Name: Lucius Harrison

Date: October 14, 2024

Course Code: MDSC1104

ID Number: 620172846

Title: Diagnostic/ QSTR

Section #1:
PART A
1.What is molecular diagnostic? (3 marks)
Molecular diagnostics is a field of medical testing which uses genetic material such as
DNA, proteins, and RNA in a fluid or tissue sample to identify diseases at a molecular level. It
utilizes techniques such as Polymerase chain reaction (PCR), Fluorescence In Situ Hybridization
(FISH) and ELISA (Enzyme-Linked Immunosorbent Assay). Apart from simply diagnosing a
disease, it also helps in planning treatments, identifying chronic diseases and examining the
effectiveness of treatment. Thus far, it has been indispensable in the diagnosis of diseases such as
Cystic fibrosis, cancers, Alzheimers and Covid-19.

2. (a) What is PCR Multiplexing? (3 marks)

PCR Multiplexing is a variation of standard Polymerase Chain Reaction which allows for
the amplification of multiple target sequences in a single PCR reaction. This is accomplished
through the use of multiple primers which amplify different regions of DNA or RNA at the same
time. This saves time and reagent resources as opposed to carrying out multiple separate
reactions for each target.
(b) State the difference between simple and complex multiplexing with examples. (10
marks)
PCR Multiplexing is a variation of standard Polymerase Chain Reaction which allows for
the amplification of multiple target sequences in a single PCR reaction. PCR multiplexing can be
separated into Simple and Complex multiplexing based on the number of DNA sequences being
tested.
Simple Multiplexing:
Simple multiplexing is designed for use with a small number of target DNA sequences in
that PCR reaction. This technique is utilized due to being easier to optimize and manage. In this
a limited amount of primer and probe sets are used, reducing the likelihood of cross-reactivity
and making it a more straightforward process in general.
Simple multiplexing is commonly used to test for specific mutations such as single
nucleotide polymorphism, typically associated with cystic fibrosis. In this case, only a couple of
primer pairs are necessary to detect a mutation hence simple multiplexing is optimal especially
due to its simplicity and minimal risk of interference.
Complex Multiplexing:
In contrast, complex multiplexing is used to detect multiple targets simultaneously,
typically reserved for larger sample sizes. This method involves the use of numerous primers
and probes, which can increase the risk of cross-reactivity and technical challenges. Regardless
of the increased risks, complex multiplexing is useful for more comprehensive analyses, such as
pathogen detection panels or large gene panels like in cancer research.
An example of complex multiplexing is in Respiratory Pathogen Detection, where a
single PCR reaction is used to identify the presence of multiple viruses, like influenza, RSV, and
SARS-CoV-2. This allows a broad testing sample enabling multiple pathogens to be identified in
a single PCR test. This is useful as opposed to simple multiplexing since it allows quick
identification of multiple diseases, especially in a situation where time may be of the essence in
detecting a disease being the difference between life and death for the patient. Although
beneficial, it also requires careful optimization as due to its complexity, errors are more likely to
occur.
(c) What is the GeneXpert system? (2 marks)
The GeneXpert system is an automated sample preparation and automated sample
preparation and real‐time PCR detection system. Studies have shown that it is capable of
simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. This capability
accompanied by its fully automated process, makes it useful for rapid detection, with high
accuracy more so, of highly infectious diseases such as COVID-19, tuberculosis and HIV.

