Polymerase chain reaction - surface-enhanced Raman spectroscopy (PCR-SERS) method for gene methylation level detection in plasma
3-methyladenine; 5-methylcytosine; aberrant promoter methylation; classification; dna methylation; hypermethylation; methylation level; multiplex detection; pcr; plasma; point mutations; profile; scattering; sers; surface-enhanced Raman spectroscopy
Gene promoter hypermethylation is a vital step in tumorigenesis. This paper set out to explore the use of polymerase chain reaction - surface-enhanced Raman spectroscopy (PCR-SERS) for the detection of gene methylation levels, with a focus on cancer diagnosis. Methods: PCR with methylation independent primers were used on DNA samples to amplify target genes regardless of their methylation states. SERS was used on the obtained PCR products to generate spectra that contained peak changes belonging to CG and AT base pairs. Multiple linear regression (MLR) was then used to deconvolute the SERS spectra so that the CG/AT ratios of the sample could be obtained. These MLR results were used to calculate methylation levels of the target genes. For protocol verification, three sets of seven reference DNA solutions with known methylation levels (0%, 1%, 5%, 25%, 50%, 75%, and 100%) were analysed. Clinically, blood plasma samples were taken from 48 non-small-cell lung cancer (NSCLC) patients and 51 healthy controls. The methylation levels of the genes p16, MGMT, and RASSF1 were determined for each patient using this method. Results: Verification experiment on the mixtures with known methylation levels resulted in an error of less than 6% from the actual levels. When applied to our clinical samples, the frequency of methylation in at least one of the three target genes among the NSCLC patients was 87.5%, but this percentage decreased to 11.8% for the control group. The methylation levels of p16 were found to be significantly higher in NSCLC patients with more pack-years smoked (p=0.04), later cancer stages (p=0.03), and cancer types of squamous cell and large cell versus adenocarcinoma (p=0.03). Prediction accuracy of 88% was achieved from classification and regression trees (CART) based on methylation levels and states, respectively. Conclusion: This research showed that the PCR-SERS protocol could quantitatively measure the methylation levels of genes in plasma. The methylation levels of the genes p16, MGMT, and RASSF1 were higher in NSCLC patients than in controls.
Li Xiaozhou; Yang Tianyue; Li Caesar Siqi; Song Youtao; Wang Deli; Jin Lili; Lou Hong; Li Wei
Theranostics
2020
2020
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
journalArticle
<a href="http://doi.org/10.7150/thno.30204" target="_blank" rel="noreferrer noopener">10.7150/thno.30204</a>
Surface Enhanced Raman Spectroscopy (SERS) for the Multiplex Detection of Braf, Kras, and Pik3ca Mutations in Plasma of Colorectal Cancer Patients.
Adult; Female; Humans; Male; Middle Aged; Aged; *colorectal cancer; *gene; *mutation; *Mutation; *PCR; *surface enhanced Raman scattering; Class I Phosphatidylinositol 3-Kinases/blood/*genetics; Cluster Analysis; Colorectal Neoplasms/blood/*diagnosis/genetics/pathology; DNA Primers/chemistry; Exons; Multiplex Polymerase Chain Reaction/methods/standards; Neoplasm Staging; Proto-Oncogene Proteins B-raf/blood/*genetics; Proto-Oncogene Proteins p21(ras)/blood/*genetics; Biomarkers; Spectrum Analysis; Raman/methods/*standards; Tumor/blood/*genetics
In this paper, we discuss the use of a procedure based on polymerase chain reaction (PCR) and surface enhanced Raman spectroscopy (SERS) (PCR-SERS) to detect DNA mutations. Methods: This method was implemented by first amplifying
Li Xiaozhou; Yang Tianyue; Li Caesar Siqi; Song Youtao; Lou Hong; Guan Dagang; Jin Lili
Theranostics
2018
1905-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.7150/thno.22502" target="_blank" rel="noreferrer noopener">10.7150/thno.22502</a>
Prenatal detection of thalassemia by cell-free fetal DNA (cffDNA) in maternal plasma using surface enhanced Raman spectroscopy combined with PCR.
(170.1470) Blood or tissue constituent monitoring; Raman; (300.6450) Spectroscopy
Thalassemias are widely occurring genetic hemoglobin disorders; patients with severe thalassemia often require regular blood transfusions for survival. Prenatal detection of thalassemia is currently invasive and carries the risk of miscarriage and infection. A polymerase chain reaction (PCR)-based surface enhanced Raman spectroscopy (SERS) technique was investigated in this paper for the purpose of detecting prenatal alpha-thalassemia Southeast Asian (SEA) type deletion using maternal plasma. Couples with the same SEA thalassemia (-SEA/alphaalpha) were selected, and the quantification of SEA and wild type (WT) alleles in the maternal plasma sample predicted the fetal genotype. PCR was performed using two pairs of fluorescence tag-labeled primers to produce tag-labeled PCR products for both the SEA (labeled with R6G) and WT (labeled with Cy3) alleles. Then, the labeled PCR products containing the two fluorescence tags were measured by SERS. The ratios between the R6G and Cy3 tags were obtained using multiple linear regressions (MLR), and these ratios corresponded with the physical ratio of WT and SEA concentrations in maternal plasma. After verifying this technique on DNA mixtures with known SEA and WT ratios, the plasma from 24 pregnant women was screened. An accuracy of 91.7% was achieved for detecting the fetal genotypes of Hb Bart's, alpha-trait, and normal trait. The results indicated that the simple PCR-SERS method may be sensitive enough for use on cell free fetal DNA (cffDNA) in maternal plasma for non-invasive prenatal detection (NIPD).
Li Xiaozhou; Yang Tianyue; Li Caesar Siqi; Jin Lili; Lou Hong; Song Youtao
Biomedical optics express
2018
2018-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1364/BOE.9.003167" target="_blank" rel="noreferrer noopener">10.1364/BOE.9.003167</a>
Detection of EGFR mutation in plasma using multiplex allele-specific PCR (MAS-PCR) and surface enhanced Raman spectroscopy.
In this study, surface enhanced Raman spectroscopy (SERS) in combination with multiplexed polymerase chain reaction (PCR) was utilized to detect mutations of exons 19 and 21 of the epidermal growth factor receptor (EGFR) gene. Through the use of multiplexed PCR, the two mutation types were amplified in a single reaction. SERS was used on the PCR products to detect mutations. DNA mixtures with increasing mutation percentages showed good linear relationship between mutation rates and peak height. Then, this PCR-SERS method was used on the plasma of 48 patients with non-small cell lung cancer (NSCLC) to detect EGFR mutations. Analysis of variance (ANOVA) and receiver operating characteristic (ROC) analysis revealed that the peak height ratios were significant for identifying different mutation types. The specificity, sensitivity and accuracy obtained were all 100%. The proposed method was then validated through comparison with high resolution melting (HRM) and showed high concordance with HRM (Pearson correlation is 0.92). Finally, logistic regression was performed on EGFR mutation status and the clinical features of the 48 patients. Our study indicates that PCR-SERS is an effective, noninvasive, and economical method for the detection and monitoring of EGFR mutations in the plasma of patients with NSCLC.
Li Xiaozhou; Yang Tianyue; Li Caesar Siqi; Wang Deli; Song Youtao; Jin Lili
Scientific reports
2017
2017-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1038/s41598-017-05050-4" target="_blank" rel="noreferrer noopener">10.1038/s41598-017-05050-4</a>