PCR-based assay to distinguish between community-acquired methicillin-resistant
Staphylococcus aureus (CA-MRSA) and hospital-acquired methicillin-resistant
Staphylococcus aureus (HA-MRSA) strains and determination of their antimicrobial
susceptibilities.
The PCR technique is a product of the current molecular diagnostics era. Real-time PCR and
qPCR have created a revolution in molecular analysis and the detection of strain-specific nucleic
acids. This report discusses the amplification of community-acquired methicillin-resistant
Staphylococcus aureus (CA-MRSA) by performing a qPCR-based assay and designing gene-
specific primers along with the determination of their antimicrobial susceptibilities against six
antibiotics. A graph plot representing the delta Rn values obtained by qPCR was analysed to
differentiate between the community-acquired methicillin-resistant Staphylococcus aureus (CA-
MRSA) and hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strains.
Disk diffusion tests and E-tests were performed to examine the susceptibilities of S. aureus
FPR3757 and S. aureus MRSA252 against primary antibiotics. The S. aureus gene LmrS appears
to be an important predictor of antibiotic resistance in Staphylococcus aureus, according to this
study. To propose how microbial isolates effect its expression and the breadth of its
antimicrobial resistance, more research into its genomic physiology is needed.
Introduction
Due to the high morbidity and mortality of infections caused by the Staphylococcus aureus
strains, the rise of methicillin-resistant strains (MRSA) and other antimicrobials has become a
significant problem, particularly in a healthcare system (CDC, 2019) (Harmsen D, 2003 ). A
variety of manual and automated systems are available for the detection of staphylococci
including a number of Polymerase chain reaction (PCR)-based methods (Francis Martineau,
2001). PCR-based diagnostics has become an intrinsic part of molecular diagnostics for
detection and identification of nucleic acids for a number of species (Mackay, 2004). PCR
conventional methods (qPCR assays) are proven to be more reliable in comparison to the
classical phenotypic approaches (such as disc-diffusion methods & e-tests) since the latter
produces information based only on the phenotypic properties of the species. PCR based
diagnostics involves nested PCR, multiplex PCR, real-time PCR and quantitative PCR methods
(Green MR, 2019) (CDC, 2021).
Real-time PCR: It has a significant advantage of switching samples around as per the need of the
experiment. By providing quantitative results, real-time PCR has revolutionized PCR-based
diagnostics by removing the need for a gel (Park, 2020).
qPCR methods: 1) Using non-specific dyes (SYBR Green): SYBR green gets excited when bound
to the double-stranded DNA, due to changes within its structure. It can bind to the wrong region
on the template DNA as long as its dsDNA. 2) Using Sequence-specific fluorescent probe
(TaqMan): DNA polymerase digests the probe present ahead in the DNA strand. It consists of a
fluorescent molecule on the 3’ end of the template strand and a quenching molecule on the 5’
end of the template strand. The quenching molecule prevents fluorophores from emitting any
light. As a result, a double-stranded copy of the DNA fragments is obtained for further
amplification (Park, 2020).
Threshold cycle value (Ct value) indicates the starting DNA in the PCR process in addition to the
efficiency of the reaction. The normalised signal of a target intercepts the threshold value of the
reaction process. Starting DNA in a cycle is inversely proportional to the Ct value, for example a
Ct value of 0.999 shows a very strong correlation to the starting DNA. Depending on how well
the primers bind to the DNA, the threshold range can be adjusted accordingly.
, Of the many unique genes of S. aureus FPR3757 obtained from CARD (al., 2020) and Virulence
finder (Finder, 2021) , Staphylococcus aureus LmrS gene was selected for designing primers for
the amplification of DNA in a qPCR-based assay. And it has been useful in the amplification of
CA-MRSA along with the determination of its antimicrobial resistance.
Hypothesis for the report: To design a PCR-based assay to amplify community-acquired
methicillin-resistant Staphylococcus aureus (CA-MRSA) and determine their antimicrobial
susceptibilities against six antibiotics. In the first reaction with test primers, CA-MRSA was
expected to be amplified. In the second reaction with S. aureus control primers, CA-MRSA and
HA-MRSA were expected to be amplified.
