Download Performance of AccuPlex rEbola GP/NP Reference

31st Clinical Virology
Symposium
USE OF RECOMBINANT VIRUS TECHNOLOGY TO PRODUCE A
NON-INFECTIOUS, WHOLE PROCESS EBOLA CONTROL
Daytona Beach, FL
April 26-29, 2015
Catherine E. Huang, Rajeswari Vemula, Cristine Chisholm and Bharathi Anekella
SeraCare Life Sciences, Gaithersburg, MD
INTRODUCTION
RESULTS AND DISCUSSION
Stability Studies
Performance of AccuPlex rEbola GP/NP Reference Material
Cepheid Xpert Ebola Assay
BM-20 ˚C
1.00E+05
1.00E+04
BM 4 ˚C
Figure 6: Real Time
Stability of recombinant
Sindbis virus at RT, 4 ˚C
and
-20 ˚C.
1.00E+03
BM RT (25 ˚C)
1.00E+02
1.00E+01
0
2
4
6
8
10
40
40
40
35
35
CONCLUSIONS
35
NP Lot 1
GP Lot 1
GP Ct
25
NP Ct
30
GP Lot 2
25
20
Ct Value
Ct Value
30
30
NP Lot 2
25
NP Lot 3
GP Lot 3
20
20
15
15
15
2
3
4
5
6
7
8
9
10
Run2
Run3
Run4
Run5
Run6
Run1
Run7
Run2
Run3
Run4
Run5
Run6
Run7
Performance runs
Performance runs
PCR Assays: Ebola reference material was tested using a lab
developed TaqMan assay targeting either GP/NP or common viral
vector sequence. Testing was also performed using assay developed
by Cepheid and RUO assay from PrimerDesign
1.00E+06
Performance on NP TaqMan Assay
Performance on GP TaqMan Assay
Run1
Figure 4a: Testing of AccuPlex rEbola GP/NP Reference
Material on Cepheid Xpert Ebola Assay Ten replicates were
tested and the assay targets GP and NP regions. All ten replicates
were tested positive and the Ct values were between 28-30.
Figure 4b: Inter-assay performance of AccuPlex rEbola
GP/NP Reference Material Three validation lots were tested on
7 different runs in triplicate on both GP and NP real time TaqMan
assay.
Figure 4c: Testing of AccuPlex rEbola GP/NP
Reference Material on PrimerDesign assay Ten
replicates were tested and the assay targets VP24 (blue
lines) and NP (green lines). All ten replicates were
tested positive and the Ct values were between 24-25.
Positive control
Positive control
Construct
Construct
Stress stability was performed on AccuPlex rEbola
GP/NP Reference Material at 37 ˚C and multiple
freeze thaws to determine a preliminary expiration
date. Based on the data, the reference material
currently has an expiration of 24 months at 2-8 ˚C
(Figure 5a and 5b). Real time stability studies are
ongoing.
Stability Studies
Stability at 37 ˚C
Stability - Freeze/Thaw
1.00E+07
1.00E+06
1. C Xiong, R Levis, P Shen, S Schlesinger, CM Rice, and HV Huang. Science 3 March 1989: 243#
(4895), 1188-1191.
2. S Altman-Hamamdzic1, C Groseclose1, J-X Ma2, D Hamamdzic3, NS Vrindavanam1, LD Middaugh1,
NP Parratto1 and FR Sallee. Gene Therapy (1997) 4, 815–822.
3. Schlesinger S. 2000. Alphavirus expression vectors. Adv Virus Res.;55:565-77.
4. Sofer G. 2003. Virus Inactivation in the 1990s —and into the 21st Century Part 4, Culture Media,
Biotechnology Products, and Vaccines. BioPharm International. January: pp50-57.
1.00E+06
Copies/mL
Additional real time studies were performed on a
different recombinant virus at room temperature, 4 ˚C
and -20 ˚C (Figure 6). No downward trends were
detected across the nine (9) months of storage, even
for samples stored at ambient temperatures. This
demonstrates that the viral coat proteins form a stable
protective barrier that prevents nucleases in complex
clinical matrices such as plasma from degrading the
target RNA sequence.
1.00E+05
1.00E+05
1.00E+04
1.00E+04
NS
1D
3D
5D
11D
22D
 SeraCare developed stable, well-characterized Ebola positive
reference material using recombinant technology
 The recombinant genome size is comparable to many human
pathogenic RNA viruses so offer a similar secondary structural
complexity to what is found in the patient specimens.
 AccuPlex recombinant Ebola GP/NP reference material is replication
defective and non-infectious to humans to ensure added safety for
laboratory staff.
