A high-throughput microsphere-based immunoassay of anti-SARS-CoV-2 IgM testing for COVID-19 diagnostics
2021 | Publications
The pandemic of novel coronavirus disease COVID-19 is rapidly expanding across the world. A positive result of antibody tests suggests that the individual has potentially been exposed to SARS-CoV-2, thus allowing to identify asymptomatic infections and determine the seroprevalence in a given population. The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgM antibody detection on the Luminex MAGPIX platform. Clinical agreement studies were performed in 42 COVID-19 patient serum samples and 162 negative donor serum/plasma samples. Positive percent agreement (PPA) was 42.86% (95% CI: 9.90% to 81.59%), 71.43% (95% CI: 29.04% to 96.33%), and 28.57% (95% CI: 13.22% to 48.67%) for samples collected on 0–7 days, 8–14 days, and 2–8 weeks from symptom onset, respectively. Negative Percent Agreement (NPA) was 97.53% (95% CI: 93.80% to 99.32%). There was no cross-reactivity with the SARS-CoV-2 IgG antibody. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL), triglyceride (250 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. In conclusion, an anti-SARS-CoV-2 IgM antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up the anti-SARS-CoV-2 IgM testing.
Authors: Dayu Zhang, Tianyang Xu, Eric Chu, Aiguo Zhang, Jinwei Du, Michael Y. Sha.
Kinetics of plasma cfDNA predicts clinical response in non‑small cell lung cancer patients
2021 | Publications
Tyrosine kinase inhibitors (TKIs), VEGF/VEGF receptor inhibitors (VEGFIs) and immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers including non‑small‑cell lung cancer (NSCLC). This study aims to evaluate the utility of plasma cell‑free DNA (cfDNA) as a prognostic biomarker and efficacy predictor of chemotherapy (CT) with or without these precision therapies in NSCLC patients. Peripheral cfDNA levels in 154 NSCLC patients were quantified before and after the first target cycle of chemotherapy. The correlations of cfDNA with tumor burden, clinical characteristics, progression‑free survival (PFS)/disease‑free survival (DFS), objective response ratio (ORR), and therapy regimens were analyzed respectively. Baseline cfDNA, but not post‑chemotherapeutic cfDNA, positively correlates with tumor burden. Notably, cfDNA kinetics (cfDNA Ratio, the ratio of post‑chemotherapeutic cfDNA to baseline cfDNA) well distinguished responsive individuals (CR/PR) from the non‑responsive (PD/SD). Additionally, cfDNA Ratio was found negatively correlated with PFS in lung adenocarcinoma (LUAD), but not lung squamous‑cell carcinoma (LUSC) which may be due to a limited number of LUSC patients in this cohort. LUAD patients with low cfDNA Ratio have prolonged PFS and improved ORR, compared to those with high cfDNA Ratio. When stratified by therapy regimen, the predictive value of cfDNA Ratio is significant in patients with chemotherapy plus VEGFIs, while more patients need be included to validate the value of cfDNA Ratio in other regimens. Thus, the kinetics of plasma cfDNA during chemotherapy may function as a prognostic biomarker and efficacy predictor for NSCLC patients.
Authors: Xiaorong Zhou, Chenchen Li, Zhao Zhang, Daniel Y. Li, Jinwei Du, Ping Ding, Haiyan Meng, Hui Xu, Ronglei Li, Effie Ho, Aiguo Zhang, Paul Okunieff, Jianwei Lu & Michael Y. Sha
Source: www.nature.com/scientificreport. (2021) 11:7633. https://doi.org/10.1038/s41598-021-85797-z.
