Basic Viro

Question 1

PCR principles

Answer 1

1985 Kerry Mullis

Primer mediated enzymatic amplification of DNA.
Using a DNA polymerase to synthesise a strand of DNA complementary to the template strand.

Question 2

Components of PCR

Answer 2

1. DNA template
2. DNA polymerase - Taq polymerase that doesn’t denature 98 C and can function at optimum temp of 70 C
3. Oligonucleotide primers (20 to 30 bp) complementary to 3’ ends of the sense and anti-sense strands of the target sequence.
4. Deoxynucleotide triphosphates: A,T,G, and C
5. Buffer: magnesium and potassium for stability

Question 3

PCR steps

Answer 3

1. Denaturation: heating mixture to 94 C for 15-30 seconds. DNA denatured into single strands.
2. Annealing: temp lowered to 54-60C for 20-40 seconds. Primers bind (anneal) to the simple mentally sequences of template DNA
3. Elongation/Extension: 72-80C. Polymerase adds bases to each 3’ primer and extends the DNA sequence in 5’ to 3’ direction.
4. DNA binding Dyes: most commonly used is SYBR green which non specifically binds to dsDNA and exhibits fluorescence 1000-fold when binding.

Advantages: simple design, can be used for multiple genes, lower cost

Disadvantages: lack of specificity - binds to all ds DNA. Cannot be used in multiplex (parallel reaction mixtures need setting up).

2. PCR primer-probe based detection: use some form of fluorescence quenching to ensure target specific fluorescence is detected only when the amplicon of interest is present.
   Primer/target-specific oligonucelotide prove us labelled with a reporter fluorophore and it’s fluorescence is quenched when the specific target is NOT present. This is by covalentlu attaching a quencher molecule to the primer or prove and there exists a mechanism by which the reporter and quencher separate when primer/probe binds to the target.

Eg: Taqman probes - hydrolysis assay. Has a fluorescent reporter at 5’ and a quencher at 3’ end. Common reporter-quencher pairs at fluorescein (FAM) enoting green fluorescence and black hole quencher 1 dye.

Question 4

Serotypes

Answer 4

A serotype is defined as a variation within a microbial species, distinguished by the humoral immune response. The serotype classification of bacteria or viruses is based on their surface antigens and was established before the availability of other techniques, such as genome sequencing or mass spectrometry. Antibodies generated to one serotype do not usually efficiently protect against another serotype. Serotypes have been described in many viral species and generally correspond to genotypes.

Question 5

Transcription vs translation

Answer 5

The purpose of transcription is to make RNA copies of genes. The purpose of translation is to synthesize proteins for cellular functions. Translation produces proteins, while transcription produces mRNA, tRNA, rRNA, and non-coding RNA. In prokaryotes, translation and transcription occurs in the cytoplasm

Question 6

LAMP

Answer 6

loop-mediated isothermal amplification of DNA or RT-LAMP for RNA.

The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets.

the target sequence is amplified at a constant temperature of 60–65 °C

Uses Bst – Bacillus stearothermophilus – DNA polymerase.

Question 7

Generations of PCR

Answer 7

The first generation of PCR relies on gel electrophoresis to analyze PCR products, but always challenged by low detection limit, laborious operation, and single application (qualitative). The second generation of PCR, also called real-time quantitative PCR (RT-qPCR), can quantify the products with standard curves, but also show low tolerance to interfering substances [1]. Digital PCR (dPCR) is the third generation of PCR that enables absolute quantification through partitioning the reaction. Highly sensitive and accurate in molecular detection, this technology has demonstrated applications like trace DNA detection, rare mutation detection and copy number variation

Question 8

Digital PCR

Answer 8

Digital PCR builds on traditional PCR amplification and fluorescent-probe–based detection methods to provide highly sensitive absolute quantification of nucleic acids without the need for standard curves.

In the Droplet Digital™ PCR System, a PCR sample is partitioned into 20,000 droplets. After amplification, droplets containing target sequence are detected by fluorescence and scored as positive, and droplets without fluorescence are scored as negative. Poisson statistical analysis of the numbers of positive and negative droplets yields absolute quantitation of the target sequence.

