Utilizing multiplex fluor LAMPs to illuminate multiple gene expressions in situ

Autoři: Diona Podushkina aff001;  Nick W. West aff001;  Edward M. Golenberg aff001
Působiště autorů: Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223333


In situ gene expression detection is the best way to determine temporal and spatial differences in gene expression. However, in situ hybridization procedures are inherently difficult to execute and typically suffer from degradation of sample tissues, limited sensitivity to genes with low expression, high background, and limitation to single gene detections. We propose to utilize an isothermal gene amplification technique, LAMP (Loop-Mediated Isothermal Amplification), to solve these problems in a novel way. LAMP greatly amplifies the signal of expressed genes and can use multiple sets of primers and different fluorescent-labeled probes to produce multiplex gene detection. LAMP is a rapid, isothermal reaction that reduces the handling and degradation of tissue by cutting down on the washing steps required by other methods. Using this technique, we have successfully amplified 3 target genes, have produced positive fluorescent in situ results simultaneously for two genes. We have also demonstrated that LAMP can be used to exploit standard NBT/BCIP (nitro-blue tetrazolium chloride/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) detection of single expression. In situ LAMP is a robust and applicable method that can be exploited for detection of gene expression in plant species, as well as in animals and bacteria.

Klíčová slova:

Antisocial behavior – Gene amplification – Gene expression – Inflorescences – Probe hybridization – In situ hybridization – RNA amplification – Spinach


1. Huber D, Voith von Voithenberg L, Kaigala GV. Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH? Micro and Nano Engineering. 2018;1:15–24. https://doi.org/10.1016/j.mne.2018.10.006.

2. Jensen E. Technical review: In situ hybridization. The Anatomical Record. 2014;297(8):1349–53. doi: 10.1002/ar.22944 24810158

3. Koch J. Primed in situ labeling. In: Fan Y-S, editor. Molecular Cytogenetics: Protocols and Applications. Totowa, NJ: Humana Press; 2003. p. 77–84.

4. Nuovo GJ. Co-labeling using in situ pcr:A review. Journal of Histochemistry & Cytochemistry. 2001;49(11):1329–39. doi: 10.1177/002215540104901101 11668186.

5. Tecott LH, Barchas JD, Eberwine JH. In situ transcription: specific synthesis of complementary DNA in fixed tissue sections. Science (New York, NY). 1988;240(4859):1661–4. Epub 1988/06/17. doi: 10.1126/science.2454508 2454508.

6. Stewart NT, Byrne KM, Hosick HL, Vierck JL, Dodson MV. Traditional and emerging methods for analyzing cell activity in cell culture. Methods in cell science: an official journal of the Society for In Vitro Biology. 2000;22(1):67–78. Epub 2000/01/29. 10650337.

7. Park K, Kim J, Lim S, Han S, Lee JY. Comparing fluorescence in situ hybridization and chromogenic in situ hybridization methods to determine the HER2/neu status in primary breast carcinoma using tissue microarray. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc. 2003;16(9):937–43. Epub 2003/09/19. doi: 10.1097/01.mp.0000086487.78558.7d 13679458.

8. Tanner M, Gancberg D, Di Leo A, Larsimont D, Rouas G, Piccart MJ, et al. Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples. The American journal of pathology. 2000;157(5):1467–72. Epub 2000/11/14. doi: 10.1016/S0002-9440(10)64785-2 11073807.

9. Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and Cellular Probes. 2002;16(3):223–9. https://doi.org/10.1006/mcpr.2002.0415. 12144774

10. Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loop-mediated isothermal amplification reaction using a nondenatured template. Clinical Chemistry. 2001;47(9):1742. 11514425

11. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 2000;28(12):e63–e. doi: 10.1093/nar/28.12.e63 10871386

12. Pfent C, Pobursky KJ, Sather DN, Golenberg EM. Characterization of SpAPETALA3 and SpPISTILLATA, B class floral identity genes in Spinacia oleracea, and their relationship to sexual dimorphism. Dev Genes Evol. 2005;215(3):132–42. doi: 10.1007/s00427-004-0459-4 15660251.

