p190: 10-4 mut into wt dilution (panel A)
P210: 10-5 mut into wt dilution (panel B)
p190: 10-4 mut into wt dilution (panel A)
P210: 10-5 mut into wt dilution (panel B)
REAL-TIME MULTIPLEX MOLECULAR DETECTION OF BCR-ABL P190 AND P210 TRANSCRIPTS
BY RT-QLAMP ON THE LIAISON IAM SEMI-AUTOMATIC INSTRUMENT
D’Agostini E1, Tettamanzi V1, Pultrone C1, Boroni C2, Spinelli O2, Rambaldi A2, Colotta F1, Minnucci G1, Amicarelli G1.1, DiaSorin SpA, Gerenzano (VA), Italy; 2, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy
INTRODUCTION
METHOD RESULTS
CONCLUSIONSCONCLUSIONS
The molecular detection of BCR-ABL transcripts is routinely performed to confirm
diagnosis of suspected Chronic Myeloid Leukemia (CML) and for risk stratification of B-
precursor Acute Lymphoblastic Leukemia (ALL). RT-PCR [1] is the method of choice for
this purpose, consisting in at least 3 hours of complex procedure.
We present a novel molecular method, based on Loop Mediated Isothermal
Amplification (LAMP) [2] 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.
Total RNA 500 ng
Fluorescence signals specific for amplification of
p190, p210 and Internal Control (GUSβ),
respectively detected in dedicated fluorescent
channels:
ISOTHERMAL ISOTHERMAL
ONE HOMOGENEOUS FORMAT ONE HOMOGENEOUS FORMAT
RNA Retro-transcription and amplification in a
single step using one single enzyme, a DNA
polymerase with RT and strand-displacement
activities
MULTIPLE PRIMER SETSMULTIPLE PRIMER SETS
3 primer sets for the simultaneous
detection of p190, p210 and GUSββββ
(Internal Control)
fluorescent channel: Result:
500 nm Positive p190
570 nm Positive p210
530 nm Negative
No amplification Invalid run
Triplex BCR-ABL RT-QLAMP reaction mix
REAL TIME MONITORINGREAL TIME MONITORING
ASSAY SENSITIVITY
CLINICAL VALIDATIONCLINICAL VALIDATIONASSAY SPECIFICITYASSAY SPECIFICITY
^ 60 Healthy
Donors
The assay specificity has been established by
testing 340 replicates of wild type RNA from 7 cell
lines (Table 1), all resulted BCR-ABL negative in
the presence of the correct amplification of the
GUSββββ internal control.
The assay sensitivity has been analytically determined by testing serial dilutions of mutated p190 and
p210 RNA (from TOM1 and K562 cell lines respectively) into wild type RNA from HL60 cell line.
A
B
Table 1
The BCR-ABL RT-QLAMP assay was validated on RNA
obtained from 125 clinical samples previously
diagnosed at Azienda Ospedaliera Papa Giovanni XXIII
(Bergamo) by using conventional RT-PCR (Biomed) [1]
Table 2
The semi-automatic detection of the p190 and p210 BCR-ABL transcripts by the triplex
RT-QLAMP performed on the Liaison IAM instrument represents a novel system for
efficient diagnosis of CML and Ph positive ALL.
The multiplex, one-step format reduces the contamination risks of conventional multi
step RT-PCR and simplifies the overall procedure.
The validation of negative results through amplification of endogenous GUSβ mRNA
ensure reliability.
The high sensitivity, specificity and rapidity significantly enhance the patient
management and improve the diagnostic laboratory routine.
REFERENCES:
[1] Van Dongen JJM et al. Leukemia (1999) 13, 1901-1928; [2] Notomi T et al. NAR (2000) 15: 28 (12): E63
BCR-ABL RT-QLAMP RT-PCR [1]
ASSAY FORMAT TRIPLEX SIMPLEX
TEMPERATURE ISOTHERMAL (65°C) PCR CYCLING
CONTROL OF REACTION INTERNAL (GUS b) 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
100% specificity on 340 replicates
50 min incubation at 65°C
Release of results in terms of “positive”,
“negative” and “invalid”:
Table 3 Comparison between the novel DiaSorin BCR-ABL RT-QLAMP system and the conventional
RT-PCR method [1]
AUTOMATIC DATA ANALYSISAUTOMATIC DATA ANALYSIS
100% agreement with conventional
RT-PCR on 125 clinical samples
100% agreement with conventional
RT-PCR on 125 clinical samples
