SLP PCR

a. What have I learned from this SLP?
 Firstly, I learned about PCR (Polymerase Chain Reaction). PCR is a revolutionary method
developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA
polymerase to synthesize new strand of DNA complementary to the offered template strand.
Because DNA polymerase can add a nucleotide only onto a pre-existing 3'-OH group, it
needs a primer to which it can add the first nucleotide.
 This requirement makes it possible to delineate a specific region of template sequence that
the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will
be accumulated in billions of copies (amplicons).
 Some of the components of PCR are DNA template which is the sample DNA that contains
the target sequence. At the beginning of the reaction, high temperature is applied to the
original double-stranded DNA molecule to separate the strands from each other.
 Next, DNA polymerase which is a type of enzyme that synthesizes new strands of DNA
complementary to the target sequence. The first and most commonly used of these enzymes
is Taq DNA polymerase (from Thermis aquaticus), whereas Pfu DNA polymerase (from
Pyrococcus furiosus) is used widely because of its higher fidelity when copying DNA.
Although these enzymes are subtly different, they both have two capabilities that make
them suitable for PCR: 1) they can generate new strands of DNA using a DNA template and
primers, and 2) they are heat resistant.
 Then, Primers a short piece of single-stranded DNA that are complementary to the target
sequence. The polymerase begins synthesizing new DNA from the end of the primer.
 Plus, Nucleotides (dNTPs or deoxynucleotide triphosphates) which is a single unit of the
bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.
 Lastly, RT-PCR (Reverse Transcription PCR) is PCR preceded with conversion of sample
RNA into cDNA with enzyme reverse transcriptase
 Limitations of PCR and RT-PCR
 Some of the limitation of the PCR and RT-PCR are PCR reaction starts to generate copies of
the target sequence exponentially. Only during the exponential phase of the PCR reaction is
it possible to extrapolate back to determine the starting quantity of the target sequence
contained in the sample. Because of inhibitors of the polymerase reaction found in the
sample, reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of
the accumulating product, the PCR reaction eventually ceases to amplify target sequence at
an exponential rate and a "plateau effect" occurs, making the end quantification of PCR
products unreliable. This is the attribute of PCR that makes Real-Time Quantitative RT-
PCR so necessary.
b. Why is it important to detect MTB and especially MDRTB?
 Unprecedented sensitivity for detecting MTB — even in smear-negative, culture positive
specimens
 Simultaneous detection of both MTB and rifampin resistance mutations, which are markers
for MDR-TB strain
 On-demand results that empowers physicians to manage patients effectively,
 Acceptable samples include raw or concentrated sediments prepared from induced or
expectorated sputum
c. What are the advantages and disadvantages of using PCR in detection of MTB?
 Some of the advantages of using PCR in detection of MTB are PCR are quick, reliable,
sensitive, relatively easy and specific.
 The disadvantages of using PCR in detection of MTB are PCR need for equipment, Taq
polymerase is expensive, easily exposed to contamination, potentially bring out false
reactions, internal control, cross-reaction, enrichment steps in (contaminated) samples.
capacity building needed and unspecific amplification.
d. What is the definition of MDRTB?

 Multidrug-resistant tuberculosis (MDR-TB) is defined as TB that is resistant to both
isoniazid [INH] and rifampicin [RMP], two of the first-line drugs used in treating
smear-positive pulmonary tuberculosis.
e. How do MDRTB appear?

 The 2 reasons why multidrug resistance continues to emerge and spread are
mismanagement of TB treatment and person-to-person transmission.
 Most people with TB are cured by a strictly followed, 6-month drug regimen that is
provided to patients with support and supervision. Inappropriate or incorrect use of
antimicrobial drugs, or use of ineffective formulations of drugs (such as use of single
drugs, poor quality medicines or bad storage conditions), and premature treatment
interruption can cause drug resistance, which can then be transmitted, especially in
crowded settings such as prisons and hospitals.
 In some countries, it is becoming increasingly difficult to treat MDR-TB. Treatment
options are limited and expensive, recommended medicines are not always available,
and patients experience many adverse effects from the drugs. In some cases even more
severe drug-resistant TB may develop.
 Drug resistance can be detected using special laboratory tests which test the bacteria for
sensitivity to the drugs or detect resistance patterns. These tests can be molecular in
type (such as Xpert MTB/RIF) or else culture-based. Molecular techniques can provide
results within hours and have been successfully implemented even in low resource
settings.
 Solutions to control drug-resistant TB are to:
o cure the TB patient the first time around
o provide access to diagnosis
o ensure adequate infection control in facilities where patients are treated
o ensure the appropriate use of recommended second-line drugs.

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a. What have I learned from this SLP?

d. What is the definition of MDRTB?

e. How do MDRTB appear?

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