baculoQUANTTM One-step titration kit
baculoQUANTTM, is a novel one-step virus titration kit which
can give an accurate result within 2 hours without the need for plaque assays
or immunoassays.
baculoQUANTTM is a fast and cost-effective method of recombinant
virus titration. It is based on a technique called quantitative PCR (QPCR). QPCR
utilizes a set of primers and a probe which are designed to specifically target
and amplify a small region of the baculovirus genome. The increase in amplification
of this small region is then correlated to virus genome copy number and can
be used to measure the number of recombinant virus particles present in an
infected insect cell culture. This novel titration method has been proven
to be a very accurate, reproducible and fast way to titrate recombinant baculoviruses1,
both for single-use titrations and for high throughput applications on automated
systems.
Compared with other methods of virus titration such as plaque
assay, end-point dilution and ELISA, which require anywhere from 24 to 120
hours to yield a result and involve specialised procedures,baculoQUANTTM can
provide a result within 2 hours, dramatically improving the productivity of
the protein production process.
- Quickest method of baculovirus titration ? results in
2 hours
- Cheapest method of virus titration - GBP 18 per
virus
- No need for plaque assays or immuno-assays
- Compatible with all AcMNPV-based
baculovirus expression systems
- Accurate titres
Patent pending - No. 0607656.7
The BaculoQuant One-Step Virus Titration Kit is the quickest method for the
titration of baculoviruses, being designed to give accurate titres within 2
- 6 hours (depending on number of viruses). Other methods typically require
24 to 48 hours (immunological assays) or 3 - 4 days (plaque assay or end-point
dilution assays). In addition, BaculoQuant titration costs only GBP 18 per
virus if the kit is used to its 26 virus titration capability.
Detection is
based on quantitative real-time PCR (QPCR) and Taqman fluorogenic probes,
specifically designed to the viral genome. Taqman technology takes advantage
of the 5 exonuclease activity of Taq polymerase to digest a probe, hybridised
between flanking PCR primers, and labelled with two fluorescent dyes (2).
The dyes undergo fluorescent resonance energy transfer (FRET) because of
their proximity to each other (3). Cleavage by Taq during primer elongation
interrupts the FRET and allows the reporter dye to fluoresce for every cycle
of the PCR. This increase in fluorescence can be analysed in real-time and
allows quantification during the exponential phase of the reaction. The system
has been calibrated against known viral titres, previously determined by
plaque assay.
Each kit contains enough forward and reverse primers, probe
and standard virus DNA to enable the user to perform up to 5 QPCR runs, with
a total maximum number of 26 viruses titrated.
NB.Virus must be no older than
3 months or an accurate titre cannot be achieved
Contents
- Forward and reverse primers
- Probe
- Control baculovirus DNA
- User manual
- Research-use licence (please contact OET for commercial use)
Storage
-20OC for all components
OET announce the release of baculoQuant? - a next-day baculovirus titration
service. The new service was officially launched at the Baculovirus Technology
Conference in Boston, U.S.A. on 25th September 2006.
‘A fast, accurate and efficient means of obtaining viral titres is
essential in the services we provide to pharmaceutical and biotechnology customers
in the area of insect cell expression and in our ongoing development of the
baculoworkstation. We have chosen to work with the quantitative Polymerase
Chain Reaction (QPCR) viral titration service from Oxford Expression Technologies
as this fulfils these criteria with knowledgeable, personable service.’
Dr. K S Richards, Senior Scientist, NextGen Sciences Ltd.
OET has developed
a rapid baculovirus titration method (1) using quantitative real-time PCR (QPCR)
and Taqman? fluorogenic probes, specifically designed to the viral genome.
Taqman technology takes advantage of the 5? exonuclease activity of Taq polymerase
to digest a probe, hybridised between flanking PCR primers, and labelled with
two fluorescent dyes (2). The dyes undergo fluorescent resonance energy transfer(FRET)
because of their proximity to each other (3). Cleavage by Taq during primer
elongation interrupts the FRET and allows the reporter dye to fluoresce for
every cycle of the PCR. This increase in fluorescence can be analysed in real-time
and allows quantification during the exponential phase of the reaction. OET
has utilised this technology to develop an accurate and rapid baculoviral titration
system. The system has been calibrated against known viral titres, previously
determined by plaque assay, and has been semi-automated using a Corbet Research
liquid handler to prepare the QPCR reactions ready for quantification on an
ABI 7500 Sequence Detection System.
Service includes:
- Viral DNA extraction
- QPCR preparation and quantification
- Viral titration determination (using
an algorithm previously determined by plaque assay)
- Email of titres (Excel
format)
Advantages of the
BaculoQuant? service
- Avoids unnecessary wastage of viral stocks
- Allows optimisation of viral
amplification and protein production
- Provides rapid and accurate titration
of multiple viruses in 96-well format
- Fast and simple to use
To order
- email/phone to check availability
- send 200ul FRESH baculovirus stock (must
be less than 3 months old or reading will be inaccurate)
- send/fax official
purchase order
- email/fax notice of despatch of virus
- NB stock received by 12.00 midday
will have results the following working day
Reference Papers
- Hitchman RB, Siaterli EA, Nixon CP, King LA. 2006. Quantitative real-time
PCR for rapid and accurate titration of recombinant baculovirus particles.
Biotechnology and Bioengineering.
- Holland PM, Abramson RD, Watson R, Gelfand
DH. 1991. Detection of specific polymerase chain reaction product by utilizing
the 5'?3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc.
Natl. Acad. Sci. U.S.A. 88: 7276?7280.
- Heid CA, Stevens J, Livak KJ, Williams
PM. 1996. Real time quantitative PCR. Genome Res. 6: 986?994.
For more information on commercial licences, please contact Helen Irving at
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