Plasma cfDNA as a Potential Biomarker to Evaluate the Efficacy of Chemotherapy in Gastric Cancers
2020 | Publications
Objective: To investigate the clinical value of plasma cell-free DNA (cfDNA) as a potential biomarker for advanced gastric cancer (GC). Patients and Methods: One hundred and six cases of advanced gastric cancer patients receiving chemotherapy were selected as study objects. Another 40 healthy volunteers were included as control groups. Plasma cfDNA concentration was detected by (SuperbDNATM) hybridization. Changes in cfDNA concentration during chemotherapy in patients with gastric cancer whose efficacy was assessed as partial response (PR), stable disease (SD) and disease progression (PD) were analyzed respectively. The relationship between the level of cfDNA and the efficacy of chemotherapy and clinical characteristics was also explored. In addition, cfDNA and other tumor markers were subjected to specificity and sensitivity analyses using ROC. Results: cfDNA concentration in advanced GC patients was significantly higher than that in healthy controls (P<0.05). The concentration of plasma cfDNA in patients with PD showed an increasing trend over time. The concentration of plasma cfDNA in patients with therapeutic effect of PR decreased over time. In patients with therapeutic effect of SD, the plasma DNA concentration showed a stable trend over time. There was no significant correlation between cfDNA concentration and factors including gender, age, pathological type, CA724, CA125,CA199, AFP and CEA. ROC results showed that the area under the curve of cfDNA was larger than other tumor markers. Conclusion: Plasma cfDNA concentration was significantly increased in patients with gastric cancer, and its diagnostic efficacy was superior to that of traditional tumor markers. It can be used as a tumor biomarker to monitor the efficacy of chemotherapy for gastric cancer. Keywords: advanced gastric cancer, cfDNA, tumor marker
Authors: Yuejiao Zhong, Qingyu Fan, Zhaofei Zhou, Yajing Wang, Kang He, Jianwei Lu
Source: Cancer Management and Research 2020:12 3099–3106
A high-throughput Anti-SARS-CoV-2 IgG testing platform for COVID-19
2020 | Publications
Background: Serology tests for detecting the antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can identify the previous infection and help to confirm the presence of current infection. Objective: The aim of this study was to evaluate the performances of a newly developed high throughput immunoassay for anti-SARS-CoV-2 IgG antibody detection. Results: Clinical agreement studies were performed in 107 COVID-19 patient serum samples and 226 negative donor serum/plasma samples. Positive percent agreement (PPA) was 46.15 % (95 % CI: 19.22 % ∼74.87 %), 61.54 % (95 % CI: 31.58 % ∼86.14 %), and 97.53 % (95 % CI: 91.36 % ∼99.70 %) for samples collected on 0−7 days, 8−14 days, and ≥15 days from symptom onset, respectively. Negative Percent Agreement (NPA) was 98.23 % (95 % CI: 95.53 % ∼99.52 %). No cross-reactivity was observed to patient samples positive for IgG antibodies against the following pathogens: HIV, HAV, HBV, RSV, CMV, EBV, Rubella, Influenza A, and Influenza B. Hemoglobin (200 mg/dL), bilirubin (2 mg/dL) and EDTA (10 mM) showed no significant interfering effect on this assay. Conclusion: An anti-SARS-CoV-2 IgG antibody assay with high sensitivity and specificity has been developed. With the high throughput, this assay will speed up anti-SARS-CoV-2 IgG testing.
Authors: Jinwei Du, Eric Chu, Dayu Zhang, Chuanyi M. Lu, Aiguo Zhang, Michael Y. Sha
Source: Journal of Virological Methods, Volume 287, January 2021, 114009
Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test
2020 | Publications
Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patient. Here we report the performance of QuantiVirus™ SARS-CoV-2 multiplex test using saliva as the testing specimens with or without pooling.
Authors: Qing Sun, Jonathan Li, Hui Ren, Larry Pastor, Yulia Loginova, Roberta Madej, Kristopher Taylor, Joseph K. Wong, Zhao Zhang, Aiguo Zhang, Chuanyi M. Lu, Michael Y. Sha
Source: medRxiv 2020.10.27.20219196; doi: https://doi.org/10.1101/2020.10.27.20219196
Evaluation of a Novel Liquid Biopsy-Based ColoScape Assay for Mutational Analysis of Colorectal Neoplasia and Triage of FIT+ Patients: a Pilot Study
2018 | Publications
Circulating cell-free tumour derived nucleic acids are becoming recognised as clinically significant and extremely useful biomarkers for the detection of cancer and for monitoring the progression of targeted drug therapy and immunotherapy. Screening programmes for colorectal cancer in Europe use the Fetal Immunochemical Test (FIT) test as a primary screener. FIT+ patients are referred to immediate colonoscopy and the positive predictive value (PPV) is usually 25%. In this article, we report a study employing the ColoScape assay panel to detect mutations in the APC, KRAS, BRAF and CTNNB1 genes, in order to collect preliminary performance indicators and plan a future, larger population study. The assay was evaluated on 52 prospectively collected whole-blood samples obtained from FIT+ patients enrolled in the CRC screening programme of ASL NAPOLI 3 SUD, using colonoscopy as confirmation. The assay’s sensitivity for advanced adenomas was 53.8% and the specificity was 92.3%. The PPV was 70.0% and negative predictive value (NPV) was 85.7%. Workflow optimisation is essential to maximise sensitivity. Of note, four of the six positive cases missed by ColoScape had a less than suboptimal DNA input (data not shown). Had they been ruled out as inadequate, sensitivity would have increased from 53.8% to 69%. However, as stated previously, this is not a clinical trial, but rather an initial, preliminary technical evaluation. In conclusion, this study shows that ColoScape is a promising tool and further studies are warranted in order to validate its use for the triage of FIT+ patients.
