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

(1)

DNA IQ

TM

Casework Pro Kit

for Maxwell

®

16 A Validation Study

(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

(3)

First Validation Study

Maxwell

®

_{16, Promega:}

DNA IQTM _{Casework Pro Kit}

(without new extraction buffer)

and

Blood LEV DNA Kit

BioRobot EZ1, Qiagen:

EZ1 DNA Investigator Kit

VS.

All tests were performed according to the manufacturer’s instructions with an incubation step of 70°(IQ Kit) / 56°C (Blood / Investigator Kit)

(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

(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

(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

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%)

(8)

Conclusions



Maxwell / Blood Kit:

all kinds of biological stains on swabs

and adhesive tapes



EZ1 / Investigator Kit:

stains on all other carrier materials

Since most stains were swabs and tapes –

(9)

Second validation study

Blood LEV DNA Kit

DNA IQ

TM

Casework

Pro Kit

(with the new extraction buffer)

VS.

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)

RT-PCR set up was performed by an automatic liquid handler elution volumes were adapted

(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:

(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: 11223 9908 / 10882 / 9968 10754

DNA amount: 52pg/µl 42pg/µl 25pg/µl

1 µl blood (1:10) on a swab, air-dried

Lot Number: 11223 9908 / 10882 / 9968 10754

(14)

Conclusions

2005:



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)

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%)

(17)

Silver bullet –

(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

(19)

Summary

Extraction strategy was not changed!



Maxwell / Blood Kit:

all kinds of biological stains on swabs

(with new extraction buffer)