(d) State how the Genexpert system works and discuss its advantages. (8 marks)
The Genexpert system is a molecular diagnostic tool used specifically in detecting
genetic material present within pathogens, this allows for identification of pathogens such as
tuberculosis through the analysis of the DNA or RNA found within samples collected from an
individual. The Genexpert system follows a series of 5 steps listed as follows;
Sample collection and preparation, Cartridge based technology and reagents, Nucleic acid
amplification and real time detection and Automated detection and reporting.
Step #1: Sample collection and preparation
Clinical samples are obtained from a patient in this step. The sample changes depending
on the disease being tested for examples being: sputum (commonly for tuberculosis), blood (for
viral load testing), urine (for urinary tract infections), and cerebrospinal fluid (for meningitis
pathogens). It can also analyze nasal swabs (for respiratory viruses) and rectal swabs (for
gastrointestinal pathogens).
After collecting the sample, they must then be prepared through the use reagents that
cause lysis of the pathogen. This is important as it allows the DNA and RNA of the pathogen to
be released, which then allows PCR testing to be a valid method to determine the type of
pathogen present. After lysis of the pathogens, stabilizing agents are added to prevent the natural
degredation of the genetic material.
Step #2: Cartridge-Based Technology and Reagents
A GeneXpert cartridge is a closed off, disposable, self contained unit with several
chambers preloaded with the necessary reagents. These reagents include Lysis reagents to break
down the cell walls of the pathogen. Binding agents to stabilize the nucleic acids. Reverse
transcriptase for RNA-based tests to transcribe RNA to DNA for cases of retroviruses such as
HIV or COVID-19 and finally, PCR reagents such as primers, nucleotides, polymerase enzymes,
and buffers for amplifying the target nucleic acid sequence. To complete this step, the prepared
sample from the previous step is inserted into the cartridge described above which is then placed
into a machine which automatically mixes, heats and processes the sample.

Title: Image of a GeneXpert cartridge

Annotation: Image above shows the cartridge along with the self contained units which
insert into them, the many holes in it can be deduced to be where the reagents are stored.

Step #3: Nucleic acid Amplification and Real time detection

Polymerase Chain Reaction or PCR is used to amplify target genetic material. A series of
thermal cycles are applied for mass target replication, Afterwards, fluorescence based markers
are added to amplify the DNA. Finally, a quantitative threshold is derived based on the amount
of cycles necessary to detect the pathogen DNA is calculated. Using a PCR is important as it
allows for the identification of a pathogen even if only in small amounts.
Step #4: Automated detection and Reporting
In this step, the GeneXpert system internal computer, automatically analyzes the signals
to determine if the target DNA is present. The machine can give qualitative and quantitative
results. In the form of a negative or positive result depending on if the DNA crosses the detection
threshold or in the form of a Ct value (cycle threshold) value which reflects how much pathogen
is present.
Advantages of GeneXpert system:
The GeneXpert system is a revolutionary technological development for the field of
medicine as it helps in the swift identification of diseases. Often times, time in diagnosis may be
the difference between whether a patient lives or dies therefore the most important benefit of the
GeneXpert system is the speed in which results are available. Furthermore, despite how fast
these results are obtained, there is relatively low error in accuracy whilst also being extremely
sensitive to any viral DNA present. Allowing for viruses to be accurately identified at even low
concentrations within the human body. This may enable detection of viruses even before they are
in high enough concentrations to cause any physical complications. Lastly, due to being
automated, the testing method is fairly easy to operate, allowing for a low skill floor in operating
which reduces the training time required to obtain qualified individuals for testing.
3. (a) What is real time PCR technology? (3 marks)
Real time PCR or Polymerase Chain Reaction is a technique used to identify and quantify
the type of DNA or RNA present in a sample. It works by using thermal cycles to rapidly
replicate sample DNA while using florescence dyes or probes to amplify the signal said DNA
strands give. These signals are amplified with each cycle, allowing for qualitative and
quantitative data to be obtained. It is notorious for its sensitivity, speed and accuracy in
diagnostics and research. PCR is used for detecting diseases such as HIV, AIDS and paternity
testing.
(b) What are the different controls utilized in PCR testing and state their importance? (6
marks)
PCR amplification is a high error and contamination prone technique which leads to
abnormal results and misinterpretations. Considering this, before accepting any data provided by
this testing method/to validate information obtained, controls need to be implemented. These
controls increase the reproducibility and accuracy of the information. There are three types of
controls which will be discussed: Positive controls, Negative controls and Internal controls
Positive controls:
This is a control sample that contains a known amount of target DNA or RNA. In this, a
detectable amplified signal should be prevalent if the PCR reaction works correctly. Positive
controls are often used to verify whether all reagents are working. When using the same positive
control during comparable (or the same type of) experiments, the cycle threshold should be very
similar, thus indicating consistency between multiple PCR runs. If a positive control fails to
produce a signal, there is likely to be an issue with the reagent or setup rather than the sample
itself making it good for detecting systematic errors within the PCR system. Example being In a
PCR test for COVID-19, the positive control included a sample with a known amount of
SARS-CoV-2 RNA.
Negative Control (No-Template Control):
A negative control is a reaction mix with all the PCR reagents but no DNA or RNA
template added. It is used to detect contamination of the reagents that would result in false
positive results being attained. In this reaction, no amplification is expected to occur. In the event
it does, then the sample is determined to be contaminated. Example being water, which was used
as a negative control in the Buzard et al test.
Internal Control:
Internal controls are essentially reference genes made from adding non-target DNA or
RNA sequences, which are added to each reaction. These genes are usually amplified alongside
the target DNA; therefore, it is important that they are not competitive with the DNA or RNA
being tested and furthermore, be easily distinguishable. Internal controls are used in nearly all
PCR reactions and serves to check the PCR reaction’s efficiency within each individual sample.
It helps confirm the lack of amplification in the test sample is due to the absence of the true
target sequence rather than PCR inhibition or reaction failure. Examples include human DNA
present in a test for HPV.

