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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↗
PubMed2002

Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (CCU) of M. pneumoniae. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 x 10(4) molecules of in vitro-generated RNA or 5 CCU of M. pneumoniae. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for M. pneumoniae by PCR were also positive by NASBA. These results indicate that the sensitivity of detection of M. pneumoniae in spiked respiratory samples by NASBA is high. Together with the use of the internal control, the assay merits evaluation as a diagnostic tool.

Humans↗
PubMed2003

Detection of Mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification.

Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of Mycoplasma pneumoniae. In vitro-generated M. pneumoniae RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of M. pneumoniae RNA in the amplification and 10(4) molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and the conventional NASBA assays corresponded to 5 color-changing units (CCU) of M. pneumoniae. In spiked throat swabs, nasopharyngeal aspirates, bronchoalveolar lavages, and sputum, the sensitivity of both NASBA assays corresponded to 5 to 50 CCU of M. pneumoniae. A total of 17 clinical specimens positive for M. pneumoniae by PCR were also positive by conventional NASBA, but one specimen was negative by RT-NASBA. These results indicate that the sensitivity of detection of M. pneumoniae by RT-NASBA in respiratory samples might be slightly reduced compared to that by conventional NASBA. However, the real-time assay is superior in speed and ease of handling.

Humans↗
PubMed2004

Construction strategy for an internal amplification control for real-time diagnostic assays using nucleic Acid sequence-based amplification: development and clinical application.

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5' end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results.

Animals↗
PubMed2004

Development of a nucleic acid sequence-based amplification assay that uses gag-based molecular beacons to distinguish between human immunodeficiency virus type 1 subtype C and C' infections in Ethiopia.

A gag-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C') circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C' by sequencing of the gag gene. All 36 isolates were correctly identified by the gag beacon test. Three isolates with genomes that were recombinant in gag were unambiguously typed as belonging to the C' subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C' sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and gag molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C' beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C' HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.

Base Sequence↗
PubMed2005

Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum.

Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 microl of venous blood to achieve this sensitivity, compared to 50 microl of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.

Animals↗
PubMed2005

Nucleic acid amplification assays for detection of La Crosse virus RNA.

We report the development of nucleic acid sequence-based amplification (NASBA) and quantitative real-time reverse transcription (RT)-PCR assays for the detection of La Crosse (LAC) virus in field-collected vector mosquito samples and human clinical samples. The sensitivities of these assays were compared to that of a standard plaque assay in Vero cells. The NASBA and quantitative real-time RT-PCR assays demonstrated sensitivities greater than that of the standard plaque assay. The specificities of these assays were determined by testing a battery of reference strain viruses, including representative strains of LAC virus and other arthropod-borne viruses. Additionally, these assays were used to detect LAC viral RNA in mosquito pool samples and human brain tissue samples and yielded results within less than 4 h. The NASBA and quantitative real-time RT-PCR assays detect LAC viral RNA in a sensitive, specific, and rapid manner; these findings support the use of these assays in surveillance and diagnostic laboratory systems.

Animals↗
PubMed2005

Development, technical performance, and clinical evaluation of a NucliSens basic kit application for detection of enterovirus RNA in cerebrospinal fluid.

The combination of nucleic acid sequence-based amplification and electrochemiluminescence detection was used to develop an internally controlled, highly sensitive and specific assay for the detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF). The analytical performance of the assay was determined using both in vitro-transcribed EV RNAs and viral culture isolates. The sensitivity of the assay was 10 EV RNA copies per amplification reaction. The assay detected all enteroviral isolates tested with no cross-reactivity to 21 nonenteroviral species, including rhinovirus and parechovirus. The clinical performance of the assay was evaluated by testing 992 CSF specimens collected from adult and pediatric patients. NucliSens EV results from a subset of 327 CSF samples were compared to viral culture of nasopharyngeal specimens and rectal swabs (n = 195) and/or CSF (n = 212). Of the 212 CSF samples, 96 samples were positive by either the NucliSens EV assay (94/96; 97.9%) or culture (63/96; 65.6%), and 61/96 (63.5%) were positive by both methods. The inclusion of an EV-specific internal control monitored the entire process, including the efficiency of nucleic acid extraction, amplification, and detection. In total, only five blood-clotted CSF samples (0.5%) were inhibited. The NucliSens EV assay demonstrated superior sensitivity over viral culture (P < 0.001), excellent specificity, clear delineation of positive samples, and minimal amplification inhibition.

Adolescent↗
PubMed2005

Real-time nucleic acid sequence-based amplification using molecular beacons for detection of enterovirus RNA in clinical specimens.

