Genotyping Performed by FRET-PCR Without DNA Extraction
|
By LabMedica International staff writers Posted on 07 Jul 2014 |
|
Image: The LightCycler 2.0 real-time polymerase chain reaction analyzer and centrifuge (Photo courtesy of Roche).”
Image: The Scepter 2.0 Automated Cell Counter (Photo courtesy of Merck Millipore).
Blood samples are extensively used for the molecular diagnosis of many hematological diseases using a variety of techniques, based on the amplification of nucleic acids.
Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC), which can be improved by a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells.
Hematologists at the Hospital Universitari Son Espases (Palma de Mallorca, Spain) studied peripheral blood from 34 patients collected into tubes containing ethylenediaminetetraacetic acid (EDTA). Among the samples, they included a mixture of mutant alleles for patients suffering from thrombosis or hereditary hemochromatosis. Red blood cells (RBCs) were lysed and white blood cells (WBCs) isolated. A real-time PCR was then performed followed by a melting curve analysis for different genes including methylenetetrahydrofolate reductase (MTHFR), hemochromatosis (HFE), coagulation factor V Leiden (F5), prothrombin factor two (F2) and coagulation factor XII (F12).
The real time PCR was performed on the LightCycler 2.0 Instrument (Roche Diagnostics Corporation, Indianapolis, IN, USA). In order to standardize the samples for the real-time PCR reaction, cells were counted in a Scepter 2.0 Automated Cell Counter (Merck Millipore, Billerica, MA, USA) and adjusted to 5×106 cells/mL. After testing 34 samples comparing the real-time crossing point (CP) values between 5×106 WBC/mL and 20 ng/µL of purified DNA, the results for F5 Leiden were as follows: CP mean value for WBC was 29.26 ± 0.57 versus purified DNA 24.79 ± 0.56. There was an observed delay of about four cycles when PCR was performed from WBC instead of DNA.
The authors concluded that their protocol obviates the DNA purification stage, thereby saving time and resources. Furthermore, since the manipulation performed on the sample is minimal, it may decrease the risk of contamination. As they reported the results from a variety of genes, they contend that their protocol will be suitable for the genotyping of almost any inherited polymorphism. The study was published on June 25, 2014, in the Journal of Blood Medicine.
Related Links:
Hospital Universitari Son Espases
Roche Diagnostics Corporation
Merck Millipore
Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC), which can be improved by a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells.
Hematologists at the Hospital Universitari Son Espases (Palma de Mallorca, Spain) studied peripheral blood from 34 patients collected into tubes containing ethylenediaminetetraacetic acid (EDTA). Among the samples, they included a mixture of mutant alleles for patients suffering from thrombosis or hereditary hemochromatosis. Red blood cells (RBCs) were lysed and white blood cells (WBCs) isolated. A real-time PCR was then performed followed by a melting curve analysis for different genes including methylenetetrahydrofolate reductase (MTHFR), hemochromatosis (HFE), coagulation factor V Leiden (F5), prothrombin factor two (F2) and coagulation factor XII (F12).
The real time PCR was performed on the LightCycler 2.0 Instrument (Roche Diagnostics Corporation, Indianapolis, IN, USA). In order to standardize the samples for the real-time PCR reaction, cells were counted in a Scepter 2.0 Automated Cell Counter (Merck Millipore, Billerica, MA, USA) and adjusted to 5×106 cells/mL. After testing 34 samples comparing the real-time crossing point (CP) values between 5×106 WBC/mL and 20 ng/µL of purified DNA, the results for F5 Leiden were as follows: CP mean value for WBC was 29.26 ± 0.57 versus purified DNA 24.79 ± 0.56. There was an observed delay of about four cycles when PCR was performed from WBC instead of DNA.
The authors concluded that their protocol obviates the DNA purification stage, thereby saving time and resources. Furthermore, since the manipulation performed on the sample is minimal, it may decrease the risk of contamination. As they reported the results from a variety of genes, they contend that their protocol will be suitable for the genotyping of almost any inherited polymorphism. The study was published on June 25, 2014, in the Journal of Blood Medicine.