(c) What are the four phases of Real Time PCR amplification? (20 marks) NB. Please
explain each phase.
PCR testing is a ground breaking technological advancement which allows for the
identification of genes present within a sample, which allows for fast analysis of genetic samples,
allowing scientists to understand everything from which diseases are present to who the parent of
a child are. Real time PCR, differs from conventional PCR as the accumulation of amplification
product is measured as the reaction progresses rather than at the end point. Real Time PCR does
this in 4 distinct phases:
 Initiation Phase
The initiation phase is the first phase of real time PCR amplification. In this phase, the
DNA is essentially doubled but the signal of the DNA in this stage is too weak. This is because it
does not surpass the threshold of background noise, essentially making it undetectable. During
this phase, there are around 1 to 15 cycles. Consequently of this low fluorescence and subsequent
merging with the background noise, DNA amount is unable to be quantified.
This phase essentially acts as a baseline, which allows us to identify the signal strength
when it does start by giving the range at which the baseline begins. This essentially increases the
sensitivity and accuracy of the PCR test, as without it we’d be unable to identify exactly where
the PCR identification started.
Exponential phase
This phase is characterized on a real time PCR graph by its logarithmic shape. This is the
first phase where amplification is detectable. This detection is possible since the DNA doubles
with each cycle. This results in a strong fluorescent signal directly proportional to the steadily
growing DNA concentration. This usually occurs between 20 and 28 cycles.
Characteristically, a cycle threshold (Ct) value, which is a set fluorescence level used to
quantify the target DNA is obtained. In this stage, amplification efficiency is at its peak, causing
this stage to also be the most reliable when quantifying the initial amount of DNA present. These
traits make the Exponential phase essential since it allows for correlation of fluorescence to DNA
concentration.
Linear phase
As suggested by the name of this phase, this phase is often characteristic of a linear
increase of ‘amplification’ versus ‘no. of cycles’. The efficiency of the reaction decreases due to
the gradual consumption of the reagents and the accumulated PCR byproducts. However,
although decreased in rate, the fluorescence signal still increases in a linear matter as mentioned
above. This phase typically occurs between 27 and 35 cycles.
This site is not used as a measure of initial DNA quantification since the DNA no longer
doubles per cycle. Furthermore, the depletion of reagents and accumulation of byproducts make
the amplification rate less and less consistent which leads into the next phase.
Plateau phase
The plateau phase is the final phase of Real time PCR testing. In this phase the reaction
nears completion as reagents are almost completely used up, indicating that the PCR has reached
its endpoint. This phase is characteristic on a graph by a plateau as suggested by its name. This
phase typically begins until the 35th cycle and extends until the end of the PCR process.
This phase is important as it acts as an indicator of the end of the reaction. Meaning that
the reaction can no longer yield useful information for quantification. At this point the data taken
from the exponential phase can be used as a reference to calculate the quantification of the DNA
present.

