Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1.
ABSTRACT We evaluated the performance characteristics of a new, real-time nucleic acid sequence-based amplification (NASBA) assay that incorporates molecular beacon technology for detection of human immunodeficiency virus type 1 (HIV-1). The quantitative results were comparable to those obtained with three leading commercially available assays. The analytical sensitivity was 37 IU/ml. The NASBA assay detected clinically relevant recombinant viruses and all group M HIV-1 subtypes.
- SourceAvailable from: Casper L Jansen[show abstract] [hide abstract]
ABSTRACT: We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.Journal of Clinical Microbiology 04/1990; 28(3):495-503. · 4.07 Impact Factor
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ABSTRACT: Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA can also be amplified. This amplification system uses a battery of three enzymes (avian myeloblastosis virus reverse transcriptase, RNase H and T7 RNA polymerase) leading to main amplification product of singlestranded RNA. Expensive equipments are not necessary to acquire a high level of precision. NASBA is an established diagnostic tool in clinical use, with a theoretically bigger analytical sensitivity than reverse transcription-polymerase chain reaction (RT-PCR) for pathogen detection. It has a potential for detection of viable cells through selective amplification of messenger RNA, even in a background of genomic DNA, which PCR does not possess. In the future, NASBA could be used to identify and subsequently quantify microorganisms (even those which cannot be readily cultured) and would be very efficient as routine diagnostic procedures.International Journal of Life Science & Pharma Research. 03/2012; 2(1-ISSN 2250-0480):106-121.
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ABSTRACT: The Nuclisens EasyQ HIV-1 v1.1 assay (Biomerieux) is a real-time detection method combined with NASBA technology designed to measure plasma HIV-RNA. Its performance was assessed in 1008 clinical specimens collected from individuals infected with clade B (774) and non-B (234) HIV-1 variants at four European laboratories. The results were compared with those obtained using three other commercial viral load assays: Cobas Amplicor Monitor HIV-1 v1.5 (Roche), Versant HIV-1 RNA assay (Bayer) and Nuclisens HIV-1 QT (Biomerieux). Overall, the linearity, specificity and reproducibility of the EasyQ assay was comparable with that from the other tests. The correlation coefficient (R) between methodologies was 0.85 for Amplicor; 0.87 for Versant; and 0.91 for Nuclisens. The specificity of the assay was 99.4%. Of note, Versant missed 17% of specimens with non-B subtypes which could be detected by EasyQ, while Amplicor provided similar results than EasyQ. HIV-1 group O specimens were only detected by the EasyQ assay. In conclusion, the performance of the EasyQ assay seems to be similar to that of other HIV-1 viral load tests currently on the market, but it is more sensitive than Versant for HIV-1 non-B subtypes and shows a wider dynamic range than Amplicor. Moreover, as it incorporates the advantage of real-time detection procedures, it facilitates high throughput and short turnaround time.Journal of Virological Methods 08/2005; 127(1):54-9. · 1.90 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, June 2006, p. 2280–2282
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 6
Evaluation of a Real-Time Nucleic Acid Sequence-Based Amplification
Assay Using Molecular Beacons for Detection of Human
Immunodeficiency Virus Type 1
D. R. McClernon,* C. Vavro, and M. St. Clair
Clinical Virology Department, GlaxoSmithKline, Research Triangle Park, North Carolina
Received 18 October 2005/Returned for modification 15 December 2005/Accepted 15 March 2006
We evaluated the performance characteristics of a new, real-time nucleic acid sequence-based amplification
(NASBA) assay that incorporates molecular beacon technology for detection of human immunodeficiency virus
type 1 (HIV-1). The quantitative results were comparable to those obtained with three leading commercially
available assays. The analytical sensitivity was 37 IU/ml. The NASBA assay detected clinically relevant
recombinant viruses and all group M HIV-1 subtypes.
Current FDA-approved human immunodeficiency virus type
1 (HIV-1) viral load assays are labor intensive and rely on
older detection technologies that limit the throughput capabil-
ities of the assays (4). Most assays use a separate detection
step, which results in additional (hands-on) time. For example,
the AMPLICOR HIV-1 Monitor test uses an initial reverse
transcription-PCR amplification reaction and a subsequent
colorimetric step performed in a microwell plate for manual
detection. With the COBAS AMPLICOR tests, the detection
step is automated on the COBAS instrument.
The NucliSens EasyQ HIV-1 assay (bioMe ´rieux, Boxtel, The
Netherlands) is a quantitative, next-generation amplification
assay designed to overcome the labor and throughput obstacles
in the earlier assays. The EasyQ HIV-1 assay uses nucleic acid
sequence-based amplification (NASBA) and provides real-
time detection by incorporating molecular beacons in the
NASBA reaction (2, 3, 6, 7). Like the AMPLICOR assay, the
NucliSens EasyQ test targets the gag region of the HIV-1
genome. Combining the amplification and detection steps fa-
cilitates high-throughput capabilities and provides greater user
convenience. In this study, we evaluated the performance char-
acteristics of the NucliSens EasyQ HIV-1 assay for its interas-
say precision, sensitivity and dynamic range, and ability to
detect HIV-1 group M subtypes and clinically relevant recom-
binant viruses and for the degree of concordance of the results
of the assay with those of three other marketed HIV-1 assay
We used the NucliSens extractor for nucleic acid isolation
and the NucliSens EasyQ HIV-1 assay (version 1.1) according
to the manufacturer’s protocols. An internal calibration stan-
dard (synthetic HIV-1 RNA) was added to the sample before
nucleic acid extraction and isolation by a silica bead procedure
(1). NASBA of the sample RNA together with the calibrator
RNA was performed by using primers specific for wild-type
HIV-1 RNA and calibrator RNA.