Related Links:
Hospital Universitari Son Espases
Roche Diagnostics Corporation
Merck Millipore
Platinum Member
Automated Coagulation Analyzer
Hemolumi H6
Gold Member
Flocked Fiber Swabs
Puritan® Patented HydraFlock®
Automated Urinalysis Solution
UN-9000
Japanese Encephalitis Test
Japanese Encephalitis Virus Real Time PCR Kit
Latest Molecular Diagnostics News
- Self-Collected HPV Testing Reaches Patients Missed by Routine Screening
- Genetic Risk Score Identifies Type 1 Diabetes in MODY Testing
- Comprehensive Genomic Testing Expands Treatment Options for Advanced Cancers
- Multi-Omics Analysis Identifies Additional Risk Genes in Hereditary Breast and Ovarian Cancer
- Blood Test for Lung Cancer Screening Receives FDA Breakthrough Device Designation
- Machine Learning Approach Expands Epigenetic Testing for Prenatal Genetic Disorders
- New Liquid Biopsy Workflow Maximizes Tumor Signals from Limited Blood Samples
- Sequencing-Based Newborn Screening Identifies Pediatric Cancer Risk at Birth
- Liquid Biopsy Combines Multiple Signals for Cancer Detection in a Single Analysis
- Newborn Screening Program Adds Spinal Muscular Atrophy Testing Across England
- Blood Test Could Help Fast-Track Lymphoma Diagnosis in Resource-Limited Settings
- Liquid Biopsy Assay Enriches Tumor DNA for Early Detection and Monitoring
- Whole-Cell Genomic Analysis Expands the Potential of Liquid Biopsy
- Point-of-Care Multiplex PCR Test Submitted to FDA for Flu A/B and RSV Detection
- New PCR Assays Expand Cyclospora Testing for Outbreak Surveillance
- Single Liquid Biopsy Predicts Early Immunotherapy Benefit in Advanced Lung Cancer
Channels
Clinical Chemistry
view channel
Blood Test Markers Could Help Identify Older Adults at Risk of Disability
Maintaining independence into very old age is a growing public health challenge as Japan’s population rapidly ages. Nearly 60% of Japanese adults aged 85 years and older already receive support through... Read more
FDA Clears Roche Blood Test for Alzheimer’s Amyloid Pathology Assessment
An estimated 75% of people living with dementia remain undiagnosed, while confirmatory tools such as positron emission tomography (PET) or cerebrospinal fluid (CSF) testing can be costly, invasive, and... Read more
Rapid Ferritin Test Enables Iron Deficiency Detection at the Point of Care
Iron deficiency is one of the world’s most common nutritional disorders and can be difficult to diagnose where access to laboratories and trained personnel is limited. Women and children in low-resource... Read more
Alzheimer’s Blood Test Becomes First FDA-Cleared Option for Adults as Young as 40
C2N Diagnostics’ PrecivityAD2 blood test was cleared by the U.S. Food and Drug Administration (FDA) on August 20, 2026, for adults as young as 40 who are experiencing signs of cognitive impairment, according... Read moreMolecular Diagnostics
view channel
Genetic Risk Score Identifies Type 1 Diabetes in MODY Testing
Accurately distinguishing maturity-onset diabetes of the young from type 1 diabetes is difficult in routine care, especially when onset occurs in adolescence or early adulthood. Although maturity-onset... Read more
Self-Collected HPV Testing Reaches Patients Missed by Routine Screening
Timely cervical cancer screening prevents progression from persistent high‑risk human papillomavirus (HPV) infection, yet many adults marginalized from care do not complete routine exams.... Read more
Comprehensive Genomic Testing Expands Treatment Options for Advanced Cancers
Selecting effective therapies for advanced cancers remains difficult because standard tumor testing often targets only a limited set of genes. As options dwindle, many patients face decisions without strong... Read more
Multi-Omics Analysis Identifies Additional Risk Genes in Hereditary Breast and Ovarian Cancer
Hereditary breast and ovarian cancer can be difficult to explain genetically, leaving many high-risk families without clear answers. Although 13 established risk genes, including BRCA1 and BRCA2, are routinely... Read moreImmunology
view channel
AI-Based Antibody Profiling Predicts Strength of COVID-19 Vaccine Response
Vaccine-induced protection can vary widely between individuals, creating uncertainty for clinicians, particularly in patients with immunosuppressive conditions. Demographic and health factors explain only... Read more
Immune Biomarkers May Predict Recurrent Checkpoint Inhibitor Arthritis
Inflammatory arthritis is a recognized immune-related adverse event in patients treated with immune checkpoint inhibitors (ICIs) for cancer. Between 20% and 50% of affected patients experience more than... Read more
Microbiology
view channel
Genetic Marker Identifies Emerging Resistance to Frontline Malaria Drugs
Artemisinin-based combination therapy remains central to controlling Plasmodium falciparum malaria, but emerging drug resistance is complicating treatment and surveillance. In Uganda and across sub-Saharan... Read more
Surveillance and Susceptibility Testing Track Rising Candida Auris in U.S.
Drug-resistant fungal infections are straining infection control and treatment in hospitals and long-term care facilities, where rapid transmission can lead to severe outcomes. Candida auris has expanded... Read more
Pathology
view channel
AI-Pathologist Framework Improves Accuracy and Reliability in Cancer Diagnosis
Ensuring reliable cancer diagnosis from digital pathology remains a critical challenge as clinical decisions rely on accurate slide interpretation. While artificial intelligence (AI) has accelerated whole-slide... Read more
New Review Highlights Intelligent Agents as Next Step for Digital Pathology
Digital pathology remains constrained by models that classify single images without mirroring how clinicians interrogate multiple slides, adjust magnification, and synthesize ancillary tests.... Read more
Simple Immunohistochemical Size Ratio Improves Classification of Primary Aldosteronism
Primary aldosteronism is a common but underdiagnosed cause of hypertension, in which excess aldosterone promotes salt retention and raises blood pressure. Identifying the dominant source of hormone overproduction... Read more
Technology
view channel
Autonomous Robotic System Receives FDA Authorization for Blood Collection
Venipuncture is central to many diagnostic pathways, but routine blood collection can be affected by staffing constraints and procedural variability that influence consistency and patient experience.... Read more
Interoperable Data Platform Standardizes Multi-Cancer Blood Test Results
Proteotype Diagnostics has introduced Alchemi, a proprietary data and clinical workflow platform being developed for Enlighten, the company’s investigational blood-based multi-cancer test.... Read more
Industry
view channel
New Collaboration Advances Precision Oncology Screening for Lung Cancer in Japan
Molecular profiling is central to precision oncology in lung cancer, but tissue samples can be limited and minimally invasive approaches are often preferable. Liquid biopsy enables blood-based genomic... Read more
