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

Jin Huang,1,2 Yu-Ligh Liou,1,2 Ya-Nan Kang,3 Zhi-Rong Tan,1,2 Ming-Jing Peng,1,2 Hong-Hao Zhou1,2 1Department of Clinical Pharmacology, Xiangya Hospital, Central South University, 2Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, 3Department of...

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Autores principales:	Huang J, Liou YL, Kang YN, Tan ZR, Peng MJ, Zhou HH
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2016
Materias:	colorimetric detection gold nanoparticles PAX1 DNA methylation cervical cancer Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/75ecaf3f3c0e41529cfb9b8901a3fae1
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spelling	oai:doaj.org-article:75ecaf3f3c0e41529cfb9b8901a3fae12021-12-02T03:56:41ZReal-time colorimetric detection of DNA methylation of the PAX1 gene in cervical scrapings for cervical cancer screening with thiol-labeled PCR primers and gold nanoparticles1178-2013https://doaj.org/article/75ecaf3f3c0e41529cfb9b8901a3fae12016-10-01T00:00:00Zhttps://www.dovepress.com/real-time-colorimetric-detection-of-dna-methylation-of-the-pax1-gene-i-peer-reviewed-article-IJNhttps://doaj.org/toc/1178-2013Jin Huang,1,2 Yu-Ligh Liou,1,2 Ya-Nan Kang,3 Zhi-Rong Tan,1,2 Ming-Jing Peng,1,2 Hong-Hao Zhou1,2 1Department of Clinical Pharmacology, Xiangya Hospital, Central South University, 2Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, 3Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China 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. Keywords: colorimetric detection, gold nanoparticles, DNA methylation, cervical cancer screening, UV-vis, high sensitivity, quantitative detectionHuang JLiou YLKang YNTan ZRPeng MJZhou HHDove Medical Pressarticlecolorimetric detectiongold nanoparticlesPAX1DNA methylationcervical cancerMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol Volume 11, Pp 5335-5347 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	colorimetric detection gold nanoparticles PAX1 DNA methylation cervical cancer Medicine (General) R5-920
spellingShingle	colorimetric detection gold nanoparticles PAX1 DNA methylation cervical cancer Medicine (General) R5-920 Huang J Liou YL Kang YN Tan ZR Peng MJ Zhou HH 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
description	Jin Huang,1,2 Yu-Ligh Liou,1,2 Ya-Nan Kang,3 Zhi-Rong Tan,1,2 Ming-Jing Peng,1,2 Hong-Hao Zhou1,2 1Department of Clinical Pharmacology, Xiangya Hospital, Central South University, 2Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, 3Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China 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. Keywords: colorimetric detection, gold nanoparticles, DNA methylation, cervical cancer screening, UV-vis, high sensitivity, quantitative detection
format	article
author	Huang J Liou YL Kang YN Tan ZR Peng MJ Zhou HH
author_facet	Huang J Liou YL Kang YN Tan ZR Peng MJ Zhou HH
author_sort	Huang J
title	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
title_short	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
title_full	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
title_fullStr	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
title_full_unstemmed	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
title_sort	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
publisher	Dove Medical Press
publishDate	2016
url	https://doaj.org/article/75ecaf3f3c0e41529cfb9b8901a3fae1
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_version_	1718401558432972800

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

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