Non-competitive Internal Control Concept for PCR-based Qualitative Assays

Transcription

1 Christian O. Simon Roche Molecular Diagnostics Rotkreuz, CH Non-competitive Internal Control Concept for PCR-based Qualitative Assays 23 rd SoGAT Meeting, 16 th 17 th April, 2012, Vilnius, Lithuania

2 Design of Controls Performance Data for prototype MPX and WNV tests Conclusions

3 Controls and their functions External Controls Positive Control + Negative Control Test specific reagents System Functions Contamination Internal Controls Blood Screening Viral Load Testing Internal Control (IC) Internal Quantitation Standard (QS) Process Control Sample effects Quantitation Process Control Sample effects

4 Evolution in Blood Screening NAT testing Single targets HIV-1, HCV, HBV Internal control: competitive Multiple targets HIV / HBV / HCV + Discriminatory tests Internal control Semi-competitive Multiple targets HIV + HBV + HCV using multiple dyes Internal control Non-competitive Universal internal control for different tests

5 For Discussion and Demonstration: Is it possible to design a non-competitive internal control with an unrelated sequence for a common multiplex assay? and expand to other assays?

6 IC Design for a prototype Multiplex (MPX) Test for HIV, HCV, HBV Design Rules for IC: 1. Unique, non-competitive sequence 2. Amplicon sizes comparable to targets 3. GC content comparable to targets 4. IC and targets have to fit into one PCR Profile (DNA and RNA targets) relative amplicon sizes HIV amplicon Detection via Channel 1 HBV amplicon Detection via channel 2 HCV amplicon Detection via channel 3 IC amplicon Detection via channel 4 Generation of a non-competitive IC molecule with a comparable amplicon size and GC content to function with one PCR Profile

7 Design of Controls Performance Data for Prototype MPX and WNV Tests Conclusions

8 Limit of Detection (LOD) Target Prototype MPX test LOD (PROBIT) Current MPX v1 test HBV 1.7 IU/mL 3.8 IU/mL HCV 6.5 IU/mL 11 IU/mL HIV IU/mL 49 IU/mL LOD performance of prototype MPX Test not adversely affected compared to current assay (MPX v1)

9 Specificity Number of plasma samples tested Target # Reactive Samples Clinical Specificity [%] HIV % 3006 HBV 1 out of % HCV 0 100% All 1 out of % Non-competitive IC does not affect Specificity of prototype MPX Test

10 Monitoring for Inhibition Sample / Hit Rate HIV Channel 1 HBV Channel 2 HCV Channel 3 IC Channel 4 without Target Donor 1: w/o hdna 0/3 0/3 0/3 3/3 Donor 2: w/o hdna 0/3 0/3 0/3 3/3 Donor 1: 4 mg/l hdna 0/2 0/2 0/2 2/3 Donor 2: 4 mg/l hdna 0/3 0/3 0/3 3/3 with HIV-1, HBV, HCV Target ~3x LOD Donor 1: w/o hdna 3/3 3/3 3/3 3/3 Donor 2: w/o hdna 3/3 3/3 3/3 3/3 Donor 1: 4 mg/l hdna 3/3 3/3 3/3 2/3 Donor 2: 4 mg/l hdna 3/3 3/3 3/3 3/3 human DNA (hdna) used as an example of a potential endogenous interference Non-competitive IC monitors for inhibition IC dropping out but target still present

11 Advantages of a non-competitive IC concept Independent target primer / probe design High flexibility during assay development Optimization of specific target primer / probe does not impact IC Fast menu expansion For example, prototype WNV test using same non-competitive IC

12 Advantages: Menu expansion using same IC molecule Performance data for prototype WNV Test: LOD Target Prototype WNV test LOD (PROBIT) Current WNV test WNV 10.9 copies/ml 40.3 copies/ml LOD performance of the prototype WNV test not adversely affected compared to current WNV test

13 Advantages: Menu expansion using same IC molecule Performance data for prototype WNV Test: Specificity Number of plasma samples tested Target # Reactive Samples Clinical Specificity [%] 1500 WNV 0 100% Non-competitive IC does not affect Specificity

14 Advantages: Menu expansion using same IC molecule Sample / Hit Rate WNV Channel 2 IC Channel 4 Performance data for prototype WNV Test: Monitoring for Inhibition without Target Donor 19: w/o hdna 0/3 3/3 Donor 20: w/o hdna 0/3 3/3 Donor 19: 4 mg/l hdna 0/3 3/3 Donor 20: 4 mg/l hdna 0/3 3/3 with WNV ~3x LOD Donor 19: w/o hdna 3/3 3/3 Donor 20: w/o hdna 3/3 3/3 Donor 19: 4 mg/l hdna 3/3 2/3 Donor 20: 4 mg/l hdna 3/3 3/3 human DNA (hdna) used as an example of a potential endogenous interference Non-competitive IC monitors for inhibition IC dropping out but target still present

15 Design of Controls Performance Data for prototype MPX and WNV tests Conclusions

16 Summary and Conclusions Multiplexed assays improve efficiency and minimize sample volume requirements Non-competitive IC is the choice for multiplexed assays Performance of Non-competitive IC was shown with two prototype blood screening assays Non-competitive IC will help in faster assay development and menu expansion to develop a new test to detect emerging viruses

17 Outlook Non-competitive concept also works for Quantitative assays Roche will show similar performance data for viral load monitoring assays More data will be presented at IPFA/PEI 19th International Workshop on Surveillance and Screening of Blood Borne Pathogens, Budapest, Hungary (23-24 May, 2012)

18 Thank you for your attention. Roche Diagnostics Ltd Rotkreuz Switzerland COBAS and LIFE NEEDS ANSWERS are trademarks of Roche This presentation is our intellectual property. Without our written consent, it shall neither be copied in any manner, nor used for manufacturing, nor communicated to third parties.