Title: Graph of Real Time PCR amplification.

Annotation: From the image above it is possible to deduce the Initiation phase characteristic of
a flat line, the exponential phase found in the steep rise at approximately the 20th cycle, the
linear phase is slightly discernible as the graph flattens off whilst the plateau phase is marked and
easily discernible as the graph flattens off.
4. Briefly describe the five subclasses of antibodies. (25 marks)
In immunology, antibodies are important for protecting organisms from invasion and
death from microbes such as bacteria, viruses or fungi. They are categorized into 5 main
subclasses each with unique locations, structures, and roles in immune defense. These subclasses
and their characteristics are as follows:
1. IgA
Primarily found in mucosal areas such as the respiratory and gastrointestinal tracts, these
antibodies are responsible for mucosal immunity. This is achieved since they prevent pathogens
from adhering to and invading mucosal surfaces, essentially serving as the body’s first line of
defense. IgA often exists as a dimer when secreted. Examples of its function would be in the gut
where it neutralizes bacteria and viruses, preventing them from crossing to and infecting deeper
tissue. Apart from in the gut, IgA is also found in other bodily secretions such as saliva, tears and
breast milk.

2. IgD
IgD are antibodies found predominantly on the surface of B cells which are found in the
bone marrow, they are also found in small amounts in the blood. Typically they are responsible
for B cell regulation and activation. Although unclear, scientists believe that there is correlation
between IgD and the early immune system, by enabling B cells to recognize antigens. IgD is
considered and outlier amongst antibodies as it has limited abundance and a relatively undefined
role in immunity.
3. IgE
IgE is commonly found attached to immune cells like basophils and mast cells. Its
primary function encompasses allergic reactions and responses to parasitic infections. It binds to
allergens, which triggers the release of histamines and basophils from the mast cells resulting in
inflammation. This ability and response account for its association with allergic reactions.
Finally, IgE activates immune cells targeting parasites such as worms, which are typically
difficult to detect by the immune system.
4. IgG

Continuing, IgG is the most abundant antibody in the blood and extracellular fluid and
plays a significant role in long-term immunity and memory responses. Its primary functions
include neutralizing pathogens, opsonizing them for phagocytosis, and activating the
complement system to destroy pathogens. IgG has multiple subtypes, like IgG1 and IgG2, which
have specialized roles in immune defense. Essentially, IgG is vital in the immune response to
vaccinations, as it helps provide long-lasting immunity by neutralizing pathogens and facilitating
their removal.

5. IgM
 Last but not least, IgM is primarily found in the blood and lymphatic fluid and is the first
antibody produced in response to an infection. It has a pentameric structure, meaning it forms a
large complex of five antibody units, which allows it to bind effectively to antigens and form
large antigen-antibody complexes. This makes IgM very effective in the initial stages of
infection, where it can activate the complement system and help clear pathogens quickly before
IgG levels rise. An example of IgM’s role is in acute infections, where its presence is often used
as an indicator of a recent infection.

