Molecular Methods in Virology

• Molecular methods are used in the Department of Virology at NHLS, Inkosi Albert Luthuli Central Hospital.
• The lecture on this topic was given by Melen Pillay on March 26th and 27th, 2025.

Overview of Molecular Methods

• The lecture covers introduction to molecular methods, target amplification, probe amplification, signal amplification, limitations, and learning objectives.

Introduction to Molecular Methods

• Effective clinical management of viral infections relies on the rapid and specific identification of the causative organism.
• Early recognition of infectious agents enables clinicians to make sound therapeutic decisions.
• Traditional methods include virus isolation by cell culture or antibody detection.
• Molecular diagnostic tools and nucleic acid amplification are used for identifying viral pathogens.

Target Amplification: Polymerase Chain Reaction (PCR)

• PCR is an efficient method to amplify low levels of specific DNA sequences to reach the threshold of detection.
• PCR uses short DNA primers (oligonucleotides) specific to the pathogenic DNA, flanking the DNA section to be amplified.
• PCR involves repeated cycles of DNA denaturation, primer annealing, and extension of the primed DNA sequence.
• PCR is used to amplify a specific region of a DNA strand, known as the DNA target.
• Most PCR methods amplify DNA fragments between 0.1 and 10 kilo base pairs (kb).
• The amount of amplified product is determined by the available substrates.

Three-Step PCR Process

• Denaturation of Template DNA: Heat causes DNA strands to separate, occurring at 94°C.
• Primer Annealing: Lower temperature allows primers to bind, typically between 37-65°C.
• Extension: Taq polymerase makes complementary strands at the optimum temperature for the enzyme, 72°C.

Key PCR Components and Their Functions

• DNA Template: Extracted from a patient specimen, contains the region of interest.
• DNA Polymerase: Incorporates dNTPs into the growing strand, crucial for strand synthesis and elongation.
• Primers: Induce PCR by annealing to the target region; are short, single-stranded DNA fragments complementary to the template.
• dNTPs: Free-floating nucleotide bases used for synthesizing complementary DNA strands.
• Buffer Solution: Maintains a pH effective for PCR.
• PCR grade Water: Must be DNAase/RNAase free, usually considered a major source of contamination.
• MgCl2: A cofactor for DNA polymerase, enhancing its activity, promoting DNA amplification, stabilizing DNA structure, and facilitating primer annealing.

PCR Detection Methods: Real-Time PCR

• Real-time PCR is the greatest leap in DNA amplification technology since the development of PCR.
• It is achieved through the use of fluorescent detection technology.
• Fluorescent molecules are added to the PCR reaction mix which interact with the PCR product, increasing the fluorescent signal when PCR amplification occurs.
• A significant benefit of real-time PCR is that it quantifies the amount of RNA or DNA in specimens.
• Quantitative PCR (qPCR) involves amplification beginning sooner in specimens with higher viral loads.
• qPCR results are expressed in absolute terms like copies per mL, using known internal quantified standards.

Limitations of End Point PCR

• End point PCR can be limited in its ability to accurately quantify the amount of amplified product.

Real Time PCR Detection Metrics

• Controls are used to validate the run, and targets are checked at 530nm and internal controls (IC) are checked at 560nm.
• Specimen Control (SC) should not validate the run, with no CT at either target or internal control.
• Quality Standard Positive (QS3) should validate the run, with a CT between 30-35 at the target, and no CT at the internal control.
• Internal Control Negative (IC2WO) should validate, with no CT at the target, and a CT at the internal control.
• Water Negative Control (RO) should validate, with no CT at the target or internal control.

