title: Real-time colorimetric detection of DNA methylation of thePAX1gene in cervical scrapings for cervical cancer screening with thiol-labeled PCR primers and gold nanoparticles.
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 methylatedPAX1gene 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-methylatedPAX1gene samples because of aggregation of AuNPs in the absence of the MSP-amplified products. Furthermore,PAX1methylation 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 thePAX1gene 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) ofPAX1were 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).
  
  
  CONCLUSIONS
  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.
authors: Department of Clinical Pharmacology, Xiangya Hospital, Central South University; Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University.
authors: Jin
authors: J
authors: Huang
authors: Department of Clinical Pharmacology, Xiangya Hospital, Central South University; Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University.
authors: Yu-Ligh
authors: YL
authors: Liou
authors: Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China.
authors: Ya-Nan
authors: YN
authors: Kang
authors: Department of Clinical Pharmacology, Xiangya Hospital, Central South University; Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University.
authors: Zhi-Rong
authors: ZR
authors: Tan
authors: Department of Clinical Pharmacology, Xiangya Hospital, Central South University; Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University.
authors: Ming-Jing
authors: MJ
authors: Peng
authors: Department of Clinical Pharmacology, Xiangya Hospital, Central South University; Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University.
authors: Hong-Hao
authors: HH
authors: Zhou
mesh_terms: D000328:Adult; D014408:Biomarkers, Tumor / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods*; D019175:DNA Methylation; D017931:DNA Primers; D055088:Early Detection of Cancer / Q000379:methods; D005260:Female; D006046:Gold; D006801:Humans; D008875:Middle Aged; D053758:Nanoparticles / Q000737:chemistry; D051761:Paired Box Transcription Factors / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011379:Prognosis; D012372:ROC Curve; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology*; D002578:Uterine Cervical Dysplasia / Q000235:genetics / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; D017931:DNA Primers; D051761:Paired Box Transcription Factors; C070835:PAX1 transcription factor; D006046:Gold
keywords: DNA methylation; UV-vis; cervical cancer screening; colorimetric detection; gold nanoparticles; high sensitivity; quantitative detection