Molecular Diagnostics and the Detection of DNA and RNA Viruses
Many molecular diagnostics techniques or tests first developed for their use in molecular biology and biotechnology laboratories are now routinely used in clinical laboratories. Molecular virology refers to the specific detection of viral infection utilizing molecular detection methods. However, to allow for transparent quantitative molecular testing and data sharing between laboratories and institutions DNA and RNA based reference materials or standards are needed. The benefit of the availability of reference materials or standards for quantitative molecular testing is now been recognized internationally. Clinical laboratories, independent control and reference material manufacturers, in vitro diagnostics (IVD) manufacturers, and regulatory and policy makers, such as the AACC and FDA, are involved in developing standardized materials and protocols. Molecular probes or primers designed using natural or artificial nucleic acids are enabling key tools for the development of sensitive molecular diagnostics assays. For example, recently, bridged nucleic acids (BNAs) have been used to allow for the quantitative and sensitive detection of different mutant alleles.
Figure 1: 3D Model of RNA and DNA structures. (Source: RCSB Protein Data Bank (PDB) DNA and RNA structural data:
Since Watson, Crick and Wilkens received the Nobel Prize in Physiology or Medicine in 1962 for solving the DNA double-helix structure, many new nucleic acid-based diagnostic tools or assays have been developed that allow analysis of DNA and RNA molecules. Over time, they have been given many different names, such as Molecular Diagnostics, Molecular Pathology, Molecular Genetics, Molecular Genetic Pathology, Molecular Hematology-Oncology, Molecular Oncology, Molecular Virology, and Molecular Microbiology. However, the term “Molecular Diagnostics” covers them all. Many of these molecular diagnostics techniques have migrated to clinical laboratories. In the early years of biotechnology, in the 1980s to the 1990s, Southern blot hybridization was the technique used the most. Since it was a time-consuming, labor-intensive, and less sensitive diagnostic method, centers of excellence performed this test exclusively. The polymerase chain reaction (PCR) invented in the 1980s, an in-vitro nucleic acid amplification technique, has now become the dominant method after several rounds of optimization. In molecular virology, molecular testing is now possible in a short period-of-time, often taking just 20 to 30 minutes. In the case of HIV/AIDS, viral load testing is now possible as well.
Figure 2:3D Model of the H1N1 Influenza Virus. (Source: Center for Disease Control and Prevention: www.cdc.gov, http://www.cdc.gov/h1n1flu/images.htm)
As many people have experienced first hand, during the flu season, many people can get infected with the flu virus. However, other viruses can infect people in many different ways. Viral infections can happen by inhaling or swallowing them, by being bitten by an insect, or through sexual contact. Examples of viruses that cause a disease are the Ebola virus, human papillomavirus (HPV), hepatitis C virus (HPC), hepatitis D virus (HPD), human immunodeficiency virus (HIV/AIDS), dengue fever virus, and many others. Often, viral infections target the nose, throat, and upper airways. Diagnosis of the infection type is based on symptoms, blood test, and cultures, as well as the examination of infected tissues. Common viral infections like measles, rubella, or chickenpox are often just diagnosed based on symptoms. However, the use of more precise tests such as blood tests and cultures offer a more accurate diagnosis.
The polymerase chain reaction or PCR allows making many copies of the viral genetic material. The PCR method enables the rapid and accurate identification of a particular virus. Therefore, PCR-based molecular detection of a virus is currently the method of choice for its correct identification.
In the last decade, the molecular diagnostics field has been growing rapidly. Tests or assays used in molecular diagnostics detected specific DNA or RNA sequences. Specific molecular probes and primers are designed for this purpose. Molecular diagnostics tests or assays are now commonly used for the diagnosis of a disease. Furthermore, these assay types can be used to monitor or detect the risk for a disease, as well as to help decide which therapies will work best for infected patients. Furthermore, molecular diagnostics now enables personalized medicine.
Many innovated assays and instruments have been developed in the last decades enabling in vitro diagnostics (IVD). Examples are real-time PCR based instruments such as thermal cyclers, genetic analyzers, Sanger sequencing and next-generation sequencing.
What is a virus?
