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Species-specific
Virus Detection by qPCR
The selection of viruses required to be tested
depends upon the origin and history of the production cell line, virus
vaccine seed and raw material used in the manufacturing process. For
example, PCR testing for the presence of human viruses is required when
cells of human/primate origin or products obtained from human blood or
tissues are involved. Examples where human virus PCR testing is advised,
include human hybridomas, stem cells, and other cell lines such as Vero,
HEK 293 and PER.C6 used in the production of Adenovirus vectors and
vaccines. In cases where viruses cannot be readily grown in culture, PCR
is currently the most effective tool to assess for contamination with
such viruses. The human viruses that should be screened for are those
associated with severe or oncogenic diseases and particularly those that
might establish latent or abortive replication in cells. Manufacturers of
certain influenza vaccines derived from cell culture are encouraged by
the EU regulatory authorities to perform testing for specific respiratory
viruses by PCR assays. Certain exogenous avian virus PCR assays are
recommended if the influenza virus vaccine seed has historical culture
within eggs, or a cell substrate is of avian origin. Due to the
specificity of PCR, it is usual to perform multiple PCR assays in order
to detect the full range of viruses of concern. The recommendation is
that PCR assays when possible should detect several agents using
degenerate or consensus primers provided the sensitivity of these assays
is sufficient to assure product safety.
Vitrology’s
real time qPCR assays contain a forward/reverse primer set and
fluorogenic-probe to detect the presence of viral pathogen target
sequences within test samples. Each virus testing qPCR package is
customised for the client’s cell line to ensure they meet
regulatory requirements. Each viral qPCR assay utilises TaqMan based
probe technology which forms part of a customised high density qPCR array
system fully compliant with the controls recommended in the regulatory
guidelines.
The
regulatory acceptance of highly sensitive virus detection methods for the
testing of biopharmaceutical products has depended on PCR assays that are
validated to ICH guidelines. All Vitrology’s assay validation, test
design and assay performance control systems are carried out essentially
as described in the regulatory guidelines, including US FDA, ICH Q2,
European Phamacopoeia and as initially published by Lovatt et al., 2002.
All assay validation parameter data including detection limit (DL),
specificity, quantitative range, 95% cut-off and quantitation limit (QL)
are described to our clients in our quality assurance approved validation
reports.
The
following control system is performed in compliance to European
Pharmacopeia 2.6.21:
- Contamination control: Separate clean rooms for reagents, test
samples, and positive controls. Each room and assay step contains a
multi-step contamination control system.
- Nucleic Acid Extraction
Interference:
Prior to extraction each test sample is diluted and spiked with
“extraction control DNA or RNA” that mimics low level
virus genome nucleic acid.
- Nucleic Acid
Amplification Interference:
Prior to testing the sample and dilutions for the presence of
“extraction control DNA or RNA” by qPCR each test sample
is spiked with a secondary “amplification internal control
nucleic acid ”.
- Optimisation of test
sample volume:
The results from the low level internal control qPCR assays
determine the optimal sample concentration for testing with the
viral target qPCR assays
- Positive control: Test sample is spiked with positive control
viral nucleic acid at the detection limit to control for the
presence of assay specific inhibitors.
We
have developed and validated in excess of 100 species specific qPCR
assays, covering viral pathogens from Human, Murine, Simian, Porcine,
Insect, Duck, Avian, Bovine species. Below is a small selection from our
list of Human virus qPCR assays.
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Study Plan Number
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Human Virus Assays
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Study Plan Number
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Simian Virus Assays
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SP-M.8101
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B19 parvovirus
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SP-M.8119
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Ebola virus*
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SP-M.8102
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CMV
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SP-M.8120
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Lymphotropic papovavirus
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SP-M.8103
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EBV
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SP-M.8121
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Monkeypox
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SP-M.8104
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HAV*
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SP-M.8122
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Simian adenoviruses
|
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SP-M.8105
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HBV
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SP-M.8123
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Simian CMV
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SP-M.8106
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HCV *
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SP-M.8124
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Simian Foamy Virus
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SP-M.8107
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HHV-6
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SP-M.8125
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Simian herpes viruses
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SP-M.8108
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HHV-7
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SP-M.8126
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SIV
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SP-M.8109
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HHV-8
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SP-M.8127
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Simian Retroviruses
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SP-M.8110
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HIV 1 & 2
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SP-M.8128
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STLV I & II
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SP-M.8111
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HSV 1 & 2
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SP-M.8128
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Simian virus 40
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SP-M.8112
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HTLV I & II
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SP-M.8135
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Porcine Bovine Circovirus
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SP-M.8113
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SV40
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|
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SP-M.8114
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AAV
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|
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SP-M.8115
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Enteroviruses *
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|
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SP-M.8116
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Papillomaviruses
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|
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SP-M.8117
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Polyoma viruses
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SP-M.8117
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Adenoviruses
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*RNA
viruses
Click here for literature references.
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