Authors: Mauro Scimia, Jinwei Du, Francesco Pepe, Maria Antonia Bianco, Silvana Russo Spena, Farah Patell-Socha, Qing Sun, Michael J Powell, Umberto Malapelle, Giancarlo Troncone
Source: Clin Pathol 2018;0:1–4. doi:10.1136/jclinpath-2018-205412
Reducing Artifactual EGFR T790M Mutations in DNA from Formalin-Fixed Paraffin-Embedded Tissue by Use of Thymine-DNA Glycosylase
2017 | Publications
Authors: Hongdo Do, Ramyar
Source: Clinical Chemistry 63:9 000–000 (2017) Molecular Diagnostics and Genetics 000–000 (2017),
Human Papillomavirus (HPV) E6/E7 mRNA Detection in Cervical Exfoliated Cells: a Potential Triage for HPV-Positive Women
Feb 8, 2017 | Publications
Cytology triage has been generally recommended for human papillomavirus (HPV)-positive women, but is highly dependent on well-trained cytologists. The present study was designed to explore whether HPV E6/E7 mRNA detection in cervical exfoliated cells can be a potential triage for HPV-positive women from a clinic-based population. Both the primary HPV testing and Papanicolaou (Pap) test were performed on all eligible HPV-positive women. HPV E6/E7 mRNA was detected by QuantiVirus® HPV E6/E7 mRNA assay in cervical exfoliated cells. All HPV-positive women underwent colposcopy and further biopsy if indicated. The data were assessed by Pearson’s Chi-squared test and the receiver operating characteristic curve. A total of 404 eligible HPV-positive women were enrolled. Positive rate of E6/E7 mRNA in high-grade squamous intraepithelial lesion (HSIL) cases was higher than that in low-grade squamous intraepithelial lesion (LSIL) or normal cases. There was no statistical difference found between mRNA and cytological testing with sensitivity (89.52% vs. 86.67%, P=0.671), specificity (48.96% vs. 48.96%, P=1.000), positive predictive value (39.00% vs. 38.24%, P=1.000), and negative predictive value (92.76% vs. 90.97%, P=0.678) for detecting ≥HSIL. HPV E6/E7 mRNA detection in cervical exfoliated cells shows the same performance as Pap triage for HSIL identification for HPV-positive women. Detection of HPV E6/E7 mRNA may be used as a new triage option for HPV-positive women.
Keywords: Human papillomavirus (HPV); HPV E6/E7 mRNA; High-grade squamous intraepithelial lesion (HSIL http://dx.doi.org/10.1631/jzus.B1600288
Authors: Ye-li Yao, Qi-fang Tian, Bei Cheng, Yi-fan Cheng, Jing Ye, Wei-guo Lu
Source: J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2017 18(3):256-262
Alu–based cell-free DNA: a novel biomarker for screening of gastric cancer
2016 | Publications
Gastric cancer (GC) is the fourth most common cancer and the second major cause of cancer-related deaths worldwide. In our previous study, a novel and sensitive method for quantifying cell-free DNA (CFD) in human blood was established and tested for its ability to predict patients with tumor. We want to investigate CFD expression in the sera of GC patients in an attempt to explore the clinical significance of CFD in improving the early screening of GC and monitoring GC progression by the branched DNA (bDNA)-based Alu assay. The concentration of CFD was quantitated by bDNA-based Alu assay. CEA, CA19-9, C72-4 and CA50 concentrations were determined by ABBOTT ARCHITECT I2000 SR. We found the CFD concentrations have significant differences between GC patients, benign gastric disease (BGD) patients and healthy controls (P < 0.05). CFD were weakly correlated with CEA (r = −0.197, P < 0.05) or CA50 (r = 0.206, P < 0.05), and no correlation with CA19-9 (r = −0.061, P > 0.05) or CA72-4 (r = 0.011, P > 0.05). In addition, CFD concentrations were significantly higher in stage I GC patients than BGD patients and healthy controls (P < 0.05), but there was no significant difference in CEA, CA19-9 and CA50 among the three traditional tumor markers (P > 0.05). Our analysis showed that CFD was more sensitive than CEA, CA19-9, CA72-4 or CA50 in early screening of GC.