Advantages:
1. Small sample quantity
2. Sensitive and specific
3. Less sensitive to inhibition
4. copy number variation (CNV) analysis

Question 9

Intrathecal Ab testing

Answer 9

Ab index = viral ab ratio/ albumin ratio

Viral ab ratio = CSF ab/serum ab in paired sample
Albumin ratio = CSF albumin/ serum albumin

Ratio < 3 is normal
Raised controls (eg rubella, VZV, HSV) indicates a polyclonal response within the CNS

Question 10

Antibody avidity testing

Answer 10

2 wells

1st well: bound antigen to plate + test serum - washed with 0.85% NACL, then anti-human IgG HRP conjugate added. In acute & chronic infection, antigen antibody complex will NOT be disrupted.

2nd well: bound antigen to plate + test serum - washed with 8M urea, then anti-human IgG HRP conjugate added. In acute infection, Ab-Ab complex will be disrupted and ab will be washed away. In chronic, this will not happen due to stronger bond.

Avidity index = OD Urea/OD NACL x 100%. Eg for CMV, 80% is the cut off used to say high vs low avidity.

Question 11

What is an open reading frame?

Answer 11

In molecular biology, reading frames are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible reading frames will be “open” (the “reading”, however, refers to the RNA produced by transcription of the DNA and its subsequent interaction with the ribosome in translation). Such an ORF may contain a start codon (usually AUG in terms of RNA) and by definition cannot extend beyond a stop codon (usually UAA, UAG or UGA in RNA). That start codon (not necessarily the first) indicates where translation may start. The transcription termination site is located after the ORF, beyond the translation stop codon. If transcription were to cease before the stop codon, an incomplete protein would be made during translation

Question 12

Sanger sequencing

Answer 12

AKA chain termination method

3 main steps:

1. DNA sequence for chain termination PCR:
   *The DNA sequence of interest is used as a template for a special PCR called chain termination PCR (CTP).
   * CTP works like a standard PCR except for the addition of modified nucleotides (dNTPs) called dideoxyribonucleotides (ddNTPs).
   *In standard PCR, during extension phase, DNA polymerase adds dNTPs to the growing chain by catalysing formation of phosphodiester bond between the free 3’OH of the last nucleotide with the 5’phosphate of the next nucleotide. SEE FIGURE IN PHONE ALBUM.
   * In CTP, ddNTPs lack the 3’OH group required for binding - so when ddNTPs are incorporated by the DNA polymerase at random, chain termination occurs.
   * Millions to billions of oligonucleotode copies of the DNA sequence of interest are made at random lengths terminated by ddNTPs.
2. Size separation by gel electrophoresis:

*The oligonucleotides are separated by gel electrophoresis.
*DNA samples are loaded into one end of a gel matrix and electric current applied.
*As DNA is negative charged, it’ll be pulled towards the positive electrode on the opposite side.
* The speed of movement will depend on the charge which is determined by the size.
* Smaller fragments move quicker.

3. Gel analysis and determination of DNA sequence

*Reading the gel to determine the sequence of the input DNA.
* As DNA polymerase only makes DNA in 5’ to 3’ direction from a starting primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence.
*Reading gel bands from smallest to largest will determine the 5’ to 3’ sequence of the original DNA.

Reading a Sanger sequence:
Depends on which of the two complementary DNA strands is of interest and what primer is available. For eg: if the strands are A & B and the strand of interest is A, but the primer is more suited to strand A, the output will be a sequence of strand B and then the computer has to convert the letters to the sequence of strand A.

Question 13

Basic DNA structure

Answer 13

1. Double helix
2. Each strand is made of string of molecules called deoxyribonucleotides (dNTPs).
3. Each dNTP contains a phosphate group, sugar group, and 1 of 4 nitrogenous bases (A,T,G,C).
4. dNTPs are strung together in a linear fashion by phosphodiester bonds between sugar of one dNTP (3’OH) and the phosphate group of the next.
5. The nucleotides are bound together by hydrogen bonds between complementary bases.

