MOLECULAR DIAGNOSTICS

 

Molecular Diagnostics testing is comprised of a variety of techniques used to assess biological markers in a patient’s unique genetic code. Molecular Diagnostics is used to diagnose disease, manage and monitor disease, detect risk and decide which therapies work best for each particular patient.

The tests are extremely useful in a wide range of medical specialization, such as infectious disease, oncology, immunology and coagulation. Molecular Diagnostics is also beneficial with pharmacogenomics, which is the genetic prediction of the success or failure of a prescribed drug or medical protocol, and clinical chemistry, which is medical testing of bodily fluids.

 

 

POLYMERASE CHAIN REACTION (PCR) TESTING

 

The most popular molecular diagnostic testing technique is PCR testing.  Polymerase chain reaction (PCR) is a technique used to “amplify” or “photocopy” small segments of a person’s DNA (or RNA).

Since a significant amount of DNA/RNA sample is necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification.

The entire amplification cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA/RNA to separate into strands and synthesize those strands into duplicates using the original strands as templates. The cycle gets repeated many times during those few hours, and results in more than one billion exact copies of the DNA/RNA segment.

Once amplified, the DNA/RNA produced by PCR can be used in many different laboratory procedures and clinical techniques. These findings include DNA/RNA fingerprinting, detection and identification of pathogenic organisms such as bacteria, fungi or viruses and diagnosis of genetic disorders.

Additionally, the PCR test was in use most commonly to identify the genetic material of the deadly SARS CoV-2 coronavirus responsible for the COVID-19 pandemic. PCR tests are also used to identify and characterize genetic mutations and rearrangements found in certain cancers.

At Convergent, we use Real-time PCR (Rt-qPCR). This method combines PCR amplification and detection into a single step. This eliminates the need to detect products using gel electrophoresis and, more importantly, it enables the method to be truly quantitative. With real-time PCR, fluorescent dyes are used to label PCR products during thermal cycling. Real-time PCR instruments measure the accumulation of fluorescent signal during the exponential phase of the reaction for fast, precise quantification of PCR products and objective data analysis.

 

NEXT GEN SEQUENCING (NGS)

 

Next generation sequencing (NGS) is technology which has revolutionized genomic research.

A great benefit of next generation sequencing is that it can detect abnormalities using less DNA/RNA than required for traditional sequencing approaches; An entire human genome can be sequenced within hours.

NGS enables the interrogation of hundreds to thousands of genes at one time in multiple samples, as well as discovery and analysis of different types of genomic features in a single sequencing run, from single nucleotide variants (SNVs), to copy number and structural variants, and even RNA fusions. NGS provides the ideal throughput per run, and studies can be performed quickly and cost-effectively. Additional advantages of NGS include lower sample input requirements, higher accuracy, and the ability to detect variants at lower allele frequencies. NGS can also detect antibiotic resistance, bacterial load, and fungi in a specimen.

Differences: NGS vs. RT-qPCR

When comparing next-generation sequencing (NGS) vs. qPCR technologies, the key difference is discovery power. While both offer highly sensitive and reliable variant detection, qPCR can detect only known sequences. In contrast, NGS is a hypothesis-free approach that does not require prior knowledge of sequence information. NGS provides higher discovery power to detect novel genes and higher sensitivity to quantify rare variants and transcripts.

NGS vs. qPCR technologies also differ in scalability and throughput. While qPCR is effective for low target numbers, the workflow can be cumbersome for multiple targets. NGS is preferable for studies with many targets or samples. A single NGS experiment can identify variants across thousands of target regions with single-base resolution.

NGS Testing

Once a specimen is received in the laboratory, Convergent will perform a standard probe(s), RT-qPCR test covering an array of the most popular (or prescribed) infectious microbes. Within 24-48 hours, the laboratory findings and antibiotic resistance gene markers will be available to you in order to begin a treatment protocol.

The DNA/RNA that was amplified during the RT-qPCR test will be used with our NGS test. NGS requires a longer testing time (usually a few weeks) to detect the precise microbe known to cause a certain infection. NGS technology allows the test to canvass the universe of pathogens and infectious microbes, attempting to match DNA/RNA sequence codes with tens of thousands of known microbes.

NGS is extremely beneficial to a physician who is concerned for chronic infection, hard to treat infections and biofilms.

Biofilm Treatment Protocol

Biofilms are an immense, complex structure that can be treated in a variety of ways. DNA sequencing technologies has allowed experts not only to identify the vast colonies of bacteria and develop strategies to address them, but to begin to examine the cells and colonies specific to the effect of the therapies they employ.

Next Generation DNA sequencing (NGS) for infectious disease provides greater sensitivity and specificity than a culture, serologic and PCR methods. NGS allows for better discrimination between strains, species, detection of novel variants and new organisms, detection of an ever-growing array of uncultivable organisms, and the ability to detect eukaryotes that were previously undetectable. Convergent Diagnostics will provide a physician with the comprehensive NGS findings reports that are needed to determine drug resistance and sensitivity.

The power of NGS resides in its ability to generate a massive amount of genetic information in a relatively simple and rapid manner, especially when combined with the most prevalently used marker to study microbiological communities (SSU rRNA). The analysis involves extensive parallel sequencing, where information is gathered from several sets of primers, targeting hypervariable regions and taking data from several sources simultaneously.

In addition to identifying components of the microbiome, NGS technology has also been utilized to identify antibiotic resistance genes (ARGs) within a given microbiome, with the intended purpose of tailoring antibiotic therapy and promoting antibiotic stewardship.

This next generation sequencing (NGS) approach can lead to more precise medicine and treatment because it has a much higher sensitivity. NGS can also detect antibiotic resistance, bacterial load, and fungi in samples. More accurate detection of the leading pathogen causing the infection allows for more accurate and effective treatment.