Development and evaluation of LAMP, CPA and IMSA methods for rapid detection of the AML1/ETO fusion gene in acute myeloid leukemia

Abstract

The objective of the present study was to determine whether the loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and/or isothermal multiple-self-matching-initiated amplification (IMSA) methods can provide rapid detection of the runt related transcription factor 1/runt related transcription factor 1 translocation partner 1 (AML1/ETO) fusion gene in acute myeloid leukemia (AML). According to the sequence of the AML1/ETO fusion gene available in GenBank and the principles of the LAMP, CPA and IMSA methods, specific primers were designed to bind a conserved region of the AML1/ETO gene in each assay. Following optimization of the conditions for the LAMP, CPA and IMSA assays, the specificity and sensitivity of the assays were examined and compared. In addition, 41 clinical samples were assayed using the three methods. It was observed that a ladder-like pattern of DNA products was produced in AML1/ETO-positive samples in all three assays, whereas no DNA product was generated with the controls. The detection limit of the LAMP and CPA assays was 50 copies/tube, and for the IMSA assay was 10 copies/tube. This sensitivity was consistent, and improved in the latter case, compared with that of the reverse transcription-polymerase chain reaction (RT-PCR) assay. Furthermore, the detection rate for bone marrow or peripheral blood samples was 9.76%, and the agreement among the LAMP, CPA, IMSA and RT-PCR methods was 100%. Therefore, the LAMP, CPA and IMSA methods optimized in the present study provided rapid detection of the AML1/ETO fusion gene for an initial clinical diagnosis of AML. In addition, the LAMP, CPA and IMSA assays are straightforward to perform and do not require specialized instruments. Therefore, these three isothermal methods may be used to perform field tests or assays at resource-limited hospitals.

Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli

“…The three isothermal amplification assays demonstrated high specificity and sensitivity when detecting heat-labile enterotoxin-producing E. coli in clinical samples [24,28]. Additionally, research by Yang, et al also confirmed that IMSA demonstrated higher sensitivity than LAMP or CPA assays [19]. However, compared with other pathogenic bacteria, ETEC is more serotyped and relatively complicated.…”

“…The primers are able to specifically recognize distinct corresponding regions of the target DNA and can typically generate multiple self-matching structures (SMS) similar to a dumbbell, which single-stranded DNA amplicons generate from the hybrid primers. This technology does not require complex instruments and the detection of target DNA sequences can be completed within 1-2 hours using isothermal conditions, for which the method is potentially rapid and simple allowing practical diagnosis of a specific disease [18,19]. Cross-priming amplification (CPA) was invented by Fang et al which uses a mechanism similar to LAMP with 4-8 nucleotide primers complementary to the DNA template and using a detection procedure achieved using isothermal conditions within 1 h [20,21].…”

The Colorimetric Isothermal Multiple-Self-Matching-Initiated Amplification Using Cresol Red for Rapid and Sensitive Detection of Porcine Circovirus 3

“…In our study, the IMSA primers were designed according to the conserved region of the PCV3 capsid gene, which was the usual detective marker of PCR-based methods (7,11). The primer design work was not as complicated as those of LAMP, because the IMSA primers consisted of seven basic primers specifically recognizing distinct regions of the target, which can be easily designed by using the software Primer Premier 5.0 (15). The results of colorimetric IMSA can be visually judged within 60 min, which was at least 60 min shorter than qPCR or PCR methods.…”

“…The conserved region of the capsid gene was determined by alignment of PCV3 strains indexed in the GenBank (accession no: MF589105.1 , MF589107.1 , MF769811.1 , MF769807.1 , MF084994.1 , KX778720.1 , KX898030.1 , MG310152.1 , MF079254.1 , and MG250187.1 ). According to the principle of IMSA assay, capsid gene sequences of PCV3-US/MO2015 strain (accession no: KX778720.1 ) were input for IMSA primer design by using the software Primer Premier 5.0 ( 15 ). Among multiple sets of primers, the primers targeting to the conserved regions of the capsid gene were selected for subsequent analysis.…”

“…This makes them to be good choice for low-cost isothermal methods. What is more, IMSA proved to be more sensitive than LAMP in the previous study (13,15). Recently, rapid, sensitive, and visual detection assay was achieved by the addition of pH-sensitive indicator dyes.…”