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