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Dual-probe ligation without PCR for fluorescent sandwich assay of EGFR nucleotide variants in magnetic gene capture platform

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Abstract

A simple, rapid, and highly efficient fluorescent detection technique without PCR through dual-probe ligation with the genetic capture of magnetic beads and reported probe was developed for determination of epidermal growth factor receptor (EGFR) gene exon 19 deletions. The EGFR exon 19 deletion mutation makes up 48% of all mutations associated with anti-tyrosine kinase inhibition sensitivity, and thus, the EGFR nucleotide variant is very important in clinical diagnosis. In this approach, the dual-probe ligation was designed to target exon 19 deletion. The magnetic genetic captured system was then applied to capture the successful dual-probe ligation. Thereafter, a reporter probe which is coupled with 6-fluorescein amidite (6-FAM) was introduced to hybridize with dual-probe ligation product on the surface of streptavidin magnetic beads, and finally, the supernatant was taken for fluorescence measurements for distinguishing mutant types from wild types. After optimization (the RSD of the fluorescent intensity was less than 4.5% (n = 3) under the optimal condition), 20 blind DNA samples from the population were analyzed by this technique and further confirmed by direct sequencing. The results of our assay matched to those from direct sequencing data, evidencing that the developed method is accurate and successful. These 20 blind DNA samples were diagnosed as wild and then spiked with different percentages of the mutant gene to quantify the ratio of the wild and mutant genes. This strategy was also successfully applied to quantify the ratio of the wild and mutant genes with good linearity at the λex/λem of 480 nm/520 nm (r = 0.996), and the limit of detection reached 1.0% mutant type. This simple fluorescent detection of nucleotide variants shows its potential to be considered a tool in biological and clinical diagnosis. Importantly, this strategy offers a universal detection capability for any kind of mutation (point, deletion, insertion, or substitution) in a gene of interest.

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Abbreviations

EGFR:

Epidermal growth factor receptor

6-FAM:

6-Fluorescein amidite

DNA:

Deoxyribonucleic acid

PCR:

Polymerase chain reaction

NSCLC:

Non-small cell lung cancer

TKI:

Tyrosine kinase inhibitors

PNA-LNA:

Peptide nucleic acid-locked nucleic acid

ARMS:

Amplification refractory mutation system

HPLC:

High-performance liquid chromatography

RFLP:

Restriction fragment length polymorphism

RCA:

Rolling circle amplification

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Acknowledgements

We deeply extend our sincere thanks to volunteers and families who kindly contributed samples that were crucial to this study.

Funding

We gratefully acknowledge the Ministry of Science and Technology of Taiwan (MOST 108-2113-M-037-018-MY2) and the Zuoying Branch of Kaohsiung Armed Forces General Hospital (ZBH108-10) who provided financial assistance for our research and the Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung, Taiwan, who offered the opportunities for collaboration in clinical application.

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  1. Hwang-Shang Kou and Kung-Hung Lin contributed equally to this work.

Authors and Affiliations

  1. School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 100, Shi-chuan 1st Rd, Kaohsiung, 807, Taiwan, Republic of China

    Hwang-Shang Kou, Kung-Hung Lin, Ravery Sebuyoya, Cheng-Wei Cheng & Chun-Chi Wang

  2. Department of Surgery, Division of General Surgery, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan, Republic of China

    Kung-Hung Lin

  3. Department of Urology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan, Republic of China

    Kuang-Shun Chueh

  4. Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, Republic of China

    Chun-Chi Wang

  5. Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China

    Chun-Chi Wang

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Kou, HS., Lin, KH., Sebuyoya, R. et al. Dual-probe ligation without PCR for fluorescent sandwich assay of EGFR nucleotide variants in magnetic gene capture platform. Microchim Acta 190, 375 (2023). https://doi.org/10.1007/s00604-023-05950-5

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