Kein Folientitel

1
Direct Amplification and Detection of Enterovirus
RNA by Nucleic Acid Sequence Based Amplification
(NASBA) A. Heim, J. Schumann, P.
Pring-Akerblom Institut für Virologie,
Medizinische Hochschule Hannover, 30623 Hannover
ahei_at_virologie.mh-hannover.de
Figure 2 depicts the positions of the primers
and the probe in a multiple alignment of the
5NTR of various enteroviruses.
Moreover, the sensitivity of
NASBA was detemined with a panel of laboratory
enterovirus strains, and a panel of clinical
samples (cerebrospinal fluid and endomyocardial
biopsies) which were also screened for
enterovirus RNA with a nested RT-PCR Heim
1997 3. Results Adjustment of the K
concentration to 50 mM gave an enhanced
sensitivity compared to the frequently
recommended K concentration of 70 mM (figure
3). Sensitivity was in the range of 10
to 100 molecules positive strand enterovirus
(CVB3) RNA with detection of NASBA amplificates
by ethidium-bromide stained agarose gel
electrophoresis. NASBA was partially selective
for positive strand RNA detecting negative
strands with an about 100-fold reduced
sensitivity (figure 4).
1. Background NASBA is an isothermal nucleic
acid amplification reaction (similar to 3SR and
TMA) using two specific oligonucleotide primers
(one of these contains a T7 promotor sequence),
and three enzymes, AMV reverse transcriptase,
RNAse H, and T7 RNA polymerase Compton 1991,
Kievits 1991. In contrast to polymerase chain
reaction (PCR), NASBA amplifies RNA directly and
does not require an extra reverse transcription
reaction. The main reaction product is negative
strand RNA. Figure 1 gives an overview of the
reaction principle. 2. Material
and Methods In order to evaluate NASBA for the
detection of genomic enterovirus RNA and to
compare its sensitivity with reverse
transcription/PCR, the reaction was set up with
enterovirus specific primer sequences identical
to those proposed by Rotbart J. Clin.
Midrobiol., 1994 for the detection of
enterovirus RNA by reverse transcription/
polymerase chain reaction. The upstream primer
(QN41, nt. 450-474) was not modified, whereas the
downstream primer (QN42, nt. 584-603) had to be
modified for NASBA by adding a 5overhang
containing an adapter segment (AGAAGG) and a T7
promotor sequence. NASBA products are mainly
negative stranded RNA, and to a minor extent
dsDNA (lt 10). These products can be detected by
ethidium bromide stained agarose gel
electrophoresis or after Northern-blot/slot-blot
hybridization with a biotinylated oligonucleotide
probe (EV3) followed by chemoluminescence. Both
primers and the probe were synthesized by
Eurogentec, Belgium, all other reaction
components were provided with the RNamplifire kit
(Qiagen, Hilden, Germany). Sensitivity of
enterovirus NASBA was determined with in vitro
transcribed enterovirus RNA (positive strand and
negative strand samples). As the KCl
concentration of the reaction mix is critical for
optimal sensitivity, this had to be adjusted
prior to determining the sensitivity.
Sensitivity was increased to about 1 to 10
molecules by detection of NASBA amplicons by
Northern-blot hybridization (figure 5). A
panel of enterovirus strains (prototype strains
and clincal isolates) was used to determine the
group specifity of enterovirus RNA detection by
NASBA (figure 6). Surprisingly, a coxsackievirus
B1 strain was not detected, perhaps due to a
mutation in the primer binding region. Moreover,
a coxsackievirus B5 strain was only amplified
faintly. Figure 6. Slot-blot
hybridization of NASBA products. Row A 1 polio1
(Droge), 2 polio2 (Schuster), 3 polio3, 4 CVA7, 5
CVA 10, 6 CVA14, 7 CVA11, 8 CVA21 row B 1 CVB1
(Schäfer), 2 CVB2 (Ohio 1), 3 CVB4 (Bensehofen),
4 CVB 5 (Kannegiesser), 5 CVB6, 6 echo1 (Farouk),
7 echo2 (Cornelis), 8 echo5 (Noyce) row C 1
echo6 (Henigst), 2 echo7 (Greon), 3 echo8
(Bryson), 4 echo 9 (Hill), 10 echo11 (Gregory),
11 echo12 (Travis), 12 echo24 (DeCamp) Applicati
on of enterovirus NASBA to 30 clinical samples
(26 cerebrospinal fluid and 4 endo-myocardial
biopsy samples) confirmed a similar sensitivity
and specifity as enterovirus RT-PCR. 4 samples
were positive by both methods. One sample was
positive in the enterovirus NASBA, but not in the
nested RT-PCR. This PCR amplicon was identified
as CVB6 by nucleic acid sequencing. Another two
samples were positive in the enterovirus NASBA,
but not in the nested RT-PCR. Allother samples
were negative by both methods. 4.
Conclusion NASBA is a simple, one step method for
the highly sensitive detection of enterovirus
RNA, and an excellent alternative method to
RT-PCR. 5. References Compton J (1991) Nature,
350, 91-92. Heim A et al. (1997) Antiviral Res.
137, 47-56. Kievits T et al. (1991) J. Virol.
Methods, 35, 273-286. Rotbart HA (1990) J. Clin.
Microbiol. 28, 438442.