For comparison, assays were also performed by using one or
more of the following HIV-1 assay systems approved for mar-
ket in the United States: NucliSens HIV-1 QT (bioMe ´rieux),
AMPLICOR UltraSensitive v1.0 (Roche Molecular Systems,
Pleasanton, Calif.), and COBAS AMPLICOR v1.5 (Roche). In
each case the samples were extracted and the assays were
performed by using the protocols provided by the manu-
To evaluate the precision of the NucliSens EasyQ HIV-1
assay, we analyzed a panel of serial dilutions of an HIV-1 RNA
reference strain calibrated against a WHO standard HIV-1
RNA (NIBSC code 97/656). The viral concentrations in the
panel ranged from 25 to 3 ? 106copies/ml, and 8 to 12 repli-
cates from a total of six runs were tested at each concentration.
Response curves were generated from the serial dilutions.
Although the assay precision was higher at concentrations
?1,000 IU/ml, the assay was linear over a range of 50 IU/ml to
3 ? 106IU/ml, with a mean standard deviation of 0.22 log10
(Fig. 1). Probit analysis of the sensitivity of the assay showed
probabilities of detection of 50% and 95% at 37 IU/ml and 200
IU/ml, respectively, with a 1-ml input volume.
For assessment of clinical reactivity of the assay, we used
repository plasma samples from clinical studies of antiretrovi-
ral therapy sponsored by GlaxoSmithKline (Research Triangle
Park, N.C.). All plasma samples used were from institutional
review board-approved studies in which subjects had provided
written informed consent approving the use of plasma samples
for research purposes. Repository plasma samples from subjects
participating in North American clinical trials were used as a
source of HIV-1 subtype B for comparison of the NucliSens
EasyQ HIV-1 assay with the NucliSens HIV-1 QT assay and
the Roche AMPLICOR UltraSensitive v1.0 assay. All reposi-
tory plasma samples used for this purpose had been verified
earlier to be infected with subtype B. For evaluation of virus
subtypes A, C, D, E F, G, and H and clinically relevant recom-
binant viruses (5), we used repository plasma samples from
antiretroviral therapy-naive subjects from different demographic
regions who were participating in clinical trials in North and
South America. The HIV-1 subtypes in these clinical samples had
already been determined by DNA sequencing of the reverse tran-
scriptase-coding region (ViroSeq HIV-1 Genotyping System v2;
Celera Diagnostics, Alameda, Calif.) and phylogenetic analysis of
* Corresponding author. Mailing address: Clinical Virology Depart-
ment, GlaxoSmithKline, P.O. Box 13398, Research Triangle Park, NC
27709-3398. Phone: (919) 483-9425. Fax: (919) 315-0068. E-mail: daniel
?600 nucleotides of the reverse transcriptase-coding region by
using the NCBI Retroviruses HIV subtyping tool (NIH, Be-
For comparisons of the NucliSens EasyQ HIV-1 assay with the
NucliSens HIV-1 QT assay and with the Roche AMPLICOR
UltraSensitive v1.0 assay, 55 and 91 samples, respectively, were
extracted and tested in parallel. Overall, the concordance of the
results of both assays was very good, with slightly better concor-
dance observed between the results of the two NucliSens assays
(Fig. 2). Eighty-three of 91 samples were positive by both the
Roche AMPLICOR UltraSensitive v1.0 and the NucliSens
EasyQ HIV-1 assays. Six samples had viral loads below the lower
detection limits of both assays. One sample was negative by the
Roche AMPLICOR UltraSensitive assay and positive by the
NucliSens EasyQ HIV-1 assay, and another was negative by
the EasyQ assay and positive by the AMPLICOR assay.
Concordance with the HIV-1 QT assay gave a correlation
coefficient (R) of 0.97, whereas with the AMPLICOR Ultra
Sensitive assay had an R value of 0.93. The log10biases were
0.04 and 0.13 for the comparisons with the NucliSens HIV-1
QT and the Roche AMPLICOR UltraSensitive assays, respec-
To assess assay reproducibility with clinical samples infected
with non-subtype B HIV-1 isolates, three plasma samples in-
fected with HIV-1 subtypes A, C, and F, respectively, were
each independently extracted and tested four times by the
NucliSens EasyQ HIV-1 assay. The EasyQ HIV-1 assay quan-
titated each of the three HIV-1 subtypes reproducibly, with
standard deviations of 0.35 for subtype A, 0.08 for subtype C,
and 0.24 for subtype F (Fig. 3).
