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Semi-Automated Molecular Detection of BCR-ABL Major and Minor Transcripts by Isothermal RT-Loop Mediated Reaction on the Liaison IAM Instrument
Elena D’Agostini1, Giulia Minnucci, PhD1, Giulia Amicarelli, PhD1, Cinzia Pultrone1, Veronica Tettamanzi1, Chiara Boroni2, Orietta Spinelli2,Francesco Colotta, MD1, Alessandro Rambaldi, MD2
1DiaSorin SpA, Gerenzano (VA), Italy; 2USC HematologyOspedali Riuniti di Bergamo, Bergamo, Italy.
The molecular detection of the BCR-ABL fusion transcripts is necessary for the genetic confirmation of Chronic Myeloid Leukemia
(CML) diagnosis and for the risk classification of Acute Lymphoblastic Leukemia (ALL) [1,2]. The molecular identification for this
purpose is actually based on conventional RT-PCR [3]; the main limitations are long time-to-result and multi-step procedures that
often cause a slow-down of diagnostic laboratories routine activity. Here we present a novel molecular method, based on Loop
Mediated Amplification (LAMP) [4] reaction that, coupled with the Liaison IAM instrument (DiaSorin SpA), ensures semi-
automated rapid detection of BCR-ABL p190 and p210 fusion transcripts and of the endogenous GUSβ mRNA.
INTRODUCTIONINTRODUCTION RESULTSRESULTSRT-LAMP is HIGHLY SENSITIVE
RNA EXTRACTION500ng
METHODSMETHODS
BCR-ABL multiplex Fluorescent RT-LAMP assay has been developed to detect and distinguish the two fusion transcripts (p190 and
p210) of the BCR-ABL translocation.
BCRBCR--ABL RTABL RT--LAMPLAMP
The assay detects p210 and p190
dilutions down to 10-5 and 10-4,
respectively (Figure 1 A,B).
The level of sensitivity was
established on 500ng RNA extracted
from p210 and p190 positive cell
lines (K562 and TOM1 respectively)
serially diluted into negative cell line
RNA (from cell line HL60).
CConventional RT-PCR
(Biomed protocol [3])
A
Results by conventional RT-PCR
(Biomed) [3]
p190* p190* p210p210§§ NegativeNegative
^̂
TotTot
p190*p190* 30 - - 30
RNA EXTRACTION500ng
Fluorescence generation from amplification of
ISOTHERMAL
Incubation at constant temperature 65°C for
50 min50 min in Liaison IAM instrument (DiaSorin)
ONE HOMOGENEOUS STEP
Retrotranscription and amplification in a single
step using one single enzyme, a DNA pol with RT
and strand-displacement activities
MULTIPLE PRIMER SETS
3 primer sets for the simultaneous detection of
p190, p210 and GUSββββ (Internal Control)
RETROTRANSCRIPTION
PCR
Control (ABL)p190 p210
Liaison IAM instrument
CLINICAL VALIDATIONCLINICAL VALIDATIONRTRT--LAMP is HIGHLY SPECIFICLAMP is HIGHLY SPECIFIC
** (29 B-ALL, 1 CML)
§ (27 CML, 3 B-ALL)
^ 60 Healthy Donors
The assay specificity was established on negative BCR-ABL RNA
extracted from 7 cell lines (Table 1). All negative results have
been validated by amplification of the Internal Control (GUSβ).
Figure 1
1000ng
Triplex BCR-ABL
RT-LAMP
reaction mix
REAL TIME MONITORING
Interestingly a clear relationship
between target dose and
amplification time is observed (Fig 1
C,D)
D
(Biomed protocol )
B
The BCR-ABL RT-LAMP assay was validated on RNA obtained
from 120 clinical samples previously diagnosed at Ospedali
Riuniti di Bergamo by using conventional RT-PCR (Biomed) [3]
Re
sults b
y
RT
-LAM
P
p210p210§§ - 30 - 30
Negative^Negative^ - - 60 60
TotTot 30 30 60 120
50’
Fluorescence generation from amplification of
p190, p210 and Internal Control (GUSββββ) detected
in dedicated fluorescent channels:
BCR-ABL
RT-LAMP
RT-PCR
(Biomed protocol[3])
ASSAY FORMAT TRIPLEX SIMPLEX
TEMPERATURE ISOTHERMAL (65°C) PCR CYCLING
CONTROL OF REACTION INTERNAL (GUS β) EXTERNAL
STEPS 1 3
TIME TO RESULTS 50 min 3 h and 30 min
TARGETS RNA cDNA
SENSITIVITYp190: 10-4
p210: 10-5
p190: 10-3
p210: 10-4
SPECIFICITY 100% n.a.
RESULTS INTERPRETATIONOBJECTIVE
(automatic elaboration)SUBJECTIVE
Amplification in Channel Results
500 nm Positive p190
570 nm Positive p210
530 nm Negative
No amplification Invalid run
Automatic elaboration of
results: GEL SEPARATION
RESULTS
100% specificity on 275 replicates
CONCLUSIONSCONCLUSIONS
100% agreement with conventional RT-PCR on 120
clinical samples
REFERENCES:
[1] National Comprehensive Cancer Network 2010
[2] National Cancer Institute 2010
[3] Van Dongen JJM et al. Leukemia (1999) 13, 1901-1928
[4] Notomi T et al. NAR (2000) 15: 28 (12): E63
The triplex p190-p210-GUSβ RT-LAMP is a one-step
procedure for specific, highly sensitive and rapid molecular
detection of the BCR-ABL fusion transcripts.
The semi-automatic approach on the instrument Liaison
IAM allow to simplify the entire procedure, reducing the
contamination risks deriving from the conventional, multi
step RT-PCR, thus resulting in a significantly improvement of
the diagnostic lab routine.
From RNA to
interpreted result in
50 minutes
From RNA to result in From RNA to result in
3 hours and 30 minutes
Table 1 Table 2