THE ISOTHERMAL LOOP MEDIATED AMPLIFICATION (LAMP) METHOD IS LESS AFFECTED BY AMPLIFICATION REACTION INHIBITORS IN RESPECT TO PCR Tettamanzi V 1 , Minnucci G 1 , Amicarelli G 1 , Pultrone C 1 , D’Agostini E 1 , Rossi V, 2 Cazzaniga G, 2 Colotta F 1 ; 1 DiaSorin SpA, Gerenzano (VA), Italy ; 2 Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza, Italy. INTRODUCTION Several compounds can interfere with the nucleic acid amplification during polymerase chain reaction (PCR) [1,2] These inhibitors can derive from: • the clinical starting specimen (i.e. heparin, haemoglobin, polysaccharides,..) • chemicals employed during extraction procedure (i.e. phenol, ethanol, guanidium isothiocyanate,..) For this reason RNA/DNA purity is an important parameter that must be evaluated before PCR analysis. It is determined measuring the ratio of spectrophotometric absorbance of the sample at 260 nm, 280 nm and 230 nm (table 1). Table 1: DNA/RNA purity index Effect of inhibitors on PCR are absence or delay in sample amplification → risk of false negative or suboptimal results LAMP METHOD [3] • Nucleic acid amplification under isothermal condition • Retro-transcription and amplification in a single homogeneous step • Employment of DNA polymerase with strand displacement activity • Real time fluorescent reaction followed by annealing analysis • Highly specific and rapid (< 60 minutes) METHOD As model assay we have employed the TRIPLEX BCR-ABL RT-LAMP (see P182) • Multiplex detection of p190 and p210 transcripts • Simultaneous detection of housekeeping GUSβ mRNA as internal control • RNA amount: 500 ng/ rx • Time to result: 50 min (RT-amplification-signal detection) • Discrimination of amplified products by annealing analysis (table 2) Table 2: melting temperature range RESULTS Loop mediated isothermal amplification (LAMP) is significantly less affected by the common inhibitors of PCR reaction: • LAMP technology is not influenced by chemicals : 20 RNA samples with suboptimal 260/230 ratio have been perfectly detected • LAMP technology is not influenced by heparin contamination: 2 clinical RNA samples have been correctly amplified; by contrast a Heparinase treatment was needed prior to PCR analysis This innovative LAMP technique allows the efficient detection of low quality samples decreasing the risk of false negative and invalid run FROM CLIN SAMPLES Figure 1: LAMP results (annealing analysis) Red, green and light-blue peaks correspond to the tested controls with optimal purity index (table 2). Both cell lines and clinical samples with suboptimal purity values produced melting peaks perfectly superimposed to the ones of controls, giving the correct expected diagnosis. A RNA FROM CELL LINES Table 3: Spectrophotometrical Abs ratio values established by Nanodrop (Thermo Scientific) on RNA extracted from cell lines (panel A) and clinical samples (panel B): all suboptimal samples present chemical contamination. LAMP PERFORMANCE ON RNA SAMPLES CHEMICALLY CONTAMINATED LAMP RESULTS ON HEPARINE CONTAMINATED CLINICAL SAMPLES (COMPARISON WITH PCR) Two clinical samples contaminated with heparine were tested with both BCR-ABL RT LAMP and RT-PCR (Biomed protocol) [4] Differently from PCR, LAMP correctly amplified these critical samples (panel A). Melting analysis of LAMP products showed two perfect peaks superimposed to the p210 control peak (panel B) References [1] W. Abu Al-Soud et al. Journal of Applied Microbiology 2001; 39, 485–93 [2] C. Schrader et al. Journal of Applied Microbiology 2012; ISSN 1364-5072 [3] T. Notomi et al. Nucleic Acid Research 2000; Vol. 28, e63. [4] JJM. Van Dongen et al. Leukemia 1999; 13, 1901-1928. B CONCLUSION B LAMP RESULTS A B A

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THE ISOTHERMAL LOOP MEDIATED AMPLIFICATION (LAMP) METHOD IS LESS AFFECTED BY AMPLIFICATION REACTION INHIBITORS IN RESPECT TO PCR

Tettamanzi V1, Minnucci G1, Amicarelli G1, Pultrone C1, D’Agostini E1, Rossi V,2 Cazzaniga G,2 Colotta F1; 1DiaSorin SpA, Gerenzano (VA), Italy ; 2Centro Ricerca Tettamanti, Clinica Pediatrica Università di Milano Bicocca, Monza, Italy.