Subtype linearity in quantitation was assessed by perform-
ing the NucliSens EasyQ HIV-1 assay by using a 10-fold
serial dilution of each of the three HIV-1 subtype-infected
samples over a dilution range from 1 to 103. Excellent lin-
earity was exhibited for all three subtypes (Fig. 4), although
the slope for subtype F seemed to deviate somewhat com-
pared to those for the other subtypes. This might be attrib-
uted to the lower viral loads and subsequent higher variabil-
ity obtained with subtype F.
We assessed the HIV-1 subtype reactivity of the NucliSens
EasyQ HIV-1 assay in parallel with the Roche AMPLICOR
COBAS v1.5 assay against a panel of clinical specimens with
different HIV-1 subtypes (subtype A, three samples; subtype C,
FIG. 1. Assessment of the NucliSens EasyQ HIV-1 assay against a
panel of serial dilutions of an HIV-1 RNA reference strain.
FIG. 2. Comparisons of the NucliSens EasyQ HIV-1 assay with
the NucliSens HIV-1 QT assay (55 samples) and with the Roche
AMPLICOR Ultrasensitive v1.0 assay (91 samples).
FIG. 3. Assessment of assay reproducibility with clinical samples
infected with non-subtype B HIV-1 isolates.
FIG. 4. Assessment of assay linearity with non-subtype B isolates
by using 10-fold serial dilutions.
VOL. 44, 2006NOTES 2281
three samples; subtype D, one sample; subtype E, two samples;
subtype F, three samples; subtype G, one sample; subtype H,
one sample). The concordance of the results of the two assays
was very good, with an R value of 0.92 (Fig. 5).
To assess assay reactivity against recombinant clinical
strains, we analyzed 30 plasma samples selected from antiret-
roviral therapy-naive subjects of multiple demographic back-
grounds in North and South America. Viruses from these 30
subjects were typed as B/C (n ? 11), B/F1 (n ? 7), B/D (n ?
4), B/F1/D (n ? 3), C (n ? 1), B/H/F1 (n ? 1), B/C/D (n ? 1),
C/H (n ? 1), and F1 (n ? 1). Samples were extracted and
assayed in parallel by the NucliSens EasyQ HIV-1 assay and
the Roche COBAS AMPLICOR v1.5 assay. Both assays de-
tected all recombinants. The concordance of the results of the
two assays was good, with an R value of 0.86 (Fig. 6).
In summary, the NucliSens EasyQ HIV-1 assay gave quan-
titative results comparable to those of the Roche AMPLICOR
UltraSensitive v1.5 assay and showed clinical sensitivity equiv-
alent to that of the Roche AMPLICOR UltraSensitive v1.5
assay. The assay was effective in detecting all group M subtypes
and clinically relevant recombinant viruses. By incorporat-
ing molecular beacon technology in the detection step, the
NucliSens EasyQ HIV-1 assay provides real-time detection
and permits a single test format to be used for samples from a
wide variety of patient populations with variable viral loads.
1. Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-van
Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purifi-
cation of nucleic acids. J. Clin. Microbiol. 28:495–503.
2. Deiman, B., P. van Aarle, and P. Sillekens. 2002. Characteristics and appli-
cations of nucleic acid sequence based amplification (NASBA). Mol. Biotech-
3. de Mendoza, C., M. Koppelman, B. Montes, V. Ferre, V. Soriano, H. Cuypers, M.
Segondy, and T. Oosterlaken. 2005. Multicenter evaluation of the NucliSens
EasyQ HIV-1 v1.1 assay for the quantitative detection of HIV-1 RNA in plasma.
J. Virol. Methods 127:54–59.
4. Murphy, D. G., L. Cote, M. Fauve, P. Rene, and J. Vincelette. 2000. Multi-
center comparison of Roche COBAS AMPLICOR MONITOR version 1.5,
Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex ver-
sion 3.0 for quantification of human immunodeficiency virus type 1 RNA in
plasma. J. Clin. Microbiol. 38:4034–4041.
5. Spira, S., M. A. Wainberg, H. Loemba, D. Turner, and B. G. Brenner. 2003.
Impact of clade diversity on HIV-1 virulence, antiretroviral drug sensitivity
and drug resistance. J. Antimicrob. Chemother. 51:229–240.
6. Stevens, W., T. Wiggill, P. Horsfield, L. Coetzee, and L. E. Scott. 2005.
Evaluation of the NucliSens EasyQ assay in HIV-1-infected individuals in
South Africa. J. Virol. Methods 124:105–110.
7. Yao, J., Z. Liu, L.-S. Ko, G. Pan, and Y. Jiang. 2005. Quantitative detection
of HIV-1 RNA using NucliSens EasyQ HIV-1 assay. J. Virol. Methods 129:
FIG. 5. Comparison of the NucliSens EasyQ HIV-1 assay and the
Roche AMPLICOR Ultrasensitive v1.0 assay for assessment of HIV-1
FIG. 6. Comparison of the NucliSens EasyQ HIV-1 and the Roche
COBAS AMPLICOR v1.5 assays for reactivities against recombinant
2282NOTESJ. CLIN. MICROBIOL.