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Biotechnology Letters

, Volume 38, Issue 10, pp 1709–1714 | Cite as

Lateral flow strip for visual detection of K-ras mutations based on allele-specific PCR

  • Cong Wang
  • Xiaomin Chen
  • Yuying Wu
  • Hao Li
  • Yu Wang
  • Xiaofu Pan
  • Tingting Tang
  • Ziying Liu
  • Xiaokun LiEmail author
Original Research Paper

Abstract

Objectives

To develop a convenient and sensitive point-of-care test for detecting gene mutations based on allele-specific PCR.

Results

To develop a lateral flow strip for visual detection of K-ras mutations based on a modified PCR, a specific DNA tag was covalently linked to the 5′-end of each primer by a nine-carbon linker to produce a sticky end. One of the sticky ends of the PCR products bound to gold nano-particles, while the other sticky end was captured onto a nitrocellulose membrane of lateral flow strips. The lateral flow strip showed a great sensitivity, which detected mutations in as low as 10 tumor cells. The positive rate and accuracy of the lateral flow strip for blood samples were over 92 and 96 %, respectively.

Conclusions

The lateral flow strip provides an easy method for sensitive detection of gene mutations based on allele specific-PCR.

Keywords

Detection of mutations Gene mutation K-ras mutations Lateral flow strip Mutation detection Point-of-care tests 

Notes

Acknowledgments

This work was supported by the Natural Science Foundation of Zhejiang Province of China (LY16H140004, Y2110492) to CW, the National Natural Science Foundation of China (81101712, 81270761, 31371470) to CW, the Natural Science Foundation of Ningbo city (2015A610198), and the Talent project foundation of Wenzhou Medical University (QTJ14030).

Supporting information

Supplementary Table 1—Primer sequence.

Compliance with ethical standard

Conflict of interest

The authors confirm that this article content has no conflict of interest.

Supplementary material

10529_2016_2161_MOESM1_ESM.doc (40 kb)
Supplementary material 1 (DOC 40 kb)

References

  1. Adjei AA (2001) Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 93:1062–1074CrossRefPubMedGoogle Scholar
  2. Amary MF et al (2007) Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool. Am J Surg Pathol 31:1299–1309CrossRefPubMedGoogle Scholar
  3. Ausch C et al (2009) Sensitive detection of KRAS mutations in archived formalin-fixed paraffin-embedded tissue using mutant-enriched PCR and reverse-hybridization. J Mol Diagn 11:508–513CrossRefPubMedPubMedCentralGoogle Scholar
  4. Buxhofer-Ausch V, Ausch C, Zeillinger R, Oberkanins C, Dandachi N, Reiner-Concin A, Kriegshauser G (2013) Duplex reverse-hybridization assay for the simultaneous detection of KRAS/BRAF mutations in FFPE-extracted genomic DNA from colorectal cancer specimens. Dis Markers 34:171–177CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chretien AS et al (2013) Optimization of routine KRAS mutation PCR-based testing procedure for rational individualized first-line-targeted therapy selection in metastatic colorectal cancer. Cancer Med 2:11–20CrossRefPubMedPubMedCentralGoogle Scholar
  6. Fang Z, Wu W, Lu X, Zeng L (2014) Lateral flow biosensor for DNA extraction-free detection of Salmonella based on aptamer mediated strand displacement amplification. Biosens Bioelectron 56:192–197CrossRefPubMedGoogle Scholar
  7. French D, Smith A, Powers MP, Wu AH (2011) KRAS mutation detection in colorectal cancer by a commercially available gene chip array compares well with Sanger sequencing. Clin Chim Acta 412:1578–1581CrossRefPubMedGoogle Scholar
  8. Harb W, Fan A, Tran T, Danila DC, Keys D, Schwartz M, Ionescu-Zanetti C (2013) Mutational analysis of circulating tumor cells using a novel microfluidic collection device and qPCR assay. Transl Oncol 6:528–538CrossRefPubMedPubMedCentralGoogle Scholar
  9. Harle A, Busser B, Rouyer M, Harter V, Genin P, Leroux A, Merlin JL (2013) Comparison of COBAS 4800 KRAS, TaqMan PCR and high resolution melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed paraffin embedded colorectal carcinomas. Virchows Arch 462:329–335CrossRefPubMedGoogle Scholar
  10. Heideman DA et al (2012) KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA. J Mol Diagn 14:247–255CrossRefPubMedGoogle Scholar
  11. Huang T et al (2014) Highly sensitive enumeration of circulating tumor cells in lung cancer patients using a size-based filtration microfluidic chip. Biosens Bioelectron 51:213–218CrossRefPubMedGoogle Scholar
  12. Jafari H, Gharemohammadlou R, Fakhrjou A, Ebrahimi A, Nejati-Koshki K, Nadri M, Sakhinia E (2013) Genotyping of human papillomavirus and TP53 mutations at exons 5 to 7 in lung cancer patients from Iran. Bioimpacts 3:135–140PubMedPubMedCentralGoogle Scholar
  13. Kawamata N et al (2009) Identified hidden genomic changes in mantle cell lymphoma using high-resolution single nucleotide polymorphism genomic array. Exp Hematol 37:937–946CrossRefPubMedGoogle Scholar
  14. Mok SC et al (1993) Mutation of K-ras protooncogene in human ovarian epithelial tumors of borderline malignancy. Cancer Res 53:1489–1492PubMedGoogle Scholar
  15. Rossle M, Sigg M, Ruschoff JH, Wild PJ, Moch H, Weber A, Rechsteiner MP (2013) Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF mutations in colorectal carcinoma. Virchows Arch 5:623–631CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Cong Wang
    • 1
  • Xiaomin Chen
    • 1
  • Yuying Wu
    • 1
  • Hao Li
    • 1
  • Yu Wang
    • 1
  • Xiaofu Pan
    • 2
  • Tingting Tang
    • 2
  • Ziying Liu
    • 1
  • Xiaokun Li
    • 1
    Email author
  1. 1.School of PharmacyWenzhou Medical UniversityWenzhouChina
  2. 2.The Third Affiliated Hospital of Wenzhou Medical UniversityRuianChina

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