction, self-sustained sequence replication, strand displacement amplification, and branched DNA signal amplification are described and the choice of technology is discussed.

Humans↗

PubMed1993

Homogeneous detection of nucleic acids by transient-state polarized fluorescence.

We describe a transient-state polarized fluorescence-based method for detecting nucleic acids. An active ester of the phthalocyanine dye La Jolla Blue was coupled to an oligonucleotide containing an amino group at its 5' end, and the conjugate was purified by HPLC chromatography. We monitored the hybridization characteristics of the conjugate with complementary oligonucleotides and RNA as targets by transient-state polarized fluorescence measurements. The method was comparable in sensitivity to isotopic and nonisotopic heterogeneous detection systems and was capable of detecting 1 fmol of a 382-base-long RNA transcript from human immunodeficiency virus type (HIV-1) generated in a self-sustained sequence replication (3SR) reaction.

Base Sequence↗

PubMed1998

In-situ polymerase chain reaction: foundation of the technology and today's options.

"In situ PCR" is the marriage of two established technologies in molecular genetics, the polymerase chain reaction (PCR) and in situ hybridization (ISH). It is based on the amplification within intact cells or tissue sections of specific gene sequences, or mRNA species, to levels detectable by ISH and/or immunohistochemistry. Methods to achieve in situ PCR, while sharing fundamental steps, have differed between different laboratories. On the basis of our own experience, in situ PCR appears to be best suited for the detection of DNA in single cell preparations, in which fixation and pre-treatments can be optimally controlled. Emphasis is placed on the requirement for appropriate and meaningful controls at the multiple steps involved. It is instructive to the view the emergence of this new technology in perspective. In situ PCR has not developed in isolation and is just one of several creative approaches that have been employed in recent years to study nucleic acids (DNA and RNA) intracellularly. Some approaches are more suitable for detection of mRNA, or viral RNA, while others are more easily applied to chromosomal DNA. Some further techniques, such as the isothermal self-sustained sequence replication (3SR), refined in-situ transcription (PRINS), or high sensitivity histochemical detection systems, will complement or even add to the potential of situ PCR. It is highly probable that tests will emerge, based on investigation of unique genetic markers, with important roles in specialized diagnostic laboratories for the evaluation of viral diseases, as well as hematological and other malignancies.

Humans↗

PubMed2001

Minisatellite associated sequence amplification (MASA) of the hypervariable repeat marker 33.15 reveals a male specific band in humans.

The 16-nt (5' CACCTCTCCACCTGCC 3') consensus sequence of the hypervariable repeat marker 33.15 was used as primer to conduct minisatellite associated sequence amplification (MASA) of human genomic DNA. MASA detected a 513-bp Y chromosome specific band in males as well as a number of variable bands ranging from 260-2700 bp in both the sexes. Overall polymorphism of the variable alleles was low with the band sharing probability of identity being 1.04x10(-4). Cloning and sequence analysis of the male specific band (GenBank accession no. AF134482) showed a high level (60-80%) of sequence homology with several human Y chromosome derived clones. Similar analysis of Indian rhinoceros (Rhinoceros unicornis) genomic DNA showed male specific isomorphic band though of different size. This assay may provide a useful method for the detection of Y chromosome related heterochromatic sequences in human and other vertebrates.

Animals↗

PubMed1996

Electron Microscopical Autometallography: Immunogold-Silver Staining (IGSS) and Heavy-Metal Histochemistry

Immunogold-silver staining (IGSS) utilizes a histochemical method called autometallography (AMG) to amplify tiny gold particles to sizes easily visible both in light and electron microscopy. In both applications it is advisable to use the smallest possible gold diameters (1-6 nm) to obtain the highest sensitivity, thus, allowing minute amounts of the target substance to be demonstrated. Gold labels smaller than 10 nm in diameter have been clearly shown to give the highest labeling densities of antigen-antibody binding sites. AMG can be used for the detection of catalytic crystal lattices of metallic gold and silver, and sulfides or selenides of mercury, silver, copper, bismuth, and zinc. The method has its roots in "physical development" technique, transplanted from photography to histology by Liesegang at the beginning of this century. In 1981, a series of papers were published by one of us with the purpose of introducing a reliable and easy-to-handle technique for light microscopical and ultrastructural studies. AMG has a multitude of applications apart from its use in detecting tissue metals. These include the highly sensitive and efficient in situ colloidal gold tracing of peptides, proteins, and amines by immunocytochemistry using the IGSS method, of carbohydrates by lectin IGSS, and of nucleic acids by IGSS in situ hybridization, IGSS in situ polymerase chain reaction, and IGSS in situ self-sustained sequence replication-based amplification (in situ 3SR) techniques, the last two even performing with single-copy sensitivity. Applications of pre- and postembedding AMG for semithin and ultrathin tissue sections are described.

