Poster FRCBS

FRCBS: WP2: the development of protocols for HLA typing and multiplex amplification protocols,
WP7: the validation of the pre-production prototype and a leader of the validation workpackage 7
Noora Alakulppi, Marjatta Hirvonen, Jarno Alen, Kari Aranko, Marja-Kaisa Auvinen, Pia Bruce, Johanna Castrén, Marianne Elmgren,
Katri Haimila, Elina Honkavaara, Minna Huovinen, Johanna Isopahkala, Taina Jaatinen, Kaija Javela, Satu Koskela, Sinikka Koskinen, Päivi
Kuronen, Erja Laatio, Eila Laitinen, Sisko Lehmonen, Enni Lindeberg, Sinikka Lopperi, Eeva Mainio, Sirkka Mannelin, Mikko Manninen,
Pekka Palomäki, Juha Peräsaari, Mika Räsänen, Inna Sareneva, Arja Siekkinen, Kaija Sopenlehto, Pirjo Vartiainen, Jukka Partanen
INTRODUCTION
The first aims of FRCBS were to develop and validated protocols for HLA typing
and multiplex amplification protocols to CD associating HLA-DQ2 and -DQ8
genotypes (D2.1, D2.3, D7.2). DQ2 is found in two haplotypes. DQ2 cis
consists of DQA1*0501-DQB1*0201. DQ2 trans consists of DQA1*0505DQB1*0301 and DQA1*0201-DQB1*0202. DQ8 consists of DQA1*0301DQB1*0302 (Figure 1).
Participants FRCBS and TATAA have developed one line of approach for CD
associated HLA typing which is based on sequence-specific oligonucleotides
(SSO)-PCR. In SSO-PCR, specific primers amplify the gene segment in
question, onto which gene probes specific for each allele or group will
hybridize.
FRCBS and TATAA developed SSO-PCR based on primers and probes for HLADQA1/DQB1 allele detection from the handbook of the 12th International
Histocompatibility Working Group (IHWG). However, these primers were
FIG 1 CD associating HLA haplotypes. Reference In Finnish: Celiac disease database in
Health gate by Markku Mäki. http://www.terveysportti.fi/terveysportti/ekirjat. koti?p_db=kel
designed to amplify whole exons, whereas in the CD-MEDICS device the aim
was to amplify only the gene region where the necessary probes align. The PCR
product length was aimed at 100 bps due to detection requirements in the
microchip. The locations of the IHWG probes were preserved, but the length of
the probes were modified to fit the requirements of real-time PCR.
The second aims of FRCBS were to be a leader of the validation workpackage 7
and review and sign off validation plans and reports in WP7.
RESULTS
FRCBS and TATAA did real-time PCR with primers and probes for HLADQA1/DQB1 allele detection.
Most of the validation runs had dilution series from positive and negative
samples where the end fluorescence ratio between 2 ng/reaction DNA positive
samples and 50 ng/reaction DNA negative samples was at least 1.5. The only
exception was probe 6DQB1*0302 which had 100% specificity and sensitivity
with complement probe 3DQB1*0201_0202 but 86% sensitivity alone or 60%
sensitivity with complement probe 1DQB1E2*0301 (in both cases 100%
specificity). No amplification differing from the background occurred in negative
control samples, when background was defined as curves staying below
deltaRn fluorescence value 0.01 in PCR cycles 20-40 (Figure 2).
FIG 2 Real-time PCR results. The ratio between positive and negative samples of the end
fluorescence ratio was at least 1.5.
MATERIALS AND METHODS
In real-time PCR (SSO) master mix was prepared from water, primers, probe and TaqMan(R) Genotyping Master Mix. 20 µl/well of master mix and 5 µl/well of
samples, two replicates per dilution, were pipetted to an optical PCR plate. Plate was covered the with optical adhesive film and the plate was centrifuged. Applied
Biosystems 7000 real-time PCR instrument programme was: 1. 10 min 95 ºC; 2. 15 s 95 ºC; 3. 1 min 60 ºC, measure fluorescence on FAM and VIC channels;
Repeat 2. + 3. 40 times. The data was analysed according to instrument protocol. The next data were recorded: the maximum delta normalized fluorescence (∆Rn)
values, slopes, R Square (R2) values and cycle threshold (Ct) values per sample. MULTI-D did statistical analysis with GenEx.
Real-time PCR (SSO): Approved validations had dilutions series from positive and negative samples where the ∆Rn ratio between 2 ng/reaction DNA positive sample
and 50 ng/reaction DNA negative sample is at least 1.5. When evaluating interlaboratory repeatability the criteria for approval was a ∆Rn ratio between 50
ng/reaction DNA positive samples and 50 ng/reaction DNA negative samples of at least 1.5, as the samples were only tested with 50 ng/reaction. The ratio was
more than 1.5 in every other probe results except 3DQB1*0202 where positive and negative samples overlap. Negative control sample was below ∆Rn value 0.01 in
every run.
Approved negative control sample (water) dis not show amplification that differs from the background where background was defined as curves staying below ∆Rn
value 0.01 in cycles 20-40.
CONCLUSIONS
FRCBS
developed
and
validated
the
CD
associated
HLA
typing
based
on
sequence-specific
oligonucleotides (SSO)-PCR with real-time PCR.
FRCBS led the validation workpackage 7 and reviewed and signed off validation plans and reports in
WP7.
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