 AccuPlex rEbola GP/NP Reference Material is a multiplex control and
designed to contain one recombinant virus bearing a portion of the GP
gene and a second recombinant virus bearing a portion of the NP and
VP24 genes.
 The virus is enveloped and must go through an extraction process prior
to RNA detection. This makes the reference material a more efficient
whole process control than plasmid or other biosynthetic material
because it better mimics patient samples.
 This reference material provides means for monitoring all steps of PCR
assays and are useful for assay validation and as run controls.
 The SeraCare AccuPlex rEbola GP/NP Reference Material shows
consistent performance on all tested platforms.
 AccuPlex rEbola GP/NP Reference Material is formulated in defibrinated
plasma allowing at least 9 months of stability at room temperature. This
corresponds to more than 2 years of stability at 2-8 ºC when using
conservative models.
REFERENCES
1.00E+07
Copies/mL
Stability Studies: Performance studies were conducted on 3 Ebola
validation lots over multiple days using TaqMan assay targeted to GP
and NP region. Stress stability studies were conducted on one lot of
Ebola product by subjecting it to freeze thaw cycles and holding the
samples at 37 ˚C for extended periods to simulate adverse shipment and
storage conditions. Real Time Stability studies on Ebola reference
material is ongoing. Here we present real time stability data for another
recombinant Sindbis virus that has been formulated in a similar matrix
and stored at ambient room temperature (approximately 25 ˚C). Vials
were tested periodically over 9 months using a laboratory developed
TaqMan real time PCR test.
1.00E+07
Storage Duration In Months
Number of datapoints
Design and Development of Recombinant Ebola Virus: synthetic
constructs were designed targeting glycoprotein gene (GP),
nucleoprotein gene (NP) and VP24 of Ebola Zaire isolate H. sapienswt/GIN/2014/Gueckedou-C05;
GenBank
accession
number
KJ660348.2. The capped RNA are introduced into baby hamster
kidney cells. The recombinant virus was cultured and purified from the
cell debris. The purified virus was heat inactivated as a further safety
precaution4. After titering the viruses using a laboratory developed
TaqMan PCR assay, the viruses were combined and diluted into
defibrinated human plasma and 0.09% sodium azide as a
preservative.
Viral Load (copies/mL)
SeraCare developed multiplex recombinant reference material, AccuPlex rEbola GP/NP Reference Material, that targets the GP/NP regions. The
reference material is formulated in defibrinated plasma with a fill volume of 250µL and can be stored at 2-8 ˚C. The Limit of Detection (LOD) was
estimated for 3 PCR assays, Cepheid Xpert® Ebola assay, PrimerDesign assay and in-house developed TaqMan assay. Based on the LOD a low
positive process control was manufactured (Figure 4). Performance studies were conducted over multiple days using multiple operators. The %CV for
AccuPlex rEbola GP/NP Reference Material on all 3 assays was <2%
1
MATERIALS AND METHODS
Real Time Stability
1.00E+08
Ct Value
Outbreaks of new viral communicable disease and emergence of new
viral strains, such as the recent outbreak of Ebola 2014 strain in
Guinea, can represent public health emergencies. This outbreak
mobilized the IVD laboratories to develop rapid and sensitive PCR
assays to help diagnose and treat those infected while helping contain
the rapid spread. As the test developers design, manufacture and
validate diagnostic assays to prepare for these threats, positive
reference materials are needed.
SeraCare has developed the
AccuPlex™ rEbola GP/NP Reference Material using recombinant virus
technology. The use of recombinant virus, containing target sequences
from Ebola virus has many advantages as a NAT quality control
material. First, it mimics clinical samples because it undergoes the
entire extraction procedure. Second, the product is non-infectious and
ensures biological safety for lab personnel. Recombinant virus vector
has deletion of about 1/3 of genome, which makes it incompetent to
replicate in human or animal host. SeraCare’s AccuPlex rEbola GP/NP
Reference Material was made by replacing structural proteins required
for replication with selected Ebola target sequences.
Finally,
recombinant viruses have an extended stability at 2-8 ˚C; control
materials do not require freezer storage.
RESULTS AND DISCUSSION
NS
1 F/T
2 F/T
3 F/T
ACKNOWLEDGMENTS
Figure 5a: Stability of AccuPlex rEbola GP/NP
Reference Material across multiple freeze thaw cycles
Figure 5b: Stability of AccuPlex
rEbola GP/NP Reference at 37 ˚C
The authors are most grateful to Dr. Powell at PrimerDesign for testing the samples and to
Medha Kulkarni at Cepheid for testing the samples on Xpert Ebola Assay.