66
Views
13
CrossRef citations to date
0
Altmetric
Original Research

Real-time colorimetric detection of DNA methylation of the PAX1 gene in cervical scrapings for cervical cancer screening with thiol-labeled PCR primers and gold nanoparticles

, , , , &
Pages 5335-5347 | Published online: 12 Oct 2016
 

Abstract

Background

DNA methylation can induce carcinogenesis by silencing key tumor suppressor genes. Analysis of aberrant methylation of tumor suppressor genes can be used as a prognostic and predictive biomarker for cancer. In this study, we propose a colorimetric method for the detection of DNA methylation of the paired box gene 1 (PAX1) gene in cervical scrapings obtained from 42 patients who underwent cervical colposcopic biopsy.

Methods

A thiolated methylation-specific polymerase chain reaction (MSP) primer was used to generate MSP products labeled with the thiol group at one end. After bisulfite conversion and MSP amplification, the unmodified gold nanoparticles (AuNPs) were placed in a reaction tube and NaCl was added to induce aggregation of bare AuNPs without generating polymerase chain reaction products. After salt addition, the color of AuNPs remained red in the methylated PAX1 gene samples because of binding to the MSP-amplified products. By contrast, the color of the AuNP colloid solution changed from red to blue in the non-methylated PAX1 gene samples because of aggregation of AuNPs in the absence of the MSP-amplified products. Furthermore, PAX1 methylation was quantitatively detected in cervical scrapings of patients with varied pathological degrees of cervical cancer. Conventional quantitative MSP (qMSP) was also performed for comparison.

Results

The two methods showed a significant correlation of the methylation frequency of the PAX1 gene in cervical scrapings with severity of cervical cancer (n=42, P<0.05). The results of the proposed method showed that the areas under the receiver operating characteristic curve (AUCs) of PAX1 were 0.833, 0.742, and 0.739 for the detection of cervical intraepithelial neoplasms grade 2 and worse lesions (CIN2+), cervical intraepithelial neoplasms grade 3 and worse lesions (CIN3+), and squamous cell carcinoma, respectively. The sensitivity and specificity for detecting CIN2+ lesions were 0.941 and 0.600, respectively, with a cutoff value of 31.27%. The proposed method also showed superior sensitivity over qMSP methods for the detection of CIN2+ and CIN3+ (0.941 vs 0.824 and 1.000 vs 0.800, respectively). Furthermore, the novel method exhibited higher AUC (0.833) for the detection of CIN2+ than qMSP (0.807).

Conclusion

The results of thiol-labeled AuNP method were clearly observed by the naked eyes without requiring any expensive equipment. Therefore, the thiol-labeled AuNP method could be a simple but efficient strategy for cervical cancer screening.

Supplementary materials

Figure S1 Characterization of self-synthesized 13 nm gold nanoparticles.

Notes: (A) TEM image of 13 nm gold nanoparticles. Scale bar: 50 nm. Inset image shows a single 13 nm gold nanoparticle. (B) UV–vis spectrum of 13 nm gold nanoparticles.

Abbreviations: TEM, transmission electron microscope; UV-vis, ultraviolet-visible spectrophotometry; OD, optical density.

Figure S1 Characterization of self-synthesized 13 nm gold nanoparticles.Notes: (A) TEM image of 13 nm gold nanoparticles. Scale bar: 50 nm. Inset image shows a single 13 nm gold nanoparticle. (B) UV–vis spectrum of 13 nm gold nanoparticles.Abbreviations: TEM, transmission electron microscope; UV-vis, ultraviolet-visible spectrophotometry; OD, optical density.

Figure S2 Influence of thiol-labeling of primer of PAX1 gene on PCR efficiency. PCR was performed by using both unlabeled primers (lane1), thiol-labeled forward primer and unlabeled reverse primer (lane 2).

Abbreviation: PCR, polymerase chain reaction.

Figure S2 Influence of thiol-labeling of primer of PAX1 gene on PCR efficiency. PCR was performed by using both unlabeled primers (lane1), thiol-labeled forward primer and unlabeled reverse primer (lane 2).Abbreviation: PCR, polymerase chain reaction.

Figure S3 MSP results in DNA agarose gel for PAX1 gene in selected individual normal, CIN1, CIN2, CIN3 and SCC samples.

Notes: 1-normal, 2-CIN1, 3-CIN2, 4-CIN3, 5-SCC.

Abbreviations: CIN1, cervical intraepithelial neoplasia type 1, CIN2, cervical intraepithelial neoplasia type 2; CIN3, cervical intraepithelial neoplasia type 3; MSP, methylation-specific polymerase chain reaction; M, methylation-specific primers; SCC, squamous cell carcinoma; U, nonmethylation-specific primers.

Figure S3 MSP results in DNA agarose gel for PAX1 gene in selected individual normal, CIN1, CIN2, CIN3 and SCC samples.Notes: 1-normal, 2-CIN1, 3-CIN2, 4-CIN3, 5-SCC.Abbreviations: CIN1, cervical intraepithelial neoplasia type 1, CIN2, cervical intraepithelial neoplasia type 2; CIN3, cervical intraepithelial neoplasia type 3; MSP, methylation-specific polymerase chain reaction; M, methylation-specific primers; SCC, squamous cell carcinoma; U, nonmethylation-specific primers.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No 81503563).

Disclosure

The authors report no conflicts of interest in this work.