A Novel, Rapid, Ultrasensitive Diagnosis Platform for Detecting Using Restriction Endonuclease-mediated Real-time Loop-mediated Isothermal Amplification

Overview

Journal Front Cell Infect Microbiol

Specialties Cell Biology
Infectious Diseases
Microbiology

Date 2024 Nov 26

PMID 39588507

Authors

Yizhe Wang

Yuhong Zhou

Jingrun Lu

Honglan Yu

Yu Wang

Affiliations

Soon will be listed here.

Abstract

Introduction: (, CA) is an essential invasive fungus in clinical diagnosis. Although several detection methods exist, none meet the need for early diagnosis. A rapid, sensitive, and specific diagnostic tool is crucial for effective prevention and control of infections.

Methods: This study aimed to develop a new, rapid, and ultrasensitive diagnostic tool for detection based on restriction endonuclease-mediated real-time loop-mediated isothermal amplification (ERT-LAMP-CA). The ERT-LAMP-CA technology combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence detection in a single reaction tube, which can complete a diagnosis of in a short time (approximately 1 h).

Results: Herein, we developed the primer sequences required for ERT-LAMP-CA based on the ITS2 gene of and found that ERT-LAMP-CA limit of detection was approximately 500 ag/μL genomic DNA and can present negative results for non- templates. We tested sputum samples from 64 patients with suspected infections to validate ERT-LAMP-CA applicability in clinical sample testing and found that ERT-LAMP-CA was consistent with multiplex PCR-capillary electrophoresis.

Discussion: In conclusion, ERT-LAMP-CA is a rapid, accurate, and sensitive assay with excellent potential for clinical and basic laboratory diagnosis and an efficient screening strategy.

References

Brown G, Denning D, Gow N, Levitz S, Netea M, White T . Hidden killers: human fungal infections. Sci Transl Med. 2012; 4(165):165rv13. DOI: 10.1126/scitranslmed.3004404. View

Arendrup M, Patterson T . Multidrug-Resistant Candida: Epidemiology, Molecular Mechanisms, and Treatment. J Infect Dis. 2017; 216(suppl_3):S445-S451. DOI: 10.1093/infdis/jix131. View

Benedict K, Jackson B, Chiller T, Beer K . Estimation of Direct Healthcare Costs of Fungal Diseases in the United States. Clin Infect Dis. 2018; 68(11):1791-1797. PMC: 6409199. DOI: 10.1093/cid/ciy776. View

Pincus D, Orenga S, Chatellier S . Yeast identification--past, present, and future methods. Med Mycol. 2007; 45(2):97-121. DOI: 10.1080/13693780601059936. View

Huang J, Yang X, Ren L, Jiang W, Huang Y, Liu Y . A novel, ultrafast, ultrasensitive diagnosis platform for the detection of SARS-COV-2 using restriction endonuclease-mediated reverse transcription multiple cross displacement amplification. J Med Virol. 2022; 95(2):e28444. PMC: 9880628. DOI: 10.1002/jmv.28444. View

Wilson L, Reyes C, Stolpman M, Speckman J, Allen K, Beney J . The direct cost and incidence of systemic fungal infections. Value Health. 2002; 5(1):26-34. DOI: 10.1046/j.1524-4733.2002.51108.x. View

Choi G, Moehling T, Meagher R . Advances in RT-LAMP for COVID-19 testing and diagnosis. Expert Rev Mol Diagn. 2023; 23(1):9-28. DOI: 10.1080/14737159.2023.2169071. View

Mori Y, Kitao M, Tomita N, Notomi T . Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods. 2004; 59(2):145-57. DOI: 10.1016/j.jbbm.2003.12.005. View

Gunasekera M, Narine M, Ashton M, Esfandiari J . Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum. J Immunol Methods. 2015; 424:7-13. DOI: 10.1016/j.jim.2015.04.014. View

10.

Dhama K, Karthik K, Chakraborty S, Tiwari R, Kapoor S, Kumar A . Loop-mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. Pak J Biol Sci. 2014; 17(2):151-66. DOI: 10.3923/pjbs.2014.151.166. 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.

Wang Y, Zhao X, Zhou Y, Lu J, Yu H, Li S . Establishment and application of loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor (LAMP-LFB) for visual and rapid diagnosis of in clinical samples. Front Bioeng Biotechnol. 2022; 10:1025083. PMC: 9676452. DOI: 10.3389/fbioe.2022.1025083. View

13.

Kasai M, Francesconi A, Petraitiene R, Petraitis V, Kelaher A, Kim H . Use of quantitative real-time PCR to study the kinetics of extracellular DNA released from Candida albicans, with implications for diagnosis of invasive Candidiasis. J Clin Microbiol. 2006; 44(1):143-50. PMC: 1351963. DOI: 10.1128/JCM.44.1.143-150.2006. View

14.

Chen X, Liao B, Cheng L, Peng X, Xu X, Li Y . The microbial coinfection in COVID-19. Appl Microbiol Biotechnol. 2020; 104(18):7777-7785. PMC: 7417782. DOI: 10.1007/s00253-020-10814-6. View

15.

Cao Q, Liang S, Wang L, Cao J, Liu M, Li S . A Rapid Detection of Using Multiple Cross Displacement Amplification Linked With Nanoparticle-Based Lateral Flow Biosensor. Front Cell Infect Microbiol. 2021; 11:721547. PMC: 8493954. DOI: 10.3389/fcimb.2021.721547. View

16.

Casalini G, Giacomelli A, Antinori S . The WHO fungal priority pathogens list: a crucial reappraisal to review the prioritisation. Lancet Microbe. 2024; 5(7):717-724. DOI: 10.1016/S2666-5247(24)00042-9. View

17.

Adams E, Quinn M, Tsay S, Poirot E, Chaturvedi S, Southwick K . Candida auris in Healthcare Facilities, New York, USA, 2013-2017. Emerg Infect Dis. 2018; 24(10):1816-1824. PMC: 6154128. DOI: 10.3201/eid2410.180649. View

18.

Vitiello A, Ferrara F, Boccellino M, Ponzo A, Cimmino C, Comberiati E . Antifungal Drug Resistance: An Emergent Health Threat. Biomedicines. 2023; 11(4). PMC: 10135621. DOI: 10.3390/biomedicines11041063. View

19.

Kainz K, Bauer M, Madeo F, Carmona-Gutierrez D . Fungal infections in humans: the silent crisis. Microb Cell. 2020; 7(6):143-145. PMC: 7278517. DOI: 10.15698/mic2020.06.718. View

20.

Jayanath N, Nguyen L, Vu T, Tran L . Development of a portable electrochemical loop mediated isothermal amplification (LAMP) device for detection of hepatitis B virus. RSC Adv. 2022; 8(61):34954-34959. PMC: 9087361. DOI: 10.1039/c8ra07235c. View