SEE FIGURE IN ALBUM

Question 14

Types of NGS

Answer 14

1. WGS
2. Targeted metagenomics
3. Shotgun metagenomics

Question 15

Generations of sequencing

Answer 15

1st gen - Sanger

2nd gen - Pyrosequencing - measuring pyrophosphate synthesis instead of fluorescently labelled nucleotides

3rd gen - ability to perform single molecule sequencing which means there is NO NEED for DNA amplification step. Higher throughput and longer reads being sequenced. Eg: Oxford nanopore

Question 16

Oxford nanopore mechanism

Answer 16

Guides ssDNA through a grid of protein nano pores that gather the DNA sequence through electrical current disruptions.

Advantages: Faster & does not require prior amplification

Disadvantages: more sequencing errors and higher per-read cost

Question 17

FRET PCR vs TAQMAN

Answer 17

FRET - Dual hybridisation using 2 probes to increase specificity. Both probes must bind to produce fluorescence - happens by energy transfer between probes.
Also give melt curves.

TAQMAN - Hydrolysis probe using quencher and reporter. More sensitive but less specific and does not have melt curve analysis.

Question 18

Immunofluorescence testing

Answer 18

Direct: Slide has fixed antigen (of interest). Primary antibody labelled with fluorphore added and fluorescence looked for. Wash step - will wash away the antibody if not compatible with antigen.

Indirect: slide fixed with antigen. Primary antibody added. Secondary anti-human antibody with attached flurophore added which binds ti primary antibody and fluorescence looked for.

Disadvantage: Reader subjectivity

See image in album

Question 19

Complement fixation assay

Answer 19

SEE ALBUM

Hemolysin sensitised RBCs lysed by unbound complement in the absence of antibodies of interest

Complement from hamster
RBC from sheep

Question 20

EIA aka ELISA

Answer 20

SEE ALBUM

1. Direct: coating an ELISA plate with samples containing potential antigens, then adding a primary antibody that is enzyme-conjugated.

This antibody will bind directly to the antigen if present. After incubating, any unbound antibodies are washed away, leaving only the antigen-primary antibody complexes. Next, a substrate is added that reacts with the enzyme linked to the antibody, causing a color change.

2. Indirect: primary antibody is unlabeled and a secondary labeled antibody is used to bind to the primary antibody. After incubating the plate with samples containing antigens and incubating with the primary antibody, labeled secondary antibodies are then added that bind to any attached primary antibody.
3. Sandwich: First, the ELISA plate is coated with capture antibodies that bind to the target protein. After introducing a sample, these antibodies grab onto any of the proteins present. Following this step, detection antibodies are added on top of the bound protein, which are typically linked to an enzyme or have a tag that allows for visualization. Next, a substrate is added into the mix, which the enzyme-linked antibody will react with to produce a measurable signal.
4. Competitive: coating an ELISA plate with these antibodies. Then, a mixture of unlabeled antigen from the sample and labeled antigen is introduced to each well. If high amounts of target protein are present in the sample, they will occupy more of the binding sites, leaving fewer sites for the labeled antigen. During detection, only bound enzymes emit signals—higher target concentration results in weaker signals because fewer enzyme-conjugated antigens remain attached to antibodies.

Question 21

Immunodiffusion & Prozone effect

Answer 21

SEE ALBUM

soluble Ag & Ab combine to form insoluble Ag-Ab combo which produces visible lines on the gel - remember mayo!

Prozone - too much Ab - false negative
Postzone - too much Ag - false negative

Question 22

ACDP categories

Answer 22

Advisory committee on dangerous pathogens

Based on:
* ability to cause infections
* risk of community spread
* availability of vaccines or treatment

1-4 groups

Question 23

Viruses replicating inside nucleus

Answer 23

All DNA viruses except Poxviruses
RNA viruses - Orthomyxo & retroviruses

Question 24

Liposome

Answer 24

Nucleic acid removed from an envelope - used for administration of drugs/genetic material

Question 25

Basic viral structure

Answer 25

Icosehedral shell - 20 equilateral triangles Envelope - lipid bilayer derived from host cells Capsid - protein shell around the nucleic acid Nucleocapsid - RNA + Capsid (word used when there is a lipid envelope around it) Tegument - protein between nucleocapsid & envelope Pox viruses - complex viruses which doesn’t follow the above typical structure

Question 26

Reverse transcriptase viruses

Answer 26

1. RNA retroviruses are pseudo diploid - has 2 identical copies of (+)ss RNA - provides 2 copies for DNA transcription if one copy is damaged 2. RNA genome transcribed into dsDNA in the CYTOPLASM 3. RdDP is very error prone - high degree of mutations Viruses with RtP: HIV, HBV

