Analytical and Bioanalytical Chemistry

, Volume 411, Issue 26, pp 6877–6887 | Cite as

Real-time fluorescence loop-mediated isothermal amplification assay for rapid and sensitive detection of Streptococcus gallolyticus subsp. gallolyticus associated with colorectal cancer

  • Qiuyuan Lin
  • Xin Ye
  • Bin Yang
  • Xueen Fang
  • Hui ChenEmail author
  • Wenhao WengEmail author
  • Jilie KongEmail author
Research Paper


The increasing threat of Streptococcus gallolyticus subsp. gallolyticus (SGG) infections has gained considerable attention for its strong association with colorectal cancer (CRC). Herein, we proposed real-time fluorescence loop-mediated isothermal amplification (LAMP) as a novel, simple, rapid, and highly sensitive assay for identifying SGG for the first time. This assay was capable of detecting SGG with initial DNA concentrations ranging from 102 to 108 copies per microliter, under isothermal conditions within 30 min via real-time fluorescence monitoring. Our method was tested for specific identification of SGG strains without cross-reaction with other Streptococcus gallolyticus subspecies and Escherichia coli. The developed LAMP shows a superior performance with shorter time and higher sensitivity compared with conventional polymerase chain reaction (PCR). Significantly, this proposed approach was successfully applied for detecting SGG in clinical urine samples, which is non-invasive diagnosis, showing excellent accuracy and reliability to discriminate healthy controls and CRC patients. For comparison, these samples were also tested against PCR assay. These results yielded an analytical sensitivity of 100% and a specificity of 100% for SGG testing using LAMP. The findings suggest LAMP can be employed for detecting SGG infections which is useful for diagnosis and screening of CRC.


Loop-mediated isothermal amplification Streptococcus gallolyticus subsp. gallolyticus Colorectal cancer Clinical diagnostic 



The authors gratefully acknowledge the Department of Clinical Laboratory, Yangpu Hospital (Shanghai, China), for the help in collecting clinical urine samples.

Funding information

This work was funded by the National Natural Science Foundation of China (21375027, 21335002, 21427806), Shanghai Pujiang Program (17PJD001, 18PJD047), and Natural Science Foundation of Shanghai (12ZR1401700, 17JC1400100).

Compliance with ethical standards

All participants in this study provided written informed consent prior to testing. The study design was approved by the institutional research commission of Fudan University (Shanghai, China). Ethical approval was obtained from the Ethics Committee of the Department of Clinical Laboratory, Yangpu Hospital (Shanghai, China).

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2019_2059_MOESM1_ESM.pdf (106 kb)
ESM 1 (PDF 76 kb)