Real-time nucleic acid sequence-based amplification (NASBA) using molecular beacon technology (NASBA-beacon) was compared to standard NASBA with postamplification hybridization using electrochemiluminescently labeled probes (NASBA-ECL) for detection of enteroviruses (EV) in 133 cerebrospinal fluid and 27 stool samples. NASBA-ECL and NASBA-beacon were similar in sensitivity, detecting 55 (100%) and 52 (94.5%) EV-positive samples, respectively. There were no false positives. Both NASBA assays were significantly more sensitive than culture. Real-time NASBA-beacon reagents and equipment rental were more expensive than those for NASBA-ECL; however, time to result was shortened by 1.5 h, hands-on time was reduced by 25 min, and the assay was much simpler for technologists to learn and perform.

Adolescent↗
PubMed2006

Detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-PCR, in children with acute respiratory infections during a winter season.

Five hundred seventeen consecutive nasopharyngeal aspirates were collected between October 1998 and May 1999 for episodes of acute respiratory tract infections in children presenting at the University Hospital of Antwerp. Culture and nucleic acid amplification techniques--nucleic acid sequence-based amplification (NASBA) and reverse transcription-PCR (RT-PCR)--were applied to detect rhinoviruses (RVs). Other respiratory viruses were detected by immunofluorescence (IF) analysis of the specimens and IF analysis of shell vial cultures. Among the 517 specimens, 219 viral agents were identified. They were, in decreasing order, rhinoviruses (93 [18.0%]), respiratory syncytial virus (76 [14.7%]), adenoviruses (16 [3.1%]), influenza viruses (15 [2.9%]), enteroviruses (15 [2.9%]), and herpes simplex virus (4 [0.8%]). For the evaluation of rhinovirus detection, culture positivity and/or a positive reaction in the two independent amplification methods was used as an expanded "gold standard." Based on this standard, the sensitivity, specificity, positive predictive value, and negative predictive value of culture were 44.7, 100, 100, and 99.8%, and those of NASBA and RT-PCR were 85.1, 98.3, 83.3, and 98.5% and 82.9, 93.4, 55.7, and 98.2%, respectively. NASBA and RT-PCR produced comparable results and were significantly more sensitive than virus culture. RVs showed the highest incidence in acute respiratory tract infections in children.

Acute Disease↗
PubMed2006

Development of conventional and real-time nucleic acid sequence-based amplification assays for detection of Chlamydophila pneumoniae in respiratory specimens.

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 mul sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.

Chlamydophila pneumoniae↗
PubMed2001

Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus.

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.

Drug Resistance, Microbial↗
PubMed2002

Development of real-time NASBA assays with molecular beacon detection to quantify mRNA coding for HHV-8 lytic and latent genes.

BACKGROUND: Human herpesvirus-8 (HHV-8) is linked to the pathogenesis of Kaposi's sarcoma (KS), and the HHV-8 DNA load in peripheral blood mononuclear cells (PBMC) is associated with the clinical stage of KS. To examine the expression of HHV-8 in PBMC, four HHV-8 mRNA specific NASBA assays were developed METHODS: We have developed four quantitative nucleic acid sequence-based amplification assays (NASBA-QT) specifically to detect mRNA coding for ORF 73 (latency-associated nuclear antigen, LANA), vGCR (a membrane receptor), vBcl-2 (a viral inhibitor of apoptosis) and vIL-6 (a viral growth factor). The NASBA technique amplifies nucleic acids without thermocycling and mRNA can be amplified in a dsDNA background. A molecular beacon is used during amplification to enable real-time detection of the product. The assays were tested on PBMC samples of two AIDS-KS patients from the Amsterdam Cohort. RESULTS: For all four assays, the limit of detection (LOD) of 50 molecules and the limit of quantification (LOQ) of 100 molecules were determined using in vitro transcribed RNA. The linear dynamic range was 50 to 10(7) molecules of HHV-8 mRNA. We found HHV-8 mRNA expression in 9 out of the 10 tested samples. CONCLUSION: These real-time NASBA assays with beacon detection provide tools for further study of HHV-8 expression in patient material.

Antigens, Viral↗
PubMed2006

Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification.

BACKGROUND: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (<20 parasites/mul) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at <100 parasites/microl. METHODS: This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. RESULTS: The lower detection limit of the assay is 100-1000 molecules in vitro RNA for all species, which corresponds to 0.01-0.1 parasite per diagnostic sample (i.e. 50 microl of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. CONCLUSION: Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 microl of blood) and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus making it an effective tool for diagnostic purposes and useful for epidemiological and drug studies.

Animals↗