Development and Comparison of Visual LAMP and LAMP-TaqMan Assays for

Overview

Journal Microorganisms

Specialty Microbiology

Date 2024 Jul 27

PMID 39065093

Authors

Shuning Cui

Haoze Ma

Xinyue Wang

Han Yang

Yuanzheng Wu

Yanli Wei

Jishun Li

Jindong Hu

Affiliations

Soon will be listed here.

Abstract

Strawberry anthracnose caused by spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the β-tubulin sequence of . Both methods can detect genomic DNA at very low concentrations (10 copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/μL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for . The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of in China and control strawberry anthracnose in the field.

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References

He J, Sun M, Li D, Zhu L, Ye J, Huang L . A real-time PCR for detection of pathogens of anthracnose on Chinese fir using TaqMan probe targeting ApMat gene. Pest Manag Sci. 2022; 79(3):980-988. DOI: 10.1002/ps.7260. View

Kim S, Lee S, Kim U, Oh S . Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: A review. Anal Chim Acta. 2023; 1280:341693. DOI: 10.1016/j.aca.2023.341693. View

Liu Z, Liang T, Kang C . Molecular bases of strawberry fruit quality traits: Advances, challenges, and opportunities. Plant Physiol. 2023; 193(2):900-914. DOI: 10.1093/plphys/kiad376. View

Liu W, Huang S, Liu N, Dong D, Yang Z, Tang Y . Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. Sci Rep. 2017; 7:40125. PMC: 5216335. DOI: 10.1038/srep40125. View

Lee S, Baek Y, Kim Y, Choi Y, Song M, Ahn J . One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV. Front Microbiol. 2017; 7:2166. PMC: 5220095. DOI: 10.3389/fmicb.2016.02166. View

Auyong A, Ford R, Taylor P . Genetic transformation of Colletotrichum truncatum associated with anthracnose disease of chili by random insertional mutagenesis. J Basic Microbiol. 2011; 52(4):372-82. DOI: 10.1002/jobm.201100250. View

Dowling M, Peres N, Villani S, Schnabel G . Managing on Fruit Crops: A "Complex" Challenge. Plant Dis. 2020; 104(9):2301-2316. DOI: 10.1094/PDIS-11-19-2378-FE. View

Holland P, Abramson R, Watson R, Gelfand D . Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991; 88(16):7276-80. PMC: 52277. DOI: 10.1073/pnas.88.16.7276. View

Tanner N, Zhang Y, Evans Jr T . Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques. 2015; 58(2):59-68. DOI: 10.2144/000114253. View

10.

Goto M, Honda E, Ogura A, Nomoto A, Hanaki K . Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques. 2009; 46(3):167-72. DOI: 10.2144/000113072. View

11.

Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N . Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000; 28(12):E63. PMC: 102748. DOI: 10.1093/nar/28.12.e63. View

12.

Fang H, Liu X, Dong Y, Feng S, Zhou R, Wang C . Transcriptome and proteome analysis of walnut (Juglans regia L.) fruit in response to infection by Colletotrichum gloeosporioides. BMC Plant Biol. 2021; 21(1):249. PMC: 8166054. DOI: 10.1186/s12870-021-03042-1. View

13.

Denoyes-Rothan B, Guerin G, Delye C, Smith B, Minz D, Maymon M . Genetic diversity and pathogenic variability among isolates of colletotrichum species from strawberry. Phytopathology. 2008; 93(2):219-28. DOI: 10.1094/PHYTO.2003.93.2.219. View

14.

Zhang L, Song L, Xu X, Zou X, Duan K, Gao Q . Characterization and Fungicide Sensitivity of Species Causing Strawberry Anthracnose in Eastern China. Plant Dis. 2020; 104(7):1960-1968. DOI: 10.1094/PDIS-10-19-2241-RE. View

15.

Toumazou C, Shepherd L, Reed S, Chen G, Patel A, Garner D . Simultaneous DNA amplification and detection using a pH-sensing semiconductor system. Nat Methods. 2013; 10(7):641-6. DOI: 10.1038/nmeth.2520. View

16.

Nagamine K, Hase T, Notomi T . Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002; 16(3):223-9. DOI: 10.1006/mcpr.2002.0415. View

17.

Tomita N, Mori Y, Kanda H, Notomi T . Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc. 2008; 3(5):877-82. DOI: 10.1038/nprot.2008.57. View

18.

Dean R, van Kan J, Pretorius Z, Hammond-Kosack K, Di Pietro A, Spanu P . The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol. 2012; 13(4):414-30. PMC: 6638784. DOI: 10.1111/j.1364-3703.2011.00783.x. View

19.

Mills P, Sreenivasaprasad S, Brown A . Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR. FEMS Microbiol Lett. 1992; 77(1-3):137-43. DOI: 10.1016/0378-1097(92)90145-e. View

20.

Yu Y, Li R, Ma Z, Han M, Zhang S, Zhang M . Development and evaluation of a novel loop mediated isothermal amplification coupled with TaqMan probe assay for detection of genetically modified organism with NOS terminator. Food Chem. 2021; 356:129684. DOI: 10.1016/j.foodchem.2021.129684. View