13. Sather DN, York A, Pobursky KJ, Golenberg EM. Sequence evolution and sex-specific expression patterns of the C class floral identity gene, SpAGAMOUS, in dioecious Spinacia oleracea L. Planta. 2005;222:284–92. doi: 10.1007/s00425-005-1544-2 15940462

14. Xu J, Ding Z, Vizcay-Barrena G, Shi J, Liang W, Yuan Z, et al. Aborted microspores acts as a master regulator of pollen wall formation in Arabidopsis. The Plant Cell. 2014;26(4):1544. doi: 10.1105/tpc.114.122986 24781116

15. West NW, Golenberg EM. unpublished.

16. Deng MH, Zhong LY, Kamolnetr O, Limpanont Y, Lv ZY. Detection of helminths by loop-mediated isothermal amplification assay: a review of updated technology and future outlook. Infectious diseases of poverty. 2019;8(1):20. Epub 2019/03/25. doi: 10.1186/s40249-019-0530-z 30905322.

17. Dhama K, Karthik K, Chakraborty S, Tiwari R, Kapoor S, Kumar A, et al. Loop-mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. Pakistan journal of biological sciences: PJBS. 2014;17(2):151–66. Epub 2014/05/03. doi: 10.3923/pjbs.2014.151.166 24783797.

18. Fernandez-Soto P, Gandasegui Arahuetes J, Sanchez Hernandez A, Lopez Aban J, Vicente Santiago B, Muro A. A loop-mediated isothermal amplification (LAMP) assay for early detection of Schistosoma mansoni in stool samples: a diagnostic approach in a murine model. PLoS neglected tropical diseases. 2014;8(9):e3126. Epub 2014/09/05. doi: 10.1371/journal.pntd.0003126 25187956.

19. Liu N, Zou D, Dong D, Yang Z, Ao D, Liu W, et al. Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus. Scientific Reports. 2017;7:45601. doi: 10.1038/srep45601 28349967

20. Maruyama F, Kenzaka T, Yamaguchi N, Tani K, Nasu M. Detection of bacteria carrying the stx(2) gene by in situ loop-mediated isothermal amplification. Applied and Environmental Microbiology. 2003;69(8):5023–8. doi: 10.1128/AEM.69.8.5023-5028.2003 12902306

21. Yoshida A, Nagashima S, Ansai T, Tachibana M, Kato H, Watari H, et al. Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Journal of Clinical Microbiology. 2005;43(5):2418–24. doi: 10.1128/JCM.43.5.2418-2424.2005 15872275.

22. Mori Y, Hirano T, Notomi T. Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnology. 2006;6:3-. doi: 10.1186/1472-6750-6-3 16401354

23. Mori Y, Kitao M, Tomita N, Notomi T. Real-time turbidimetry of LAMP reaction for quantifying template DNA. Journal of Biochemical and Biophysical Methods. 2004;59(2):145–57. https://doi.org/10.1016/j.jbbm.2003.12.005. 15163526

24. Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocols. 2008;3:877. doi: 10.1038/nprot.2008.57 18451795

25. Tucker JD. Reflections on the development and application of FISH whole chromosome painting. Mutation research Reviews in mutation research. 2015;763:2–14. Epub 2015/03/22. doi: 10.1016/j.mrrev.2014.11.006 25795112.

26. Bagasra O. Protocols for the in situ PCR-amplification and detection of mRNA and DNA sequences. Nat Protoc. 2007;2(11):2782–95. Epub 2007/11/17. doi: 10.1038/nprot.2007.395 18007614.

27. Meng Q, Wang S, Zhang L, Huang X, Bao Z. Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences. Chinese Journal of Oceanology and Limnology. 2013;31(1):128–33. doi: 10.1007/s00343-013-2070-x

28. Ganguli A, Ornob A, Spegazzini N, Liu Y, Damhorst G, Ghonge T, et al. Pixelated spatial gene expression analysis from tissue. Nat Commun. 2018;9(1):202-. doi: 10.1038/s41467-017-02623-9 29335461.

Článek vyšel v časopise


2019 Číslo 10