A virus, in biology, is a small infectious agent that can only replicate inside living cells of other organisms. Viruses are ultramicroscopic, metabolically inert, agents that can infect all kinds of species, such as animals, plants, and microorganisms, including bacteria, single-celled microorganism (archaea), and humans. Viruses typically consist of a nucleic acid molecule, DNA or RNA, within a protein coat, or sometimes a surrounding envelope. Viruses are quite small and tiny. Therefore, they can not be observed in a light microscopy. Viruses are only able to multiply within living cells of a host. Diseases caused by a virus are known as a “viral infections.”
Types of Molecular Diagnostics Assays
A typical molecular diagnostic assay requires the following three basic steps:
(1) Extraction and purification of DNA or RNA.
(2) Amplification or copying targeted nucleic acid sequences. Often fluorescent dyes
are also attached to the amplified oligonucleotides during this step.
(3) Detection of amplified target sequences using real-time PCR, microarrays,
multiplexing, or sequencing.
The following categories of molecular assays are available for detection of a viral infection in tissue and exfoliated cell samples. The basis for the tests is the detection of DNA or RNA sequences:
(1) Assays using Southern Blotting or Southern Transfer Hybridization, (STH),
Dot Blot Hybridization (DB), and In Situ Hybridization (ISH).
(2) Signal amplified hybridization assays, for example, a hybrid capture assay.
(3) Target amplification assays. For example, PCR and in situ PCR.
Southern blot hybridization and in situ hybridization
Originally Southern blot hybridization (STH) and in situ hybridization (ISH) were used and can still be used. Unfortunately, these techniques often have serious shortcomings. Southern blot hybridization assays require large amounts of DNA and are laborious tasks, and are often not reproducible. On the other hand, in situ hybridization or ISH base assays often show only moderate sensitivity for the virus tested.
PCR and in situ PCR
PCR and in situ PCR, techniques that amplify the target sequences, are extremely sensitive and specific. Using this method, viral DNA or RNA is amplified in vitro by a DNA polymerase or RNA polymerase to generate adequate amounts of target nucleic acids. Amplified nucleic acids are then either directly visualized on gels, or detected by specific hybridization probes.
Theoretically, PCR can detect one copy of a target sequence in a given sample. However, in practice, a PCR-based method can detect approximately 10 to 100 viral genomes in a background of 100 ng cellular DNA. Using PCR, small amounts of DNA in the range of 10 to 100 ngs can be detected. PCR is, therefore, the ideal method for the analysis of specimens with low DNA or RNA content. However, proper sample collection and handling is essential to achieve maximal sensitivity. Brush-sampling devices have been shown to be optimal for sample collection.
Most assays allow differentiating between high and low-risk virus infections. However, individual virus types cannot be identified without additional more specific tests or assays.
Examples of testing types or assays
Examples of testing types or assays used for virus detection are:
(1) Detection of proviral DNA by real-time PCR.
(2) Detection of viral infection and load using branched DNA (bDNA) tests.
(3) Detection of viral load by nucleic acid sequence-based amplification (NASBA)
(4) Detection of viral vectors such as adenovirus, MVA, AAV, and others, by
quantitative PCR and RT-PCR.
(5) Detection of cytokines and other biomarker responses.
(6) Detection of host restriction factors.
(7) Examination of virus-host interactions and global gene expression profiles.
These types of tests help to understand molecular mechanisms of immune
response and protection.
(8) Virus load determination.
Bio-Synthesis can help you synthesizing your synthetic standards and standardized material according your specifications. Bio-Synthesis has been successful using chemical and enzymatic synthesis for long DNAs and RNAs of up to 400bases. Oligonucleotides between 80-400 bases are synthesized using the latest nucleic acid technology and purified using DNAse and RNase free PAGE electrophoresis combined with HPLC. Our oligonucleotide synthesis services include mass spectrometry analysis. Purified oligonucleotides undergo stringent analytical HPLC or gel electrophoresis to determine final purity.
American Association for Clinical Chemistry (AACC) (https://www.aacc.org/store/cds/10100/viral-load-testing-diagnostic-principles-and-clinical-practice---cd).
U.S. Food and Drug Administration (FDA) – Guidelines for Standardized Assays and Protocols: http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm180306.htm
Center for Disease Control and Prevention:
Info on currently approved molecular tests at the Association of Molecular Pathology website: http://www.amp.org/
Roberta M. Madej, Jack Davis, Marcia J. Holden,Stan Kwang, Emmanuel Labourier, and George J. Schneider; Review; International Standards and Reference Materials for Quantitative Molecular Infectious Disease Testing. Journal of Molecular Diagnostics, , Vol. 12, No. 2, March 2010