Authors: Chen Qian, Shaoqing Ju, Jing Qi, Jianmei Zhao, Xianjuan Shen, Rongrong Jing, Juan Yu, Li Li, Yingjuan Shi, Lurong Zhang, Zhiwei Wang, Hui Cong
Source: Oncotarget, Advance Publications 2016
Genetic Alteration and Mutation Profiling of Circulating Cell-free Tumor DNA (cfDNA) for Diagnosis and Targeted Therapy of Gastrointestinal Stromal Tumors
2016 | Publications
Keywords: Gastrointestinal stromal
Authors: Weixin Yan, Aiguo Zhang and Michael J. Powell
Source: Chinese Journal of Cancer, Chin J Cancer (2016) 35:68 DOI 10.1186/s40880-016-0131-1
The Application of Molecular Diagnostics to Stained Cytology Smears
2016 | Publications
Detection of mutational alterations is important for guiding treatment decisions of lung
Authors: Maja H. Oktay, Esther Adler, Laleh Hakima, Eli Grunblatt, Evan Pieri, Andrew Seymour, Samer Khader, Antonio Cajigas, Mark Suhrland, Sumanta Goswami
Source: The Journal of Molecular Diagnostics, May 2016Volume 18, Issue 3, Pages 407–415, DOI: https://doi.org/10.1016/j.jmoldx.2016.01.007
Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas
2016 | Publications
Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measurable levels of at least three of the five mutations. HRAS G12D was significantly increased inDCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10−3 to 4.6 × 10−2) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047Rmutant cells have a selective advantage in
Authors: Meagan B. Myers, Malathi Banda, Karen L. McKim, Yiying Wang, Michael J. Powell and Barbara L. Parsons
Source: Neoplasia (2016) 18, 253–263
An Evaluation of the Cobas4800 HPV Test on Cervico-Vaginal Specimens in Liquid versus Solid Transport Media
Feb 1, 2016 | Publications
Objectives: Determine the ability of the Cobas 4800 assay to detect high-risk human papillomavirus (HrHPV) and high-grade cervical lesions when using
Authors: Hongxue Luo, Hui Du, Kathryn Maurer, Jerome L. Belinson, Guixiang Wang, Zhihong Liu, Lijie Zhang, Yanqiu Zhou, Chun Wang, Jinlong Tang, Xinfeng Qu, Ruifang Wu
Evaluation of a New Solid Media Specimen Transport Card for
High Risk HPV Detection and Cervical Cancer Prevention
2015 | Publications
Background: Solid media transport can be used to design adaptable cervical cancer screening programs but currently is limited by one card with published data.Objective: To develop and evaluate a solid media transport card for use in high-risk human
Keywords: Cervical cancer screening, Solid media transport, High-risk HPV testing
Authors: Kathryn Maurer, Hongxue Luo, Zhiyong Shen, Guixiang Wang, Hui Du
Source: Journal of Clinical Virology 76 (2016) 14–19
High Sensitivity Detection of Tumor Gene Mutations
Feb 16, 2015 | Publications
To reduce the wild-type background and improve sensitivity, a molecular clamp has been designed to hybridize selectively to wild-type template DNA and block its amplification. This molecular clamp consists of a synthetic, sequence-specific Xeno-nucleic acid (XNA) probe. It is called QClamp™. In the presence of a mutation such as a single nucleotide polymorphism (SNP) gene deletion, insertion, or rearrangement in the region of the XNA probe sequence, the XNA probe molecule melts off the mutant template DNA during the PCR cycling process, and only mutant templates are amplified efficiently. QClamp has been shown to be a sensitive and precise quantitative PCR (qPCR) technology. It is able to block the amplification of wild-type DNA from samples. In addition, it can detect low-frequency genetic mutations (<0.05%) in DNA samples obtained from
Authors: Michael Powell J, Madhuri Ganta, Elena Peletskaya, Larry Pastor, Melanie Raymundo
Source: et al. (2015) High Sensitivity Detection of Tumor Gene Mutations.
Diagnostic Validity of Human Papillomavirus E6/E7 mRNA Test in Cervical Cytological Samples
2013 | Publications
Human papillomavirus (HPV) DNA tests tend to show high sensitivity, but poor specificity in detecting high-grade cervical lesions. This study aimed to explore the clinical performance of QuantiVirus®HPVE6/E7 mRNA in identifying ≥Grade 2 cervical intraepithelial neoplasia. Thin-prep®liquid based cytology test (LBC) samples were collected from October 2009 to October 2011 from women who underwent out-patient hospital-based
Keywords: HPV E6/E7 mRNA, Human papillomavirus, Cervical intraepithelial neoplasia
Authors: Tong-Yu Liu, Rong Xie, Li Luo, Kathleen H. Reilly, Cheng He, Yu-Zhen Lin, Gang Chen, Xiong-Wei Zheng, Lu-Lu Zhang, Hai-Bo Wang
Source: J. Virol. Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.10.032
QuantiVirus® HPV E6/E7 RNA 3.0 Assay (bDNA) is as Sensitive, but Less Specific Than Hybrid Capture 2 Test
Nov 11, 2012 | Publications
Human papillomavirus (HPV) infection is the primary cause of cervical cancer. The Quantivirus® HPV E6/E7 RNA 3.0 assay (DiaCarta, CA, USA) detects E6/E7 mRNA of 13 high-risk subtypes and 6 low-risk subtypes. Cervical specimens collected in PreservCyt were processed for HPV detection. Cervical biopsies were taken only from those women with abnormal colposcopy. 200 out of 272 (73.5%) cases were mRNA positive. The percentage of HPV E6/E7 mRNA positive samples increases with the severity of the cytological diagnosis, but not in histological diagnosis. In 146 patients with both tests, the E6/E7 mRNA assay had significant higher positivity rate than the Hybrid Capture 2 assay (75.3% versus 62.3%). The HPV mRNA assay and the HC2 assay had the same sensitivity of high grade cervical intraepithelial neoplasia (CIN 2+), 82.4% (14/17) (95% confidence interval [CI], 64.3, 100). However, the specificity of CIN 2+ for the HPV mRNA assay was significantly lower than HC2 assay. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the E6/E7 mRNA and HC2. E6/E7 mRNA achieved 58.8% sensitivity with 74.1% specificity, HC2, achieved 47.1% sensitivity with 70.7% specificity. The overall performance of HPV E6/E7 mRNA assay for detecting CIN 2+ was lower than HC2. This study does not support the use of this assay in screening for cervical cancer prevention alone.
Keywords: Human papillomavirus, E6/E7 mRNA, HPV DNA, Cervical lesion, Liquid-based cytology triage
Authors: Yong Shen, Jiaomei Gong, Yanxia He, Guomei Cheng, Paul Okunieff, Xiaofu Li
Source: of Virological Methods 187 (2013) 288– 293
LOH and Copy Neutral LOH (cnLOH) Act as
Alternative Mechanism in Sporadic Colorectal Cancers with Chromosomal and Microsatellite Instability
2011 | Publications
Background and aims: Tumor suppressor genes are often located in frequently deleted chromosomal regions of colorectal cancers (CRCs). In contrast to microsatellite stable (MSS)
Authors: Ralph Melcher, Elena Hartmann, Waltraud Zopf, Sabine Herterich, Philipp Wilke, Ludwig Muller, Eduard Rosler, Theodor Kudlich, Oliver Al-Taie, Andreas Rosenwald, Tiemo Katzenberger, Bettina Scholtka, Stefan Seibold, Dorothee Rogoll, Wolfgang Scheppach, Michael Scheurlen and Hardi Luhrs
Source: Carcinogenesis vol.32 no.4 pp.636–642, 2011
A Gene Marker Panel Covering the Wnt and the Ras-Raf-MEK-MAPK Signalling Pathways Allows to Detect Gene Mutations in 80% of Early (UICC I) Colon Cancer Stages in Humans
2009 | Publications
Background: Very recently a gene marker panel that allows the mutation analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/ or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientiﬁc literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early-stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No signiﬁcant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.
Keywords: APC B-RAF CTNNB1 Colorectal carcinomas K-RAS Microsatellite instability Oncogenes Tumour suppressor genes
Authors: Bettina Scholtka, Mandy Schneider, Ralph Melcher, Tiemo Katzenberger, Daniela Friedrich, Kornelia Berghof-Jager, Wolfgang Scheppach, Pablo Steinberg
Source: Cancer Epidemiology 33 (2009) 123–129
Detection of the Hereditary Hemochromatosis Gene Mutation by Real-Time Fluorescence Polymerase Chain Reaction and Peptide Nucleic Acid Clamping
Feb 10, 1998 | Publications
Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new doublestranded DNA-selective ﬂuorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that ﬂuorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.
Authors: Erich M. Kyger, Mark D. Krevolin, and Michael J. Powell
Source: Analytical Biochemistry 260, 142–148 (1998) Article No. AB982687