  1. 1.
    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424.CrossRefGoogle Scholar
  2. 2.
    Sears CL, Garrett WS. Microbes, microbiota, and colon cancer. Cell Host Microbe. 2014;15(3):317–28.CrossRefGoogle Scholar
  3. 3.
    Louis P, Hold GL, Flint HJ. The gut microbiota, bacterial metabolites and colorectal cancer. Nat Rev Microbiol. 2014;12(10):661–72.CrossRefGoogle Scholar
  4. 4.
    Pasquereau-Kotula E, Martins M, Aymeric L, Dramsi S. Significance of Streptococcus gallolyticus subsp. gallolyticus Association With Colorectal Cancer. Front Microbiol. 2018;9:614.CrossRefGoogle Scholar
  5. 5.
    Boleij A, Tjalsma H. Gut bacteria in health and disease: a survey on the interface between intestinal microbiology and colorectal cancer. Biol Rev Camb Philos Soc. 2012;87(3):701–30.CrossRefGoogle Scholar
  6. 6.
    Abdulamir AS, Hafidh RR, Bakar FA. Molecular detection, quantification, and isolation of Streptococcus gallolyticus bacteria colonizing colorectal tumors: inflammation-driven potential of carcinogenesis via IL-1, COX-2, and IL-8. Mol Cancer. 2010;9(1):249.CrossRefGoogle Scholar
  7. 7.
    Boleij A, Muytjens CM, Bukhari SI, Cayet N, Glaser P, Hermans PW, et al. Novel clues on the specific association of Streptococcus gallolyticus subsp gallolyticus with colorectal cancer. J Infect Dis. 2011;203(8):1101–9.CrossRefGoogle Scholar
  8. 8.
    Boleij A, van Gelder MM, Swinkels DW, Tjalsma H. Clinical importance of Streptococcus gallolyticus infection among colorectal cancer patients: systematic review and meta-analysis. Clin Infect Dis. 2011;53(9):870–8.CrossRefGoogle Scholar
  9. 9.
    Boleij A, Roelofs R, Danne C, Bellais S, Dramsi S, Kato I, et al. Selective antibody response to Streptococcus gallolyticus pilus proteins in colorectal cancer patients. Cancer Prev Res (Phila). 2012;5(2):260–5.CrossRefGoogle Scholar
  10. 10.
    Butt J, Romero-Hernandez B, Perez-Gomez B, Willhauck-Fleckenstein M, Holzinger D, Martin V, et al. Association of Streptococcus gallolyticus subspecies gallolyticus with colorectal cancer: serological evidence. Int J Cancer. 2016;138(7):1670–9.CrossRefGoogle Scholar
  11. 11.
    Butt J, Werner S, Willhauck-Fleckenstein M, Michel A, Waterboer T, Zornig I, et al. Serology of Streptococcus gallolyticus subspecies gallolyticus and its association with colorectal cancer and precursors. Int J Cancer. 2017;141(5):897–904.CrossRefGoogle Scholar
  12. 12.
    Butt J, Jenab M, Willhauck-Fleckenstein M, Michel A, Pawlita M, Kyro C, et al. Prospective evaluation of antibody response to Streptococcus gallolyticus and risk of colorectal cancer. Int J Cancer. 2018;143(2):245–52.CrossRefGoogle Scholar
  13. 13.
    Kumar R, Herold JL, Schady D, Davis J, Kopetz S, Martinez-Moczygemba M, et al. Streptococcus gallolyticus subsp. gallolyticus promotes colorectal tumor development. PLoS Pathog. 2017;13(7):e1006440.CrossRefGoogle Scholar
  14. 14.
    Abdulamir AS, Hafidh RR, Mahdi LK, Al-jeboori T, Abubaker F. Investigation into the controversial association of Streptococcus gallolyticus with colorectal cancer and adenoma. BMC Cancer. 2009;9:403.CrossRefGoogle Scholar
  15. 15.
    Corredoira-Sánchez J, García-Garrote F, Rabuñal R, López-Roses L, García-País MJ, Castro E, et al. Association between bacteremia due to Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis I) and colorectal neoplasia: a case-control study. Clin Infect Dis. 2012;55(4):491–6.CrossRefGoogle Scholar
  16. 16.
    Farooq U, Yang Q, Ullah MW, Wang S. Bacterial biosensing: recent advances in phage-based bioassays and biosensors. Biosens Bioelectron. 2018;118:204–16.CrossRefGoogle Scholar
  17. 17.
    Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology. 2002;148(1):257–66.CrossRefGoogle Scholar
  18. 18.
    Zhao Y, Chen F, Li Q, Wang L, Fan C. Isothermal amplification of nucleic acids. Chem Rev. 2015;115(22):12491–545.CrossRefGoogle Scholar
  19. 19.
    Reid MS, Le XC, Zhang H. Exponential isothermal amplification of nucleic acids and assays for proteins, cells, small molecules, and enzyme activities: an EXPAR example. Angew Chem Int Ed. 2018;57(37):11856–66.CrossRefGoogle Scholar
  20. 20.
    Notomi T, Mori Y, Tomita N, Kanda H. Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. J Microbiol. 2015;53(1):1–5.CrossRefGoogle Scholar
  21. 21.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):e63.CrossRefGoogle Scholar
