Strategies for Preventing Amplicon Contamination in Molecular Laboratory !

The Primer

The high sensitivity of the polymerase chain reaction (PCR)—theoretically with lower limits of detection as little as a single template molecule; practically, 10 to 100 copies for many assays as run—is one of its greatest strengths, but also its greatest weakness. As the method works through creating copies of its target, any positive sample can lead to large numbers of molecules which can in turn contaminate subsequent reactions and cause false positive results.

To get a sense of the scope of this, consider a successful “average” 25μl PCR somehow getting opened and spilled in the lab. This would contain on the order of 10^12 template copies (amplicons); in other words, if a thorough cleaning reduced this by a million fold, you’d still have a million amplicon copies “floating around,” each of which could contaminate a reaction. If you’re fortunate enough to have never experienced this first-hand, you can thank the widespread acceptance of real-time PCR methods, which do away with having to open reaction tubes post amplification, and perhaps gain an appreciation of why anyone who has been through the experience treats the risk as real and ever-present.

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FISHing in the Genomic Testing Age !

Genetic analysis has come a long way; we now have an ever-expanding collection of analytical tools in the diagnostic laboratory. So why do we still need a technique that usually only looks at one or two loci? The simple answer is that results from fluorescence in-situ hybridization (FISH) can quickly confirm diagnoses, guide clinicians’ judgements regarding differential diagnoses, and correlate results with clinical risk—thus enabling an informed choice of treatment type and intensity.

FISH employs fluorescently-labeled DNA probes to bind complementary DNA sequences within an interphase cell, or onto metaphase chromosomes. These sequences can then be visualized using fluorescent microscopy. The number, location and relative positions of the probe signals indicate chromosomal changes in a particular cell (Figure 1).

Many clinical trials use cytogenetic and FISH data to stratify patients according to specific risk factors. FISH is often used as a stand-alone technique for investigating abnormalities and following-up such patients, which, alongside its relatively low expense, makes it a very convenient investigative tool.

In this article we will explore the utility of FISH in today’s clinical laboratory and the future of the technique in the evolution of molecular testing.

Figure 1. The FISH process.

Figure 2. Interphase cell showing amplified HER2 signal pattern.

Read more: FISHing in the Genomic Testing Age !

Source: MedicalLaboratoryObserver

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MicroRNA: A Tiny Molecule Yields Big Insights Into Disease States

Regular readers of this column will know that there are two main categories of nucleic acids—DNA and RNA. They’ll also know that while for living organisms DNA acts as the genetic data repository, RNA has a messenger role (mRNAs, transcribed from DNA to direct protein synthesis). Most will also recall that there are other classes of RNA molecules, particularly tRNAs (used to tag and identify amino acids for protein synthesis) and rRNAs (structural components of the ribosome, the cellular “machinery” for protein synthesis). In addition to these, there’s increasing interest in the molecular diagnostics community in a less widely known but no less common RNA form, the microRNA or miRNA.

Read more: MicroRNA: A Tiny Molecule Yields Big Insights Into Disease States

Source: MedicalLaboratoryObserver

Diagnosis of Pneumocystis jirovecii pneumonia

Diagnosis of Pneumocystis jirovecii pneumonia (PCP) is conventionally based on direct staining and visualization. Challenges in obtaining alveolar samples have stimulated interest in techniques for detection of Pneumocystis DNA in non-invasive samples, which can give good sensitivity and specificity. Robust diagnosis is key to ensuring appropriate therapy.

Introduction

Pneumocystis jirovecii (previously Pneumocystis carinii) is a pathogen capable of causing life threatening Pneumocystis pneumonia (PCP) in the immunocompromised with case fatality rates among those hospitalized of around 10%. PCP typically occurs in individuals with hematological malignancies on chemotherapy or with other causes of acquired cellular immunodeficiency or, most frequently, in human immunodeficiency virus (HIV)-positive individuals with CD4 T-cell counts <200 cells/µL or <14% of total white cell count. First-line treatment is co-trimoxazole, a combination of the antibiotics sulfamethoxazole and trimethoprim, at high dose for 3 weeks, which has the clinically significant potential side effects of bone marrow suppression, rash and bronchial hypersensitivity.