INTRODUCTION Several compounds can interfere with the nucleic acid amplification during polymerase chain reaction (PCR) [1,2]

These inhibitors can derive from: • the clinical starting specimen (i.e. heparin, haemoglobin, polysaccharides,..) • chemicals employed during extraction procedure (i.e. phenol, ethanol, guanidium isothiocyanate,..)

For this reason RNA/DNA purity is an important parameter that must be evaluated before PCR analysis. It is determined measuring the ratio of spectrophotometric absorbance of the sample at 260 nm, 280 nm and 230 nm (table 1).

Table 1: DNA/RNA purity index

Effect of inhibitors on PCR are absence or delay in sample amplification → risk of false negative or suboptimal results

LAMP METHOD [3]

• Nucleic acid amplification under isothermal condition • Retro-transcription and amplification in a single homogeneous step • Employment of DNA polymerase with strand displacement activity • Real time fluorescent reaction followed by annealing analysis • Highly specific and rapid (< 60 minutes)

METHOD

As model assay we have employed the TRIPLEX BCR-ABL RT-LAMP (see P182) • Multiplex detection of p190 and p210 transcripts • Simultaneous detection of housekeeping GUSβ mRNA as internal control • RNA amount: 500 ng/ rx • Time to result: 50 min (RT-amplification-signal detection) • Discrimination of amplified products by annealing analysis (table 2)

Table 2: melting temperature range

RESULTS

Loop mediated isothermal amplification (LAMP) is significantly less affected by the common inhibitors of PCR reaction:

• LAMP technology is not influenced by chemicals : 20 RNA samples with suboptimal 260/230 ratio have been perfectly detected • LAMP technology is not influenced by heparin contamination: 2 clinical RNA samples have been correctly amplified; by contrast a Heparinase treatment was needed prior to PCR analysis

This innovative LAMP technique allows the efficient detection of low quality samples decreasing the risk of false negative and invalid run

FRO

M C

LIN

LES

Figure 1: LAMP results (annealing analysis) Red, green and light-blue peaks correspond to the tested controls with optimal purity index (table 2). Both cell lines and clinical samples with suboptimal purity values produced melting peaks perfectly superimposed to the ones of controls, giving the correct expected diagnosis.

A F

M C

ELL

LIN

Table 3: Spectrophotometrical Abs ratio values established by Nanodrop (Thermo Scientific) on RNA extracted from cell lines (panel A) and clinical samples (panel B): all suboptimal samples present chemical contamination.

LAMP PERFORMANCE ON RNA SAMPLES CHEMICALLY CONTAMINATED

LAMP RESULTS ON HEPARINE CONTAMINATED CLINICAL SAMPLES (COMPARISON WITH PCR)

Two clinical samples contaminated with heparine were tested with both BCR-ABL RT LAMP and RT-PCR (Biomed protocol) [4]

Differently from PCR, LAMP correctly amplified these critical samples (panel A). Melting analysis of LAMP products showed two perfect peaks superimposed to the p210 control peak (panel B)

References

[1] W. Abu Al-Soud et al. Journal of Applied Microbiology 2001; 39, 485–93

[2] C. Schrader et al. Journal of Applied Microbiology 2012; ISSN 1364-5072

[3] T. Notomi et al. Nucleic Acid Research 2000; Vol. 28, e63.

[4] JJM. Van Dongen et al. Leukemia 1999; 13, 1901-1928.

CONCLUSION

LAMP RESULTS