  22. 22.
    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.CrossRefGoogle Scholar
  23. 23.
    Zhao Y, Chen F, Qin J, Wei J, Wu W, Zhao Y. Engineered Janus probes modulate nucleic acid amplification to expand the dynamic range for direct detection of viral genomes in one microliter crude serum samples. Chem Sci. 2018;9(2):392–7.CrossRefGoogle Scholar
  24. 24.
    Yu J, Wang F, Zhan X, Wang X, Zuo F, Wei Y, et al. Improvement and evaluation of loop-mediated isothermal amplification combined with a chromatographic flow dipstick assay and utilization in detection of Vibrio cholerae. Anal Bioanal Chem. 2019;411(3):647–58.CrossRefGoogle Scholar
  25. 25.
    Loo JF, But GW, Kwok HC, Lau PM, Kong SK, Ho HP, et al. A rapid sample-to-answer analytical detection of genetically modified papaya using loop-mediated isothermal amplification assay on lab-on-a-disc for field use. Food Chem. 2019;274:822–30.CrossRefGoogle Scholar
  26. 26.
    Zahradnik C, Martzy R, Mach RL, Krska R, Farnleitner AH, Brunner K. Detection of the food allergen celery via loop-mediated isothermal amplification technique. Anal Bioanal Chem. 2014;406(27):6827–33.CrossRefGoogle Scholar
  27. 27.
    Wang H, Ma Z, Qin J, Shen Z, Liu Q, Chen X, et al. A versatile loop-mediated isothermal amplification microchip platform for Streptococcus pneumoniae and Mycoplasma pneumoniae testing at the point of care. Biosens Bioelectron. 2019;126:373–80.CrossRefGoogle Scholar
  28. 28.
    Zhang L, Tian F, Liu C, Feng Q, Ma T, Zhao Z, et al. Hand-powered centrifugal microfluidic platform inspired by the spinning top for sample-to-answer diagnostics of nucleic acids. Lab Chip. 2018;18(4):610–9.CrossRefGoogle Scholar
  29. 29.
    Phillips EA, Moehling TJ, Bhadra S, Ellington AD, Linnes JC. Strand displacement probes combined with isothermal nucleic acid amplification for instrument-free detection from complex samples. Anal Chem. 2018;90(11):6580–6.CrossRefGoogle Scholar
  30. 30.
    Du Y, Pothukuchy A, Gollihar JD, Nourani A, Li B, Ellington AD. Coupling sensitive nucleic acid amplification with commercial pregnancy test strips. Angew Chem Int Ed. 2017;56(4):992–6.CrossRefGoogle Scholar
  31. 31.
    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.CrossRefGoogle Scholar
  32. 32.
    Lopes PG, Cantarelli VV, Agnes G, Costabeber AM, d'Azevedo PA. Novel real-time PCR assays using TaqMan minor groove binder probes for identification of fecal carriage of Streptococcus bovis/Streptococcus equinus complex from rectal swab specimens. J Clin Microbiol. 2014;52(3):974–6.CrossRefGoogle Scholar
  33. 33.
    Hatrongjit R, Akeda Y, Hamada S, Gottschalk M, Kerdsin A. Multiplex PCR for identification of six clinically relevant streptococci. J Med Microbiol. 2017;66(11):1590–5.CrossRefGoogle Scholar
  34. 34.
    Cao G, Kong J, Xing Z, Tang Y, Zhang X, Xu X, et al. Rapid detection of CALR type 1 and type 2 mutations using PNA-LNA clamping loop-mediated isothermal amplification on a CD-like microfluidic chip. Anal Chim Acta. 2018;1024:123–35.CrossRefGoogle Scholar
  35. 35.
    Dong J, Xu Q, Li CC, Zhang CY. Single-color multiplexing by the integration of high-resolution melting pattern recognition with loop-mediated isothermal amplification. Chem Commun. 2019;55(17):2457–60.CrossRefGoogle Scholar
  36. 36.
    Jans C, Meile L, Lacroix C, Stevens MJ. Genomics, evolution, and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC). Infect Genet Evol. 2015;33:419–36.CrossRefGoogle Scholar
  37. 37.
    Schlegel L, Grimont F, Ageron E, Grimont PA, Bouvet A. Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. gallolyticus subsp. pasteurianus subsp. nov. Int J Syst Evol Microbiol. 2003;53(3):631–45.CrossRefGoogle Scholar
  38. 38.
    Chen J, Xu Y, Yan H, Zhu Y, Wang L, Zhang Y, et al. Sensitive and rapid detection of pathogenic bacteria from urine samples using multiplex recombinase polymerase amplification. Lab Chip. 2018;18(16):2441–52.CrossRefGoogle Scholar
  39. 39.
    Li C, Li Z, Jia H, Yan J. One-step ultrasensitive detection of microRNAs with loop-mediated isothermal amplification (LAMP). Chem Commun. 2011;47(9):2595–7.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistryFudan UniversityShanghaiChina
  2. 2.Department of Clinical Laboratory, Yangpu HospitalTongji University School of MedicineShanghaiChina

Personalised recommendations