PCRmax MRSA—Methicillin-resistant Staphylococcus aureus Instruction Manual

mecA (penicillin binding protein

2)
&
S. aureus FEMB gene
(chromosomal gene)

Methicillin-resistant
Staphylococcus aureus

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

1

Quantification of Methicillin-resistant Staphylococcus aureus genomes.

Advanced kit handbook HB10.07.08

Published Date: 20/09/2016

Methicillin-resistant Staphylococcus aureus (MRSA) is a specific strain of the
Staphylococcus aureus bacterium that has developed antibiotic resistance to all penicillins,
including methicillin and other narrow-spectrum β-lactamase-resistant penicillin antibiotics.
[1] The resistant strain, MRSA was first discovered in the UK in 1961 and is now
widespread, particularly in the hospital setting where it is commonly termed a superbug.
MRSA may also be known as oxacillin-resistant Staphylococcus aureus (ORSA) and
multiple-resistant Staphylococcus aureus, while non-methicillin resistant strains of S.
aureus are sometimes called methicillin-susceptible Staphylococcus aureus (MSSA) if an
explicit distinction must be made.
Although MRSA has traditionally been seen as a hospital-associated infection, community­acquired MRSA strains have appeared in recent years, notably in the US and Australia.[2]
The abbreviations CA-MRSA (community-associated MRSA) and HA-MRSA (hospital­associated MRSA) are now commonly seen in medical literature.
Methicillin resistance arises by acquisition of a staphylococcal cassette chromosome
SCCmec, and is conferred by the mecA gene. Expression of this gene yields PBP2a, a
penicillin binding protein with reduced affinity for β-lactam rings (the primary active-site of
the β-lactam antibiotics).[6]
Some strains of S. aureus over-express β-lactamase and appear to be resistant to
oxacillin and, rarely, methicillin despite being mecA-negative. They have slightly raised
minimum inhibitory concentrations (MICs) and may thus be described as "minimally
resistant". Other strains express modified PBPs (not PBP2) and exhibit varying degrees of
β-lactam antibiotic resistance.
Not only are MRSA strains resistant to the usual antibiotics, but a curious interbreeding
with community staph has led to an additional worry. Many MRSA isolates found outside
of medical facilities, and referred to as CAMRSA (community-acquired MRSA), have
acquired the Panton-Valentine leukocidin factor,[7] a gene that produces a series of
chemicals that make these MRSA particularly invasive as well as resistant.

Reference
J Clin Microbiol. 2002 May;40(5):1821-3.

Introduction to Methicillin-resistant Staphylococcus
aureus

2

Quantification of Methicillin-resistant Staphylococcus aureus genomes.

Advanced kit handbook HB10.07.08

Published Date: 20/09/2016

MIN

MAX

The mecA gene is a plasmid based gene responsible for antibiotic resistance. FEMB is a
chromosomal gene specific to S. aureus. A positive result for both markers has been
been validated as a reliable test for MRSA. The primers and probe have 100% homology
with all reference sequences in the NCBI data. base including those listed below.

MecA sequence specificity
AM292304, AB266532, AB266531, AB245471, AB245470, AB236888, AY894415,
DQ106887, AY786579, AY271717, AB221124, AB221123, AB221122, AB221121,
AB221120, AB221119, BA000017, AP006716, CP000046, AM048803, AM048802,
CP000255, AJ810121, AJ810120, AB047089, Y14051.1, Y13095.1, Y13096.1,
Y00688.1, X52593.1, BA000033, BA000018, AB121219, CP000029, X52592.1,
AB063172, AB033763, D86934.2, AB096217, AB063173, AB037671,

FEMB sequence specificity
CP000703.1, BA000017.4, CP000046.1, CP000253.1, CP000255.1, X17688.1,
X571857.1, BA000033.2, BA000018.3, BX571856.1

The PCRmax qPCR Kit for Methicillin-resistant Staphylococcus aureus (MRSA) genomes
is designed for the in vitro quantification of MRSA genomes. The kit is designed to have
the broadest detection profile possible whilst remaining specific to the MRSA genome.

The primers and probe sequences in this kit have 100% homology with a broad range of
MRSA sequences based on a comprehensive bioinformatics analysis.

If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to [e-mail] and our bioinformatics team will
answer your question.

Specificity

3

Quantification of Methicillin-resistant Staphylococcus aureus genomes.

Advanced kit handbook HB10.07.08

Published Date: 20/09/2016

Kit Contents

• mecA (penicillin binding protein 2) primer/probe mix (150 reactions BROWN)

FAM labelled

• FEMB gene (chromosomal gene) primer/probe mix (150 reactions BROWN)

FAM labelled

• mecA (penicillin binding protein 2) positive control template (for Standard curve
RED)

• FEMB gene (chromosomal gene) positive control template (for Standard curve
RED)

• Internal extraction control primer/probe mix (150 reactions BROWN)

VIC labelled as standard

• Internal extraction control DNA (150 reactions BLUE)

• Endogenous control primer/probe mix (150 reactions BROWN)

FAM labelled

• RNAse/DNAse free water (WHITE)

for resuspension of primer/probe mixes and internal extraction control DNA

• Template preparation buffer (YELLOW)

for resuspension of positive control templates and standard curve preparation

Reagents and equipment to be supplied by the user

Real-Time PCR Instrument

DNA extraction kit

This kit is designed to work well with all processes that yield high quality DNA with minimal
PCR inhibitors.

Lyophilised 2x qPCR Mastermix

This kit is designed to be compatible with all commercially available Mastermixes that run
with standard cycling conditions.

Pipettors and Tips

Vortex and centrifuge

Thin walled 1.5 ml PCR reaction tubes

4

Quantification of Methicillin-resistant Staphylococcus aureus genomes.

Advanced kit handbook HB10.07.08

Published Date: 20/09/2016