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
New
Gold Member
Serum Indices Control
Acusera Serum Indices Control
Hematology Consumables
Bioblood Devices
Gold Member
Pre- Eclampsia Control
Acusera Pre-Eclampsia Control
Latest Molecular Diagnostics News
- Urine-Based RNA Improves Bladder Cancer Detection and Monitoring
- Blood RNA Panel Predicts Lung Cancer Risk Years Before Diagnosis
- Facilitated Cascade Program Increases Genetic Testing in Hereditary Cancer Families
- Bile DNA Testing Detects Tumor Signals Before Conventional Cancer Diagnosis
- Blood-Based Test Broadens Colorectal Cancer Screening Options
- New Blood Test Could Help Predict Severe Scoliosis in Adolescents
- Single Assay Enables Simultaneous Newborn Screening for Three Severe Genetic Disorders
- Genetic Testing Identifies Inherited Predisposition in Children With Acute Leukemia
- Pooled Saliva PCR Screening Identifies Congenital CMV Missed by Targeted Testing
- Portable Test Differentiates Seven Mosquito-Borne Viruses at Point of Care
- Rapid Genetic Test Aims to Shorten Brain Tumor Diagnosis Time
- Long-Read Sequencing Test Identifies Genetic Causes in Inherited Muscle Disease
- Genetic Testing Program Helps Uncover Inherited Risk in Pediatric Cancer
- Genomic Test Guides Extended Endocrine Therapy in Early Breast Cancer
- Genomic Breast Cancer Study Adds Real-World Evidence Across Diverse Patient Populations
- New Oncology Assays Deliver Precise Molecular Counts with MRD-Grade Sensitivity
Channels
Clinical Chemistry
view channel
UK Project Advances Blood-Based Dementia Testing Toward Routine Clinical Care
Dementia is the leading cause of death in the UK, accounting for almost one in eight deaths, with more than 72,000 people dying from dementia, including Alzheimer’s disease, in 2025. Despite this burden,... Read more
Noninvasive AI-Enabled Raman Probe Distinguishes Skin Cancers from Normal Tissue
Skin cancer is the most common cancer in the United States and among the most common worldwide. Nearly 1.5 million new cases were diagnosed globally in 2024, including nearly 340,000 melanomas.... Read more
Screening After Pneumococcal Disease May Reveal Undiagnosed Blood Cancer or Immune Disorders
Severe pneumococcal disease requiring hospitalization often presents as pneumonia, particularly in older adults and people with cancer or compromised immune function. Because M protein testing and antibody... Read more
Molecular Diagnostics
view channel
Facilitated Cascade Program Increases Genetic Testing in Hereditary Cancer Families
Hereditary cancer risk can extend beyond an individual patient to parents, siblings, and children. Although BRCA1 and BRCA2 mutations are linked to elevated risks of breast, ovarian, prostate, and other... Read more
Urine-Based RNA Improves Bladder Cancer Detection and Monitoring
Bladder cancer affects about 85,000 people in the United States each year and is prone to recurrence. Diagnosis and surveillance commonly rely on cystoscopy, an endoscopic examination that can miss up... Read more
Immunology
view channel
Routine Newborn Blood Spots Can Measure GBS Antibody Levels
Group B Streptococcus (GBS) can cause serious infections in newborns. Protecting infants remains challenging because immune protection may depend on antibodies transferred from mother to baby before birth.... Read more
Blood Test Differentiates Bacterial and Viral Infections in Febrile Infants
Fever in infants younger than 3 months is treated as a medical emergency because infections can become life-threatening while the immune system is still developing. Emergency department teams must quickly... Read more
Microbiology
view channel
Genomic Workflow Identifies Fungal Pathogens Before Blood Cultures Turn Positive
Fungal bloodstream infections pose a major threat to hospitalized patients. Candida species cause most invasive fungal infections worldwide and are among the leading causes of hospital-acquired bloodstream... Read more
FDA-Cleared Multiplex PCR Test Detects 13 Respiratory Pathogens in a Single Sample
Respiratory tract infections can be difficult to distinguish at presentation because many cause overlapping, nonspecific symptoms and are initially grouped as influenza-like illnesses. Causes span a range... Read more
One-Hour Molecular Panel Expands Bloodstream Infection Testing for Gram-Negative Pathogens
Bloodstream infections can progress rapidly and lead to sepsis, organ failure, and death. In the United States, about 1.7 million adults develop sepsis each year, and at least 350,000 die during hospitalization... Read more
FDA Clears Rapid Phenotypic Antimicrobial Susceptibility System for Positive Blood Cultures
Bloodstream infections require prompt treatment, but antimicrobial susceptibility results often lag behind a positive blood culture. Conventional testing can take another 24 to 48 hours after a culture... Read morePathology
view channel
Rapid Mass Spectrometry Test May Aid Glioma Margin Decisions
Glioma brain tumors are highly infiltrative and can extend into nearby healthy brain tissue, making tumor margins difficult to define during surgery. Residual tumor cells may contribute to recurrence and... Read more
Genomic Classifier Predicts Benefit From Adding Hormone Therapy to Salvage Prostate Radiation
Men who have undergone prostatectomy for prostate cancer may later develop a detectable or rising prostate-specific antigen, prompting salvage radiation therapy. A key challenge is determining who is most... Read more
Technology
view channel
Laser-Based Swab Analysis Shows Promise for Detecting Disease-Linked Odor Patterns
Disease-related changes in volatile organic compounds can alter body odor, producing measurable patterns in exhaled breath and bodily fluids. Current analytical methods can be complex, time-consuming,... Read more
Laser-Enhanced Assay Boosts Sensitivity for Colorectal Cancer Biomarker Detection
Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide. Early detection remains critical, but cancer biomarkers can produce only faint... Read more
Industry
view channel
Collaboration Advances Automated Benchtop Platform for Routine Blood Testing
Routine blood testing is central to clinical decision-making, but access can vary across laboratory and healthcare settings. Broader use of automated benchtop platforms may help integrate testing more... Read more
Expanded Partnership Supports AI Biomarker Validation and Clinical Trial Deployment
CellCarta (Montreal, Canada) and Imagene AI (Miami, FL, USA) have expanded their collaboration to provide biopharma companies with a coordinated pathway for biomarker strategy, assay development, validation,... Read more
Collaboration Combines AI Cognitive Assessment and RNA Blood Testing for Earlier Alzheimer’s Detection
Alzheimer’s disease is often identified only after substantial neurodegeneration, partly because current diagnostic pathways are fragmented and difficult to scale. As treatment shifts toward earlier intervention,... Read more