VERSANT HIV-1 RNA 1.0 (kPCR) - genafor.orgCOBAS AmpliPrep/COBAS TaqMan Versus VERSANT HIV RNA kPCR...

Copyright © Siemens Europe 2011. All rights reserved.

Siemens Healthcare Diagnostics / Molecular

VERSANT HIV-1 RNA 1.0 (kPCR)

May 2011

Answers for life.

Protection notice / © 2010 Siemens Healthcare Diagnostics Inc.Siemens Healthcare Diagnostics / EMEA / Molecular

Anne Beyou-Zuliani, PhD

Scientific marketing manager

Agenda

Technology

Standardization

Clinical performance

Report

Assay design



VERSANT™ kPCR Molecular System

Sample Preparation (SP)

Amplification Detection (AD)



Siemens’s Real Time PCR: kPCR

Kinetic PCR detection



Black Hole Quencher

No native fluorescence, resulting in lower background fluorescence

Increases signal-to-noise ratios, providing higher sensitivity

Maximizes spectral overlap, increasing efficiency of quenching

kPCR technology: TaqMan Probe



VERSANT kPCR Contamination Control and Robustness

Uracil is incorporated during PCR

UNG cleaves uracil bases from the uracil-containing DNA from PCR products

UNG does not react with RNA or thymine-containing target DNA.

UNG is inactivated by heat denaturation prior to PCR.

Uracil DNA Glycosylate (UNG)



Agenda

Technology

Standardization


Report

Assay design



VERSANT HIV-1 RNA 1.0 Assay (kPCR) Design

The VERSANT HIV-1 RNA 1.0 Assay (kPCR) targets a highly conserved

portion of the pol integrase gene.



VERSANT HIV-1 RNA 1.0 Assay (kPCR) Design

Lower annealing temperature tolerates mismatches to allow detection of all Group M and O subtypes

Dual primer design to increase mismatches tolerance

probe


VERSANT HIV-1 RNA 1.0 Assay (kPCR)Feature summary

Wide Dynamic Range: 37 to 11,000,000 copies/mL

0.5 mL sample input volume

Non-competitive Internal Control

Calibrators A & B in duplicate

High and Low Positive Controls

Negative control

Up to 89 samples per run in less than 6 hours



Agenda

Technology

Standardization


Report

Assay design



VERSANT® HIV-1 RNA 1.0 Assay (kPCR) Standardization system

The goal of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) standardization system is to ensure accurate and reproducible quantitation of HIV-1 RNA in each run over time and across reagent lots.



Reference Standard3.6 Kb RNA transcript of

HIV-1SF2 pol gene

Reference Standard Used for

Quantitation of

QuantitationPhosphate ContentTraceable to NIST

Quality AssessmentOD260 - Digested RNA

Hyperchromicity

Reference HIV Standard: Assessment and Role in Product Standardization

Stability TestingMonitor Every 6 mo. with Quantitative Northern Blot

Qualification and Quantitationof Reference Standard

Role in Standardization

Virus Stock

Calibrators

Positive Controls

QC Panels

Quality AssessmentOD260 - Intact RNA

Virus stock used to produce Calibrators, Positive Controls, and QC Panels.



Agenda

Technology

Standardization


Report

Assay design



VERSANT HIV-1 RNA 1.0 Assay (kPCR) Method comparison – Roche TaqMan

TaqMan HIV 1.0N= 176

R2 0.89

TaqMan HIV 2.0N= 136

R2 0.94

Wagner et al. 2008 ISHEID Troppan et al. 2009 J. Clin. Virology



N= 109R2 0.94

y = 1.0009x - 0.2441R2 = 0.9368

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00

Abbott RealTime HIV-1 (Log Copies/mL)

VER

SAN

T H

IV-1

RN

A k

PCR

(Log

Cop

eis/

mL)

N= 182R2 0.96

VERSANT HIV-1 RNA 1.0 Assay (kPCR) Method comparison – Abbott Real Time

Log

Qua

nt (k

PCR

)1

2

3

4

5

6

7

Log Quant (Abbott)1 2 3 4 5 6 7

Gorrin et al. 2008 AMPInternal data - Random Samples from BioCollections

Y=099x-018



J. Ruelle et al. 2009 Journal of Clinical Virology

VERSANT HIV-1 RNA 1.0 Assay (kPCR) Reproducibility

Intra run and inter run coefficient variation shows excellent assay reproducibility



