10th EPFA/NIBSC Workshop on Nucleic Acid Amplification Testing and the Detection of Pathogens in Blood and SoGAT Meeting
Langen, Germany, July 3, 2003 “Use of a Quantitative NAT Assay to Correlate West Nile Virus Titration Bioassays (pfu/mL) with Genomic Copy Numbers (geq/mL)”
Blood Testing R&D/Chiron Corporation
West Nile Virus Transcripts
West Nile Virus Genome
(AF196835; Lanciotti et al.; Science 286, 2333-2337, 1999)
C
97-465
Pr
466-741
M
742-966
E
967-2469
NS1
2470-3525
NS2a
3526-4218
NS2B
4219-4611
NS3
4612-6468
NS4a
6469-6915
NS4b
6916-7680
NS5
7681-10395
Transcripts
0.9 kb
1.5 kb
1 kb
Alternative NAT West Nile Virus Assay
(Qualitative and Quantitative)
• A sensitive qualitative WNV Taqman assay was developed at Chiron to be implemented as a supplemental test to confirm Procleix WNV positive specimens
•
A quantitative WNV Taqman assay was also configured at Chiron to support Procleix WNVTM assay development, and to qualify Calibrators and Proficiency Panels
Qualitative WNV Taqman Assay
Performance Criteria
(a) RNA isolation • Establish efficient target isolation and use of sufficiently large sample volumes
•
Use single tube target isolation with minimal handling
(b) Amplification efficiency • Amplify and compare capsid, NS1/NS2 and 3’UTR regions for efficiency (c) Internal control (IC) • Prepare transcripts modified for probe binding sequence to determine false negatives
Amplification and Detection Limits of WNV Capsid Internal Control Transcript
Comparative amplification efficiencies of capsid, NS1/NS2 and 3’UTR indicated that capsid is the most robust
Amplification efficiency of Capsid, NS1 and 3’UTR of WNV.
Of the three regions amplification of Capsid was the most robust.
Region Capsid
NS1/NS2
3'UTR
Ct 32.43 32.63 32.61 33.90 32.93 33.10 32.97 33.76 33.56
Ct Average= Std Dev= %CV= Average= Std Dev= %CV= Average= Std Dev= %CV=
32.56 0.11 0.34 33.31 0.52 1.56 33.43 0.41 1.22
Target Isolation and Detection
Key Features of the Protocol
• Use of 0.5 mL of sample and magnetic bead based capture of the target with complementary oligonucleotides Addition of an Internal Control transcript that is captured and amplified by the same primers but detected by an altered probe
The captured target and IC are amplified and detected simultaneously by Taqman assay Lineage 1 and 2 are detected with two separate probes. A second discriminatory assay can be performed to determine lineage
•
• •
Sensitivity of Alternative NAT WNV Assay
Detection of BBI WNV RNA Qualification Panel-QWN702
Member ID Target WNV 1 2 3 4 5 6 7 8 9 10 11 12 13 Cps/ml 100 0 10000 30 1000 300 100 0 30 1000 100 10000 0 43.1 50 36.4 43.6 39 42.3 43 50 44.2 38.1 43 35.2 50 Target Ct Target with IC Target Ct 44.9 50 35.4 50 39 47.5 42.8 50 47.3 38.2 41.1 34.4 50 IC Ct 44.5 42.2 49 41.9 43.3 41.8 41.1 43 40.3 41.3 43.7 45.9 41.6
100% positivity at 30 cps/ml without IC 66% positivity at 30 cps/ml with IC
Quantitative WNV Taqman Assay
Standard Panel
•
A seven member panel prepared by serial dilution of a viral suspension of West Nile virus propagated in Vero cells
The copy number in each of these panel members has been established using the BBI WNV RNA qualification panel The panel members are assayed as triplicates to obtain a standard graph and ascribe copy numbers to unknowns which are also tested in triplicates
•
•
Alternative NAT WNV Quantitative Assay
BBI panel members 30-10,000 copies/mL tested in triplicates to generate a standard graph
Quantitation of Vero Cell Cultured WNV
(Live and Inactivated*)
Live WNV *Heat Inactivated WNV Cps/ml Not Tested 1.6 - 3.0 x 1010 1.59 - 2.3 x 1010 1.88 - 2.3 x 1010 2.6 - 2.8 x 1010 4.1 x 1010
# # Culture Dilution Cps/dilution Cps/ml Cps/dilution Direct Not Tested Not Tested Not Tested 1 x 10-4 0.52 - 1.1 x 107 0.52 - 1.1 x 1011 1.6 - 3.0 x 106 1 x 10-5 0.66 - 1.1 x 106 0.66 - 1.1 x 1011 1.59 - 2.3 x 105 1 x 10-6 0.70 - 1.0 x 105 0.79 - 1.0 x 1011 1.88 - 2.1 x 104 1 x 10-7 0.72 - 0.74 x 104 0.72 - 0.74 x 1011 2.6 - 2.8 x 103 1 x 10-8 0.87 - 1.0 x 103 0.87 - 1.0 x 1011 4.1 x 102
#
* Heat inactivation was performed at 62.5 C for 65 minutes Experimentally determined number for samples tested in triplicates
The range represents the results from two versions of the Standard graph obtained with BBI panel members.
Based on pfu determination by AppTec Laboratory Services, which is 7.07 x 107 pfu/mL,
1 pfu = ~1000 copies
Alternative NAT WNV Taqman Assay
Summary of Performance
(a) Qualitative assay • Highly sensitive assay capable of detecting 30 cps/mL • Results from 96 samples can be obtained in four hours • The assay incorporates an IC to determine false negatives • A discriminatory assay distinguishes between lineages 1 and 2
(b) Quantitative assay • A standard panel of seven members from Vero cell suspensions has been established to assign numbers in the range of 100 - 1011 copies