Journal Article↗

PubMed2001

RANTES and MIP-1beta mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology.

The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.

Blood Circulation↗

PubMed2001

Quantitative competitive NASBA for measuring mRNA expression levels of the immediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus.

Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.

Cells, Cultured↗

PubMedDate not supplied

Bead-based sandwich hybridization characteristics of oligonucleotide-alkaline phosphatase conjugates and their potential for quantitating target RNA sequences.

The hybridization characteristics of oligonucleotide-alkaline phosphatase conjugate probes were examined in bead-based sandwich hybridization reactions using single-stranded nucleic acid targets and oligonucleotide-polystyrene capture beads. Enzymatic activity was monitored using a chemiluminescent substrate and calibration plots of chemiluminescent signal versus conjugate concentration were used to estimate the sandwich hybridization efficiencies. Improved hybridization behavior was noted using glycerol as an additive and by increasing the length of the probe and alkyl spacer of the conjugates. The chemiluminescent assay is at least as sensitive as those employing 32P-labeled probes and can detect as little as 10-20 amol of target RNA. The linear relationship of chemiluminescent signal versus target assayed provides a method for quantitating unknown target samples. A single human immunodeficiency virus type 1 infected cell in a background of 10(6) uninfected cells is facilely detected when this enzyme-based detection assay is prefaced with a self-sustained sequence-replication amplification reaction.

Alkaline Phosphatase↗

PubMed2001

Detection of human cytomegalovirus (HCMV) pp67-mRNA and pp65 antigenemia in relation to development of clinical HCMV disease in renal transplant recipients.

OBJECTIVE: To evaluate the performance of the recently introduced method based on detection of human cytomegalovirus (HCMV) pp67 mRNA in blood by the nucleic acid sequence-based amplification (NucliSens), in comparison to semiquantitative detection of pp65 HCMV antigen in white blood cells, in relation to development of clinical HCMV disease. METHODS: Thirty patients, recipients of renal transplants, were monitored prospectively for the presence of pp67 mRNA, the presence and level of pp65 antigenemia, IgG and IgM antibodies, and the development of clinical HCMV disease. A total of 148 samples were examined during the observation period. RESULTS: Twenty-five samples were positive for pp67-mRNA and 45 samples contained at least one pp65 positive cell, with 68% agreement between the two assays. Both assays predicted correctly the development of clinical disease in five patients, giving a sensitivity of 100%. However, the specificity of the pp67-mRNA test was 72%, and of the pp65 antigenemia test from 20 to 64%, depending on the level of antigenemia chosen for cut-off. pp67-RNA appeared somewhat earlier than pp65 antigenemia, and responded earlier to treatment. Sero-conversion and appearance of IgM antibodies were of very little clinical value. CONCLUSION: Both the pp67-mRNA and the pp65 antigenemia assay predicted correctly the development of clinical HCMV disease in renal transplant recipients. However, the specificity of both tests with respect to development of HCMV disease, especially the pp65 antigen test was moderate. Significantly positive tests not necessarily prove the development of clinical disease. Testing for pp67-mRNA may improve the diagnosis and management of HCMV disease in renal transplant patients.

Antigens, Viral↗

PubMed2000

Natural residues versus antiretroviral drug-selected mutations in HIV type 1 group O reverse transcriptase and protease related to virological drug failure in vivo.

