DNA IQ TM Casework Pro Kit for Maxwell 16 A Validation Study

Transcription

1 DNA IQ TM Casework Pro Kit for Maxwell 16 A Validation Study K. Anslinger, B. Bayer

2 The search for a alternative DNA extraction and purification method In comparison with EZ1 / M48 (Qiagen): Sufficient DNA quantity and quality Usable for all kinds of different forensic biological stains relevant to various carrier materials and cheaper!

3 First Validation Study Maxwell 16, Promega: BioRobot EZ1, Qiagen: DNA IQ TM Casework Pro Kit (without new extraction buffer) and VS. EZ1 DNA Investigator Kit Blood LEV DNA Kit All tests were performed according to the manufacturer s instructions with an incubation step of 70 (IQ Kit) / 56 C (Blood / Investigator Kit) for > 2 hour and the corresponding reagents.

4 Sensitivity Study blood, diluted 1:10, 1 µl on a swab, air-dried 5 samples / series all tests were carried out in parallel Quantification with RT-PCR (Quantifiler Human DNA Quantification Kit, AB) each sample was quantified twice RT-PCR set up was performed by an automatic liquid handler all samples were quantified within one RT-PCR run elution volumes were adapted for each series, the average amount of yielded DNA was calculated

5 Sensitivity Study Results General remarks: yielded values only comparable within one series results only give tendencies but these tendencies could be confirmed by repeat testing Average DNA Amount: Blood Kit > Investigator > IQ Kit 100 % 72% 25%

6 Inhibition study 1 µl diluted blood (1:10) on different papers and adhesive tape - air-dried- Swabs are the most common stains in our lab stamps, paper from cigarette butts, paper towels and newspaper adhesive tape (10cm long/2cm broad), swabbed with extraction buffer Once more: all tests were carried out in parallel and within one RT-PCR run Quantifiler Human DNA Quantification Kit, AB each sample was quantified twice RT-PCR set up was done by a automatic liquid handler elution volumes were adapted

7 Results: Inhibition study Number of successful investigated stains Maxwell EZ1 Blood Kit IQ Pro Kit Investigator Cellulose 5 / 5* 0 / 5 5 / 5* Newspaper 1 / 5 1 / 5 5 / 5 Stamps 1 / 6 not tested 6 / 6 Cig.-butts 2 / 4 not tested 4 / 4 Adhesive tape 5 / 5** not tested 5 / 5** Average amount of yielded DNA: * Blood Kit > Investigator (~ 60%) ** Blood Kit Investigator

8 Conclusions Maxwell / Blood Kit: EZ1 / Investigator Kit: all kinds of biological stains on swabs and adhesive tapes stains on all other carrier materials Since most stains were swabs and tapes Costs could be reduced!

9 Second validation study Blood LEV DNA Kit VS. DNA IQ TM Casework Pro Kit (with the new extraction buffer) All tests were performed according to the manufacturer's instructions with an incubation step of 70 (IQ Kit) / 56 C (Blood Kit) for > 2 hour. Investigator Kit/EZ1 tested in parallel as positive control for inhibition study.

10 Sensitivity study 1 µl diluted blood (1:10 and 1:100) 6 samples for each dilution step Two different dilution series: blood directly added to buffer air dried blood stains (1:10), on swabs Once more: all tests were carried out in parallel and within one RT-PCR run (Quantifiler Human DNA Quantification Kit, AB) each sample was quantified twice RT-PCR set up was performed by an automatic liquid handler elution volumes were adapted Whole Test was repeated 2 weeks later

11 Results: Sensitivity study 1:10 Blood Kit < IQ Pro Kit < EZ1 1:100 Blood Kit < IQ Pro Kit < EZ1 Swabs Blood Kit < IQ Pro Kit = EZ1 Test repeated 2 weeks later showed comparable results!

12 Self designed lot test with dried blood stains on swabs The 19 different lots of cartridges tested in this study revealed remarkable differences in the amount of yielded DNA and obtained profiles. 9 lots: no signal at all for all nine tested STR loci 6 lots: profiles with more less allelic or locus drop outs 4 lots: full profiles all alleles of the 9 loci were detected Conclusion: fluctuating quality of the chemical reagents used..

13 Lot test (Blood Kit) 1µl blood 1:10 and 1µl blood 1:10 on a swab, air-dried 1 µl blood (1:10) Lot Number: / / DNA amount: 52pg/µl 42pg/µl 25pg/µl 1 µl blood (1:10) on a swab, air-dried Lot Number: / / DNA amount: 30pg/µl 22-24pg/µl 14pg/µl

14 2005: Conclusions each new lot was checked with a self designed test system before its use in routine casework Qiagen improved the quality checks of the different reagents comparable quality, only small differences were observed 2012: Possible differences between the lots in use complicate a comparison between the different kits and methods!

15 Inhibition study 1 µl diluted blood (1:10) and 1cm 2 paper different papers: stamps, paper from cigarette butts, cellulose 5 samples for each kind of paper Once more: all tests were carried out in parallel and within one RT-PCR run (Quantifiler Human DNA Quantification Kit, AB) each sample was quantified twice RT-PCR set up was performed by an automatic liquid handler elution volumes were adapted

16 Results: Inhibition study Number of successful investigated stains Maxwell EZ1 Blood Kit IQ Pro Kit Investigator Cellulose 5 / 5 5 / 5 5 / 5 Stamps 0 / 5 5 / 5 5 / 5 Cig.-butts 0 / 5 5 / 5 5 / 5 Average amount of yielded DNA: Cellulose: Blood Kit (84%) < Investigator (90%) < IQ (100%) Cig.-butts/stamps: Blood Kit (0%) < IQ (41% / 60%) < Investigator(100%)

17 Silver bullet New Extraction buffer???

18 New extraction buffer for both kits 1µl blood 1:10 on a swab, air-dried Maxwell Blood Kit Blood Kit IQ Pro Kit corresponding buffer new extraction buffer new extraction buffer pg/µl pg/µl pg/µl

19 Summary Extraction strategy was not changed! Maxwell / Blood Kit: (with new extraction buffer) all kinds of biological stains on swabs and adhesive tapes EZ1 / Investigator Kit: stains on all other carrier materials