The organisms used in this study are:
Test Organism A: S. aureus FPR3757
Test Organism B: Staphylococcus epidermidis (control)
Test Organism C: S. aureus MRSA252
Methodology
PRIMER DESIGNING: A bioinformatic web-tool called Primer3input (Primer3Web, 2021) is used
for designing primers for PCR assays. The unique gene sequence is obtained from CARD and
uploaded on Primer2input. A few parameters such as size range (given as 100-200 base pairs)
are modified to design the required primer. Clicking on ‘Pick primers’ generates a list of relative
primers that have been already designed for that specific sequence. If there are no results
shown, that means a unique set of primers have been obtained which have not been
designed/used previously. Integrated DNA technologies is another web-tool used to analyse
various sets of primer sequences to establish their significance for subsequent research. A sub-
tool called OligoAnalyzer is used to examine the primer sequences by uploading its
forward(left) and reverse(right) ATGC sequences (Technologies, 2021). ‘Analyze’ tool produces
a plethora of results for analytical examination, and to stipulate certain parameters such as
melting temperature (expected to be 60°C for both forward and reverse primers). Additionally,
the difference in the absolute melting temperatures of both the primers should not exceed 5°C.
‘Hairpin’ tool indicates the ∆ G for the given set of primers. All numerical values of hairpin
should be >-3, only then can the primers be used for experimentation. ‘Self-Dimer’ tool indicates
the ∆ G for the given set of primers. All numerical values of self-dimer should be expected to be
>-5 for generating suitable primers.
2. Suspension and Dilution of Primers: Primers are supplied at a concentration of 100 µM
(freezer stock). 10 µL of freezer stock primer and 90 µL of PCR grade water is pipetted out into a
1.5 mL Eppendorf tube, to create a 1:10 dilution. It is Vortexed briefly to obtain the working
stock of primer, at 10 µM.
3. SETTING UP qPCR using primers designed in week 14 workshop.
Here, a qPCR has been used to differentiate between CA-MRSA and HA-MRSA.
Test Primer PCRs: In an Eppendorf tube, a Test primer qPCR master mix was prepared
comprising of: 40 µl SYBR Green, 20 µl PCR grade water, 6 µl of the Forward primer designed
and 6 µl of the Reverse primer designed. 18 µl of this master mix was aliquoted into the first
three wells of a PCR strip and 2 µl of each gDNA was added according to the following table:
Staphylococcus aureus (CA-MRSA) and hospital-acquired methicillin-resistant
Staphylococcus aureus (HA-MRSA) strains and determination of their antimicrobial
susceptibilities.
The PCR technique is a product of the current molecular diagnostics era. Real-time PCR and
qPCR have created a revolution in molecular analysis and the detection of strain-specific nucleic
acids. This report discusses the amplification of community-acquired methicillin-resistant
Staphylococcus aureus (CA-MRSA) by performing a qPCR-based assay and designing gene-
specific primers along with the determination of their antimicrobial susceptibilities against six
antibiotics. A graph plot representing the delta Rn values obtained by qPCR was analysed to
differentiate between the community-acquired methicillin-resistant Staphylococcus aureus (CA-
MRSA) and hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strains.
Disk diffusion tests and E-tests were performed to examine the susceptibilities of S. aureus
FPR3757 and S. aureus MRSA252 against primary antibiotics. The S. aureus gene LmrS appears
to be an important predictor of antibiotic resistance in Staphylococcus aureus, according to this
study. To propose how microbial isolates effect its expression and the breadth of its
antimicrobial resistance, more research into its genomic physiology is needed.
Introduction
Due to the high morbidity and mortality of infections caused by the Staphylococcus aureus
strains, the rise of methicillin-resistant strains (MRSA) and other antimicrobials has become a
significant problem, particularly in a healthcare system (CDC, 2019) (Harmsen D, 2003 ). A
variety of manual and automated systems are available for the detection of staphylococci
including a number of Polymerase chain reaction (PCR)-based methods (Francis Martineau,
2001). PCR-based diagnostics has become an intrinsic part of molecular diagnostics for
detection and identification of nucleic acids for a number of species (Mackay, 2004). PCR
conventional methods (qPCR assays) are proven to be more reliable in comparison to the
classical phenotypic approaches (such as disc-diffusion methods & e-tests) since the latter
produces information based only on the phenotypic properties of the species. PCR based
diagnostics involves nested PCR, multiplex PCR, real-time PCR and quantitative PCR methods
(Green MR, 2019) (CDC, 2021).