Read more: Diagnosis of Pneumocystis jirovecii pneumonia

Source: ClinicalLaboratoryInternational

Breathing New Life into Cystic Fibrosis Genetic Testing

New Research Has Broadened the Number of Clinically Relevant CFTR Gene Mutations.

Imagine yourself suspended a couple of hundred feet below the surface of the ocean. The sheer weight of the immense column of water pushing down on your chest makes each breath a harrowing task. Now picture that your only recourse to collect vital oxygen is to breathe laboriously throw a narrow straw that connects you to the atmosphere above. You slowly draw in air, cautiously trying not to collapse the straw from too forceful of suction—struggling just as much to exhale the expired air. Now repeat the entire cycle for the rest of your life.

If you were able to envision how the immense difficulties of breathing in this manner would be for just a few minutes, let alone your entire life, then you may have a minute sense of what a person afflicted with cystic fibrosis (CF) endures. Gasping for air while thick, sticky mucus lines the pulmonary system, seemingly threatening to drown and suffocate patients with each inhale.

Read more: Breathing New Life into Cystic Fibrosis Genetic Testing

Accurate genetic testing for cystic fibrosis can determine if a patient has the disease and provide
needed information for potential carrier couples, clinical actionability of identified mutations, and
potential severity. [Krishna Kumar/Getty]

Source: genengnews

Rising Liquid Biopsy Tide Lifts Cancer Dx

Traditional Tissue Biopsies Are Not Providing the Type of Real-Time Monitoring Necessary for Effectively Catching Relapse

As molecular diagnostics for oncology shifts into clinical practice, efforts have not only focused on genotyping patient-specific tumors to initiate targeted therapies, but on early detection, availability, improved quality control, and refined workflow.

One major push, that exemplifies the concomitance of these trends, has been the rise of the less-invasive and more cost-effective liquid sampling for cancer diagnosis; sometimes called a liquid biopsy.

Read more: Rising Liquid Biopsy Tide Lifts Cancer Dx

The FDA recently approved Hologic’s prostate cancer test, which improves upon the less sensitive
and less specific standard PSA tests via examination of the PCA3 gene in urine.

Source: genengnews

Molecular Diagnostics in the Microbiology Laboratory

A look at some of the newest generation ‘load and go’ molecular microbiology analyzers.

For decades, pathogens have been isolated and grown in blood cultures, and detected using microscopes, serology and biochemical techniques. However the last few years have seen a revolution in modern microbiology.

The above tests still form the core work of most routine microbiology labs, but modern analytical techniques such as molecular diagnostics and mass spectrometry are increasingly being incorporated, to varying degrees, in laboratories around the world.

Molecular diagnostics refers to the analysis of nucleic acid from DNA or RNA. In the clinical microbiology lab, scientists are looking for the nucleic acid of microorganisms to confirm or exclude a diagnosis.

The molecular diagnostic work undertaken in the lab can vary from a simple, manual monoplex polymerase chain reaction (PCR) based test to complex automated, multiplex testing (testing for multiple pathogens simultaneously). Some of the newest generation ‘load and go’ molecular analyzers are detailed below.

VERIS Mdx Molecular Diagnostics System

The DxN VERIS combines sample prep and sample analysis steps into a single workflow. The automation of DNA extraction, purification, assay set-up and analysis saves the user time and also prevents user error and the risk of contamination. Using real-time PCR, the system is designed for multiplex assays and uses magnetic particle separation for nucleic acid extraction and purification. The initial test menu includes Cytomegalovirus, Hepatitis B, Hepatitis C and HIV-1 ......

Read more: Molecular Diagnostics in the Microbiology Laboratory

Microbiology has traditionally involved use of blood cultures, however molecular methods are
increasingly employed in modern laboratories;
Beckman Coulter's VERIS Mdx Molecular Diagnostics System

Source: SelectScience