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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↗
PubMed2002

Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons.

For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay.

Base 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

Detection of hepatitis A virus by the nucleic acid sequence-based amplification technique and comparison with reverse transcription-PCR.

A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 x 10(2) PFU/ml) was obtained with NASBA, compared to 50 PFU (1 x 10(4) PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

Blueberry Plants↗
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↗
PubMed2001

Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses.

The development and application of nucleic acid sequence-based amplification (NASBA) assays for the detection of West Nile (WN) and St. Louis encephalitis (SLE) viruses are reported. Two unique detection formats were developed for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these NASBA assays were compared to those of a newly described standard reverse transcription (RT)-PCR and TaqMan assays for SLE virus and to a previously published TaqMan assay for WN virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA-beacon assay yielding results in less than 1 h. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methodologies and as a tool in conducting flavivirus surveillance in the United States.

Animals↗
PubMed2001

Comparison of three nucleic acid amplification assays of cerebrospinal fluid for diagnosis of cytomegalovirus encephalitis.

The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (n = 15) or other central nervous system conditions (n = 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions.

AIDS-Related Opportunistic Infections↗
PubMed2001

Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups M, N, and O.

Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies a part of the long terminal repeat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monitored in a fluorimeter with a thermostat. The lower level of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of quantification was 100 copies of HIV-1 RNA with a dynamic range of linear quantification between 10(2) and 10(7) RNA molecules. All HIV-1 groups, subtypes, and CRFs could be detected and quantified with equal efficiency, including the group N isolate YBF30 and the group O isolate ANT70. To test the clinical utility of the assay, a series of 62 serum samples containing viruses that encompassed subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed, and these results were compared to the results of a commercially available assay. This comparison showed that the quantification results correlated highly (R(2) = 0.735) for those subtypes that could be well quantified by both assays (subtypes B, C, D, and F), whereas improved quantification was obtained for subtypes A and G and CRFs AE and AG. A retrospective study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group M, who were treated with highly active antiretroviral therapy, revealed that the assay was well suited to the monitoring of therapy effects. In conclusion, the newly developed real-time monitored HIV-1 assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all subtypes and groups of HIV-1.

Anti-HIV Agents↗
PubMed2001

Evaluation of the NucliSens Basic Kit for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genital tract specimens using nucleic acid sequence-based amplification of 16S rRNA.

We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of C. trachomatis and N. gonorrhoeae and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of C. trachomatis, 1 CFU of N. gonorrhoeae, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for N. gonorrhoeae by culture and NASBA and a total of 96 specimens for C. trachomatis by PCR and NASBA. The Basic Kit detected 139 of 142 N. gonorrhoeae culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. For C. trachomatis, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both N. gonorrhoeae and C. trachomatis, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.

Cervix Uteri↗
PubMed2001

Nucleic acid sequence-based amplification of Aspergillus RNA in blood samples.

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant Aspergillus species, including Aspergillus fumigatus. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of Aspergillus RNA with potential for routine diagnosis, including the possibility to test the viability of cells.

Aspergillosis↗
PubMed2001

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG.

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.

Base Sequence↗
PubMed2001

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

Animals↗