Real-time PCR: It has a significant advantage of switching samples around as per the need of the
experiment. By providing quantitative results, real-time PCR has revolutionized PCR-based
diagnostics by removing the need for a gel (Park, 2020).
qPCR methods: 1) Using non-specific dyes (SYBR Green): SYBR green gets excited when bound
to the double-stranded DNA, due to changes within its structure. It can bind to the wrong region
on the template DNA as long as its dsDNA. 2) Using Sequence-specific fluorescent probe
(TaqMan): DNA polymerase digests the probe present ahead in the DNA strand. It consists of a
fluorescent molecule on the 3’ end of the template strand and a quenching molecule on the 5’
end of the template strand. The quenching molecule prevents fluorophores from emitting any
light. As a result, a double-stranded copy of the DNA fragments is obtained for further
amplification (Park, 2020).
Threshold cycle value (Ct value) indicates the starting DNA in the PCR process in addition to the
efficiency of the reaction. The normalised signal of a target intercepts the threshold value of the
reaction process. Starting DNA in a cycle is inversely proportional to the Ct value, for example a
Ct value of 0.999 shows a very strong correlation to the starting DNA. Depending on how well
the primers bind to the DNA, the threshold range can be adjusted accordingly.
, Of the many unique genes of S. aureus FPR3757 obtained from CARD (al., 2020) and Virulence
finder (Finder, 2021) , Staphylococcus aureus LmrS gene was selected for designing primers for
the amplification of DNA in a qPCR-based assay. And it has been useful in the amplification of
CA-MRSA along with the determination of its antimicrobial resistance.
Hypothesis for the report: To design a PCR-based assay to amplify community-acquired
methicillin-resistant Staphylococcus aureus (CA-MRSA) and determine their antimicrobial
susceptibilities against six antibiotics. In the first reaction with test primers, CA-MRSA was
expected to be amplified. In the second reaction with S. aureus control primers, CA-MRSA and
HA-MRSA were expected to be amplified.
The organisms used in this study are:
Test Organism A: S. aureus FPR3757
Test Organism B: Staphylococcus epidermidis (control)
Test Organism C: S. aureus MRSA252
Methodology
PRIMER DESIGNING: A bioinformatic web-tool called Primer3input (Primer3Web, 2021) is used
for designing primers for PCR assays. The unique gene sequence is obtained from CARD and
uploaded on Primer2input. A few parameters such as size range (given as 100-200 base pairs)
are modified to design the required primer. Clicking on ‘Pick primers’ generates a list of relative
primers that have been already designed for that specific sequence. If there are no results
shown, that means a unique set of primers have been obtained which have not been
designed/used previously. Integrated DNA technologies is another web-tool used to analyse
various sets of primer sequences to establish their significance for subsequent research. A sub-
tool called OligoAnalyzer is used to examine the primer sequences by uploading its
forward(left) and reverse(right) ATGC sequences (Technologies, 2021). ‘Analyze’ tool produces
a plethora of results for analytical examination, and to stipulate certain parameters such as
melting temperature (expected to be 60°C for both forward and reverse primers). Additionally,
the difference in the absolute melting temperatures of both the primers should not exceed 5°C.
‘Hairpin’ tool indicates the ∆ G for the given set of primers. All numerical values of hairpin
should be >-3, only then can the primers be used for experimentation. ‘Self-Dimer’ tool indicates
the ∆ G for the given set of primers. All numerical values of self-dimer should be expected to be
>-5 for generating suitable primers.
2. Suspension and Dilution of Primers: Primers are supplied at a concentration of 100 µM
(freezer stock). 10 µL of freezer stock primer and 90 µL of PCR grade water is pipetted out into a
1.5 mL Eppendorf tube, to create a 1:10 dilution. It is Vortexed briefly to obtain the working
stock of primer, at 10 µM.
3. SETTING UP qPCR using primers designed in week 14 workshop.
Here, a qPCR has been used to differentiate between CA-MRSA and HA-MRSA.
Test Primer PCRs: In an Eppendorf tube, a Test primer qPCR master mix was prepared
comprising of: 40 µl SYBR Green, 20 µl PCR grade water, 6 µl of the Forward primer designed
and 6 µl of the Reverse primer designed. 18 µl of this master mix was aliquoted into the first
three wells of a PCR strip and 2 µl of each gDNA was added according to the following table:
