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Acta Histochem Cytochem
2017 Dec 26;506:169-176. doi: 10.1267/ahc.17024.
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Novel Application of Loop-mediated Isothermal Amplification for Rapid Detection of Gene Translocation.
Matsuzaki I
,
Iguchi H
,
Mikasa Y
,
Morishita H
,
Okuda K
,
Nakaguchi K
,
Mori Y
,
Iwahashi Y
,
Warigaya K
,
Fujimoto M
,
Kojima F
,
Murata SI
.
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Identification of fusion genes in cancer is essential for pathological diagnosis and clinical therapy. Although methods for detection of fusion genes, such as fluorescence in situ hybridization (FISH) and real-time polymerase chain reaction (PCR), have been developed in last two decades, these methods are not ideal for detection of these genetic alterations owing to their high cost and time-consuming procedures. In this study, we developed novel application for detection of gene translocations using loop-mediated isothermal amplification (LAMP). We verified the amplified DNA products of echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK), synaptotagmin and synovial sarcoma, X breakpoint (SYT-SSX), and immunoglobulin heavy chain gene and B cell leukemia/lymphoma 2 (IgH/BCL2) by real-time PCR, agarose-gel electrophoresis, and the naked eye after the LAMP procedure. Fusion genes were detected in samples diluted 103 times within 60 min. Because of the advantages of rapid amplification, simple operation, and easy detection without requiring sophisticated equipment or technical skill, LAMP may have potential applications as an on-site analytical approach in hospitals for pathological diagnosis and decision making regarding appropriate therapeutic approachs.
Fig. 1. Schematic representation of detection of fusion genes by LAMP assays. A: cDNA from cells lacking a fusion gene cannot react in LAMP assays. B: cDNA from cells having a fusion gene can be analyzed by LAMP assays. The inner primers FIP (BIP) were composed of F1c (B1c) and F2 (B2). The outer primers were F3 and B3. Because dumbbell-like structures cause cyclic reactions, the LAMP reaction synthesizes various products of different sizes. The schematic shows a case of detection of the IgH/BCL2 fusion gene.
Fig. 2. Detection of gene rearrangements with FISH. A: The EML4-ALK fusion gene in H2228 cells. The arrows indicate the merged red signal (telomeric side of ALK) and green signal (telomeric side of EML4) as a yellow signal. C: SYT-SSX FISH using break-apart probes was positive in HS cells. Arrows indicating split orange and green signals showed the rearrangement of SYT-SSX. E: IgH/BCL2 FISH-positive results in TK cells. Arrows indicate the merged red signal (BCL2) and green signal (IgH) as a yellow signal. B, D, and F: EML4-ALK FISH-negative (B), SYT-SSX FISH-negative (D) and IgH/BCL2 FISH-negative (F) results in HuH cells. Original magnification: 600×.
Fig. 3. Detection of fusion genes using real-time PCR. Amplification curves obtained from real-time PCR assays for EML4-ALK (A), SYT-SSX (B), and IgH/BCL2 (C). H2228 cells (A), HS cells (B), and TK cells (C) were diluted from 100- to 103-fold.
Fig. 4. Specificity of the LAMP assay. LAMP assays for EML4-ALK (A–C), SYT-SSX (D–F), and IgH/BCL2 (G–I). LAMP products were detected using real-time PCR equipment (A, D, G), agarose gel electrophoresis (B, E, H), and the naked eye (C, F, I). M: size marker, NC: negative control without cDNA.
Fig. 5. Sensitivity of the LAMP assay. LAMP assays for EML4-ALK (A), SYT-SSX (B), and IgH/BCL2 (C). H2228 cells (A), HS cells (B), and TK cells (C) were diluted from 100 to 103-fold.
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