HIV-1 group O viruses were first recognized as a distinct subgroup of HIV-1 with the isolation and characterization in 1990 of a virus (ANT70) from a woman (individual A) and her spouse (individual B), both from Cameroon (De Leys R, et al.: J Virol 1990;64:1207-1216). During the 5-6 years before treatment, individual A remained asymptomatic, with viral RNA levels between 2.5 and 2.8 log10 copies/ml, as measured by a newly developed group O-specific quantitative NASBA-based RNA assay. Individual B developed mild clinical symptoms, with 3.1 to 3.6 log10 copies of viral RNA per milliliter. HIV-1 sequences obtained from both individuals showed pretreatment residues in protease that confer resistance to protease inhibitors in group M viruses (10I, 36I, and 71V). Individual A showed an initial response to AZT, but shortly after addition of ddC and saquinavir, the RNA levels returned to baseline, while subsequent treatment with d4T, 3TC, and indinavir reduced the RNA level to less than 50 copies/ml for the time of follow-up. Individual B showed no response to AZT or ddC monotherapy, and a change to d4T, 3TC, and indinavir had, in contrast to individual A, only a temporary effect. While a multitude of mutations in HIV-1 group O reverse transcriptase (RT) and protease appeared that are associated with drug resistance in group M viruses, the observed T215N mutation in RT and the V15I and V22A mutations in protease have not previously been described and may represent resistance-conferring mutations specific to group O viruses. These results indicate that treatment of HIV-1 group O-infected individuals with antiretroviral drug regimens that include protease inhibitors might lead to rapid selection for resistance-conferring mutations. This probably results from preexisting protease residues contributing to reduced sensitivity of group O viruses to protease inhibitors, as is observed in vitro.

Amino Acid Sequence↗

PubMed2000

Evaluation of human cytomegalovirus gene expression in thoracic organ transplant recipients using nucleic acid sequence-based amplification.

BACKGROUND: Human cytomegalovirus (CMV) infection is a major cause of morbidity in transplant patients. Early diagnosis and treatment have been shown to improve outcome. We evaluated the suitability of CMV immediate early, early, and late gene expression detected by nucleic acid sequence-based amplification (NASBA) as markers of CMV infection. METHODS: Blood samples were taken immediately before transplant and every one to two weeks after transplantation for 12 weeks from 50 patients undergoing thoracic organ transplantation. CMV-NASBA was performed and results compared with serology, CMV pp65 antigenaemia (CMV-AG) and the development of clinical CMV infection. Patients received "preemptive" anti-CMV therapy with ganciclovir based on the CMV-AG results. RESULTS: CMV immediate early and early gene expression were detected in 87 and 47%, respectively, of patients without other evidence of CMV infection. CMV late gene expression had a sensitivity of 97% for infection (compared with 83% for CMV-AG P=0.06) and a specificity of 93% (compared with 100% P=NS). Late gene expression occurred at the same time as CMV antigenaemia but 1.1 weeks earlier than the threshold of antigenaemia (CMV-AG>10) used to initiate preemptive therapy. CONCLUSION: NASBA provided a standardized tool for the detection of CMV transcripts with a greater sensitivity than the standard antigenemia test. Detection of immediate early and early gene transcripts was not specific for subsequent infection. CMV late gene expression determined by NASBA was an accurate and early marker of CMV infection. Detection of CMV late gene expression could be used to trigger "preemptive" anti-CMV therapy.

Adult↗

PubMed2001

Direct quantification of human cytomegalovirus immediate-early and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification.

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10(4) copies per 100 microl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.

Antibodies, Viral↗

PubMed2001

Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection.

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons. A major advantage of our assay is the one-tube, isothermal nature of the method, which allows high-throughput applications for nucleic acid detection. The homogeneous real-time detection allows a closed-tube format of the assay, avoiding any postamplification handling of amplified material and therefore minimizing the risk of contamination of subsequent reactions. The assay has a detection range of 10(3) to 10(9) HBV DNA copies/ml of plasma or serum (6 logs), with good reproducibility and precision. Compared with other HBV DNA assays, our assay provides good sensitivity, a wide dynamic range, and high-throughput applicability, making it a viable alternative to those based on other amplification or detection methods.

Centrifugation, Density Gradient↗

PubMed2001

Longitudinal variability of human immunodeficiency virus type 1 RNA viral load measurements by nucleic acid sequence-based amplification and NucliSens assays in a large multicenter study.

Human immunodeficiency virus type 1 (HIV-1) RNA measurements were evaluated within an externally controlled multilaboratory program. Three external standards (1.5 x 10(3) to 1.5 x 10(6) copies/ml) were included in 814 assay runs by four laboratories. Results indicate that HIV-1 RNA levels can be measured with a precision equal to that of the pre-highly active antiretroviral therapy era (standard deviations, +/-0.16 to 0.25 log10 units).

HIV Infections↗