Target Amplification Methods (Continued)

• Nucleic Acid Sequence-Based Amplification (NASBA)
• Transcription-mediated amplification (TMA)
• Rolling circle amplification (RCA)
• Loop-Mediated Isothermal Amplification (LAMP)

Nucleic Acid Sequence-Based Amplification (NASBA)

• NASBA, initially known as transcription-based amplification, amplifies a target sequence via in vitro transcription.
• Isothermal nucleic acid amplification of RNA uses simultaneous action of 3 enzymes.
• These enzymes are avium myeloblastosis virus reverse transcriptase, T7 RNA polymerase, and RNASE H.
• NASBA amplifies RNA from an RNA target using a dual-function reverse transcriptase to produce multiple copies of single-stranded RNA.

Transcription-Mediated Amplification (TMA)

• TMA is a variation of NASBA, using one enzyme, RNA polymerase.
• The RNA polymerase has reverse transcriptase and RNaseH activity, enabling RNA transcription.
• TMA can target either DNA or RNA, leading to RNA amplification and DNA synthesis.
• TMA has a lower possibility of carryover contamination because RNA is more labile compared to DNA.

Rolling Circle Amplification (RCA)

• RCA amplifies a circular target molecule by extending a single forward primer with DNA polymerase for many rounds.
• Polymerase displaces upstream sequences, yielding long, single-stranded DNA with multiple repeat copies of the target sequence.
• Modification of RCA uses two primers for exponential, hyper-branched, or cascade amplification.

Loop-Mediated Amplification (LAMP)

• LAMP involves auto-cycling strand-displacement DNA synthesis.
• LAMP uses strand-displacing DNA polymerase and two pairs of target-specific primers.
• Inner primers have two sequences corresponding to the sense and anti-sense (rc) strands of the target DNA.
• Inner primers initiate a complementary synthesis of the target DNA.
• Outer primers (FP1 & RP3) initiate strand-displacement synthesis which releases single-stranded DNA linked by inner primers.
• Single-stranded DNA forms stem-loops by self-annealing, templating exponential amplification.

Probe Amplification Methods: Ligase Chain Reaction (LCR)

• LCR uses two pairs of probes that are complementary to each other.
• Unlike PCR, the probes or primers do not flank the target sequence; they cover the target DNA immediately adjacent to one another, typically leaving a gap of 1-3 bases.
• This gap between probes acts as a template for ligation by thermostable DNA ligase, joining the two probes.

Cycling Probe Technology

• Cycling Probe Technology detects nucleic acid target sequences without target amplification.
• RNA-DNA chimeric probe - RNA sequence hybridizes to complementary target DNA sequence flanked by two DNA sequences.
• Fluorescence signal increases proportionally as probe is cleaved, allowing for measurement of amplified product.
• Probe amplification is linear, not exponential, which eliminates carryover contamination.

Signal Amplification Methods: Branched DNA (bDNA)

• bDNA involves simultaneous hybridization steps using many types of oligonucleotide probes.
• Capture probes include target probes, novel branched secondary probes, and short enzyme-linked tertiary probes.
• Enhancement is achieved through probe redesign using novel nucleotides (isoC & isoG) in probes.

Hybrid Capture

• Target DNA and specific RNA probes combine to form an RNA:DNA hybrid, captured onto a solid phase using antibodies specific to RNA:DNA hybrids.
• Captured hybrids are detected by a secondary antibody attached to an enzyme, which cleaves a chemiluminescent substrate, producing a light signal.
• Each hybrid combines with many antibody-enzyme molecules, amplifying the resultant signal.
• The major benefit of hybrid capture is that amplification reduces the possibility of cross-contamination.

Limitations of Molecular Methods

• Molecular tests using nucleic acid amplification have many advantages, but caution should still be exercised.
• If only one copy of DNA from a laboratory contaminant or previous experiment is present, a false-positive result will occur.
• Conversely, inhibitors in specimens or nucleic acid degradation may lead to false-negative results.

Learning Objectives

• Describe PCR process
• List PCR component and their functions
• Describe the Principle of PCR
• List the enzymes used in NASBA and TMA
• Describe rolling cycle amplification
• Describe Hybrid Capture
• Limitations of molecular amplification methods