5. Determine the STI pathogens infecting each patient (See STI Activity sheet). Tabulate
your response, table should be appropriately labelled. (70 marks)

Table #1: Showing results for tests for UT1 PP and UT2 PP STI presence
within each patient

Dye colors indicating pathogen presence

UT1 PP UT2 PP

Patient Neisseria Mycopla Chlamyd mCMV Patient HSV1/2 Ureaspla Tromona Gardene
sample gonorrho sma ia (IC) sample (Green) sma s rella
number eae genitaliu Trachom (Red) number urealytic vaginalis vaginalis
(Green) m atis um/parv (Orange) (Red)
(Yellow) (Orange) um
(Yellow)

24-0210 Negative Negative Negative Positive 2101.2 Negative Negative Negative Negative
1 STI9

24-0210 Negative Negative Negative Positive 2102.2 Negative Positive Negative Positive
2 STI9
24-0210 Negative Negative Negative Positive 2103.2 Negative Negative Negative Negative
3 STI9

24-0210 Negative Negative Negative Positive 2104.2 Negative Positive Negative Positive
4 STI9

24-0210 Negative Negative Positive Positive 2105.2 Negative Positive Negative Positive
5 STI9

24-0210 Negative Negative Negative Positive 2106.2 Negative Negative Negative Negative
6 STI9

24-0210 Negative Negative Negative Positive 2107.2 Negative Negative Negative Negative
7 STI9

24-0210 Negative Negative Negative Positive 2108.2 Negative Negative Negative Negative
8 STI9

24-0210 Negative Negative Positive Positive 2109.2 Negative Positive Negative Positive
9 STI9

24-02113 Negative Negative Negative Positive 2113.2 Negative Negative Negative Negative
STI9

24-02114 Negative Negative Negative Positive 2114.2 Negative Positive Negative Positive
STI9

24-02115 Negative Negative Positive Positive 2115.2 Positive Negative Negative Positive
STI9

24-02116 Negative Negative Negative Positive 2116.2 Negative Negative Negative Negative
STI9

24-02117 Negative Negative Negative Positive 2117.2 Negative Negative Negative Negative
STI9

24-02118 Negative Negative Negative Positive 2118.2 Negative Positive Negative Negative
STI9

24-02119 Negative Negative Negative Positive 2119.2 Negative Positive Negative Negative
STI9

24-0212 Negative Negative Negative Positive 2120.2 Negative Negative Negative Negative
0 STI9

24-0212 Negative Negative Negative Positive 2121.2 Negative Negative Negative Negative
1 STI9

-VE#3 Negative Negative Negative Negative -VE#3.2 Negative Negative Negative Negative
24-0212 Negative Negative Negative Positive 2122.2 Negative Positive Negative Negative
2 STI9

24-0212 Negative Negative Negative Positive 2129.2 Negative Negative Negative Negative
9 STI9

24-0214 Negative Negative Negative Positive 2144.2 Negative Negative Negative Negative
4 STI9

24-0214 Negative Negative Negative Positive 2145.2 Negative Positive Negative Positive
5 STI9

24-0214 Negative Negative Negative Positive 2146.2 Negative Positive Negative Negative
6 STI9

24-0214 Negative Negative Negative Positive 2149.2 Negative Negative Positive Positive
9 STI9

24-0215 Negative Negative Negative Positive 2150.2 Negative Positive Negative Negative
0 STI9

24-0215 Negative Negative Negative Positive 2151.2 Negative Negative Negative Positive
1 STI9

24-0215 Negative Negative Negative Positive 2152.2 Negative Positive Negative Positive
2 STI9

24-0215 Negative Negative Negative Positive 2153.2 Negative Positive Negative Positive
3 STI9

24-0215 Negative Negative Negative Positive 2154.2 Negative Positive Negative Negative
4 STI9

24-0215 Negative Negative Negative Positive 2155.2 Negative Positive Negative Positive
5 STI9

24-0215 Positive Negative Negative Positive 2156.2 Negative Positive Negative Positive
6 STI9

24-0215 Negative Negative Negative Positive 2157.2 Negative Positive Negative Positive
7 STI9

24-0215 Negative Negative Negative Positive 2158.2 Negative Negative Negative Negative
8 STI9

NTC.1 Negative Negative Negative Negative NTC.2 Negative Negative Negative Negative

PTC.1 Positive Positive Positive Negative PTC.2 Positive Negative Positive Positive
Appendix:
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