Geographic distribution of HIV-1 groups, subtypes and CRFs

CRF_02AG CRF_02AG

A, C, D, F, G, H, J, K, O, N

A, C, D, F, G, H, J, K, O, N BB

BBC, A,

BC, A,

B

AA

BB

B,F1, A, C, G,

B,F1, A, C, G,

CRF_02AGCRF_07BC

CRF_08BC

CRF_03AB

A, G,

B,C, D, Group 0

B,C, D, Group 0

CRF_12BF CRF_12BF

B, F1

A, D, C

C, B

MOST CRF

CRF_01AE CRF_01AE CRF_06cpx

CRF_14BG



VERSANT HIV-1 RNA versus CAP/CTM for Non-B Viral Load Comparison (Log Copies/mL)

COBAS AmpliPrep/COBAS TaqMan Versus VERSANT HIV RNA kPCR Subtype Data Log Copies/mL Comparison

1,5

2,5

3,5

4,5

5,5

6,5

7,5

A1

A1

A1 C C D F1 G G G G H AE

AE

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG

AG DF

06-C

PX

09-C

PX

11-C

PX

11-C

PX

11-C

PX

Rec

omb.

Rec

omb.

COBAS AmpliPrep HIV-1 kPCR VERSANT HIV-1 RNA 1.0 kPCR

Internal data



Method comparison Abbott RealTime™ HIV-1 Assay (ART)

Subtype Analysis

Presented at the Presented at the Association for Molecular Pathology (AMP) 14th Annual Meeting, October 29 –November 2, 2008, Grapevine, Texas USA



Method Comparison with Abbott RealTime HIV-1 n = 16 Random Samples from Beijing

Method Comparison HIV-1 RNA Viral Load Assay Quantitations:VERSANT kPCR, VERSANT bDNA and Abbott RealTime

2,0

2,5

3,0

3,5

4,0

4,5

5,0

5,5

6,0

B; CRF0

3CRF08

; CNot d

eterm

ined

B; CRF1

2B; C

RF12

CRF01; C

RF15

B;CRF1

2CRF01

;CRF15

CRF01B; C

RF07

B; CRF1

2B; C

RF07

B; CRF0

7B; C

RF07

B; CRF0

7B; C

RF07

HIV

-1 R

NA

Log

Cop

ies/

mL

SIEMENS VERSANT® HIV-1 RNA 3.0 Assay (bDNA) SIEMENS VERSANT® HIV-1 RNA 1.0 Assay (kPCR)Abbott HIV-1 RealTime PCR



Clinical performance - Summary

The VERSANT HIV-1 RNA 1.0 Assay (kPCR) showed good correlation with the Roche CAP/CTM HIV-1 and Abbott RealTime HIV-1

The VERSANT HIV-1 RNA 1.0 Assay (kPCR) is able to detect and quantify all HIV-1 subtypes, circulating recombinant forms (CRF) and complex recombinant forms (CPX) tested in those studies.

The VERSANT HIV-1 RNA 1.0 Assay (kPCR) shows excellent reproducibility intra and inter run



Agenda

Principle of Assay

Standardization


Report

Assay design



VERSANT HIV-1 RNA 1.0 Assay (kPCR)

TraceabilityLot

Traceabilitysample/well

ValidationInternal control

TraceabilityOperator

Validation

3 controls



VERSANT HIV-1 RNA 1.0 Assay (kPCR)

Choice of reporting unit

Value below LOQ



VERSANT kPCR HIV-1 (kPCR) Summary

High throughput with flexible run size on open system

Fast turn around time

Optimized assay design

High sensitivity with 0.5 ml input volume

Chemical contamination prevention (UNG)

State of the art standardization system

Excellent precision

Good correlation with other methods

Comprehensive & flexible report



Thank you for your attention!

Protection notice / © 2010 Siemens Healthcare Diagnostics Inc.Siemens Healthcare Diagnostics / Region / Department

VERSANT HIV-1 RNA 1.0 (kPCR) - genafor.orgCOBAS AmpliPrep/COBAS TaqMan Versus VERSANT HIV RNA kPCR...

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