Question 27

HERVS & retrotransposons

Answer 27

Human endogenous retroviruses - defective integrated retrovirus sequences which cannot make viruses Retrotransposons - sequences from F retroviruses integrated in human genomes

Question 28

Isolate vs Variant vs Strain vs Clade vs lineage vs Serotype vs Genotype

Answer 28

Isolate - isolated from an infected host & propagated in culture Variant - isolate whose genome differs from a reference virus (even by 1 nucleotide change). Strain- variant that’s possesses unique & stable phenotypic characteristics. New strains are very uncommon and takes time to develop. Clade - group of viruses composed of an ancestor & its descendants. Lineage: A lineage is a single line of descent or linear chain within the tree Serotype - viruses of the same species that are antigenically different - infection by one will not afford protection from another. Genotype - Genetic make up of the virus.

Question 29

qPCR calculations - How to evaluate the performance of a real-time PCR reaction

Answer 29

High efficiency = higher sensitivity at lower concentrations of sample. Ideal target efficiency = 90 to 110% How to evaluate Efficiency 1. Dynamic range - minimum of 3 replicates and a minimum of 5 logs of template concentration are run. Target: A slope of –3.3 ±10% reflects an efficiency of 100% ±10%. 2. R2 (squared) - statistical term that indicates how good one value is at predicting another. R2 of 1 = one value can be used to predict another. R2 of 0 means one value cannot predict another. 3. Precision - The standard deviation (square root of the variance) is the most common measure of precision. If many data points are close to the mean, the standard deviation is small; if many data points are far from the mean, the standard deviation is large. If a PCR is 100% efficient, the Ct difference between two successive concentrations in a 2-fold dilution is 1. 4. sensitivity - efficiency is a key factor in determining the sensitivity of a reaction. Another important consideration when detecting very low copy numbers is that normal distribution of template is not expected. Instead, a Poisson distribution is followed, which predicts that in a large number of replicates containing an average of one copy of starting template, approximately 37% should actually have no copies, only 37% should contain one copy, and 18% should contain two copies. Thus, for a reliable low copy detection, a large number of replicates are necessary to provide statistical significance and to overcome the Poisson distribution limitation. https://www.thermofisher.com/uk/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/real-time-pcr-understanding-ct.html#:~:text=R2%20value,value%20of%20(Figure%207A).

Question 30

Delta Ct vs delta delta ct

Answer 30

The delta-delta Ct method, also known as the 2–∆∆Ct method, is a simple formula used in order to calculate the relative fold gene expression of samples when performing real-time polymerase chain reaction (also known as qPCR). The method was devised by Kenneth Livak and Thomas Schmittgen in 2001. ∆Ct = Ct (gene of interest) – Ct (housekeeping gene) ∆∆Ct = ∆Ct (Sample) – ∆Ct (Control average)

Question 31

Southern vs Northern vs Western vs Eastern blots

Answer 31

All similar - done on agarose gel with electrophoresis Southern - detects DNA - paternity tests Northern - detects RNA - HIV Western (aka Immunoblot) - detects proteins - Lyme, CJD Eastern - detect post-translational modifications of proteins, - carbohydrates

Question 32

VOI/VOC analysis for existing NAAT

Answer 32

In silico refers to scientific discoveries that are made using computer simulation instead of biological studies. 34A of the Medical Devices Regulations 2002 If a test is re-designed, changed, or an IFU is updated, to retain UKCA/CE marking the manufacturer may also need to perform a conformity assessment and possibly gain approval from its Approved Body or Notified Body, depending upon the classification of the IVD under the UK MDR 2002. To address this change to the IVD or to correct another problem, the manufacturer may also need to submit a Field Safety Notice (FSN) and Field Safety Corrective Action (FSCA) form to the MHRA, and to send the FSN to their customers to alert them of any changes and of corrective actions to mitigate risk Wet lab: Primer & probe tested against new variant done in addition to in-silico - Where sequence variation is detected in primer or probe target sequences and this is predicted to impact assay performance, manufacturers shall conduct confirmatory wet lab testing of their product or assay against the variant sequence in question.