METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS DETECTION BY PCR

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About Author:
Mariz Sintaha
Lecturer,
School of Life Sciences
Independent University, Bangladesh
sintu.bmb@gmail.com

Introduction:
MRSA refers to strain of Staphylococcus aureus which is resistant to methicillin. MRSA infection causes Red bump (may be pus filled), warmth, pain, swollen and red and tender skin lesions and are resistant to various antibiotics used for treatment including cephalosporins and penicillins. Rapid and accurate detection of MRSA has great significance since is needed for proper treatment of staph diseases. It is difficult to detect MRSA in media using phenotypic methods such as disk diffusion, oxacillin agar screening test, agar dilution, MIC determination by broth dilutionsince expression of metcillin resistance is not uniform in all cells. The genotypic method is now taken as the gold standard to detect MSRA because of its accuracy.

Reference Id: PHARMATUTOR-ART-1969

Polymerase Chain Reaction (PCR)

Principle- Polymerase chain reaction is a technique widely used in field of Molecular Biology for various purposes. The technique is used to amplify a specific segment of DNA across several orders of magnitude. The DNA to be amplified is mixed with all the materials needed for synthesizing new DNA strand and a pair of primer to specify the region of DNA to be amplified. Temperature of the mixture is fixed into three steps one after another- the denaturation temperature (94oC), the annealing temperature (55oC to 65oC) and primer extension temperature (72oC) (William B. Coleman 2006). These three steps constitute a cycle. In each cycle the copy number of duplex DNA doubles (theoretically). The cycles are repeated for many times by the aid of an automated temperature regulating machine “Thermocycler”. After few cycles, size of the newly synthesized DNA is fixed as the distance between 5’ ends of primers (Lo 1998). Thus, at the end of the reaction (after completion of many cycles) millions copies of specific segment of template DNA are obtained.

Reagents- The PCR mixture contains (William B. Coleman 2006)-
1. Heat resistant DNA synthesizing enzyme- Taq polymerase, collected from the bacteria T. aquaticus.
2.Two short segments of DNA (18-30 bp long) named forward and reverse primer, that is complementary to the 3’ ends of the region of DNA to be amplified of the sense and anti-sense strand respectively.
3. Deoxy nucleotides triphosphates (dTTP, dGTP, dCTP and dTTP), which are building blocks of DNA
4. Tris-Cl buffer of PH 8.3 in which taq polymerase shows maximum activity.
5. Divalent ions, such as Mg+ which is required for dissociation Taq polymerase from DNA.
6. KCl which promotes primer annealing to template DNA.
7. Template DNA

Mechanism- In denaturation step, high temperature causes melting of DNA from double strand to single strand.  Generally in first cycle a higher denaturation temperature is used to cause complete melting of duplex DNA and activation of taq polymerase which remains in inactivated state in commercially available solution. A Lower annealing temperature allows primers to bind with the separated single stranded DNAs. At primer extension temperature, Taq polymerase starts adding dNTPs  at the 5’ end of the primer complimentary to the template strand. Thus a new DNA strand is synthesized which is complimentary to the template. The product of first cycle provides template for second cycle. After completion of all cycles, often temperature is hold at 70-74o C for 7-10 minutes so that remaining single stranded DNA can be extended and then hold at 4-15o C until it is removed for further work (Michael A. Innis 1999).

Visualization of PCR Product- For visualization, PCR product is resolved through Gel Electrophoresis. Million copies of similar sized DNA produced by PCR, concentrate in a location of the gel. The gel is then stained with a fluorescent dye “ethidium bromide”. The dye intercalates in to the duplex DNA. After washing the gel with distilled water to remove unbound ethidium bromide, DNA band is visualized under UV light (Mathews n.d.).

Methicillin-resistant Staphylococcus aureus (MRSA)
Staphylococcus aureus (S. aureus)
is a facultative pathogenic Gram-positive bacterium that inhabits in human skin and mucosa. It causes disease occasionally by getting into the body. The causes disease in virtually any tissue diseases including wound, skin and urinary tract infection, pneumonia, sepsis and osteomyelitis (Chambers 2009). These diseases can be treated successfully by beta-lactum antibiotics since most strains of Staphylococcus aureus are sensitive to it. But the strains resistant to methicillin (Methicillin-resistant Staphylococcus aureus  or MRSA) are resistant to various antibiotics used for treatment including cephalosporins and penicillins. Clinical use of methicillin is responsible of increasing the incidence of acquiring methicillin resistance by Staphylococcus aureus(MRSA). Other factors responsible include use of intravenous drug, uncontrolled use of antibiotics, nose infection of Methicillin-resistant Staphylococcus aureus (MRSA)and prolonged stay in hospital (Lu PL 2005). Methicillin-resistant Staphylococcus aureus (MRSA)is the major cause of community acquired and hospital-acquired infection in recent decades (Martins 2007). The symptoms of MRSAinfection include Red bump (may be pus filled), warmth, pain, swollen and red and tender skin lesions.

Polymerase Chain Reaction (PCR) to identify MRSA
Methicillin-resistant Staphylococcus aureus (MRSA) causes serious infection that is difficult to treat. For effective treatment and prevent spread of the disease, Methicillin-resistant Staphylococcus aureus must be identified in clinical specimen rapidly and accurately. Methcillin resistance occurs in Staphylococcus aureusby acquiring a gene called MecA (Hartman BJ 1984). This gene encodes an altered form of penicillin binding protein  which has low affinity for beta lactum antibiotics (Lim 2002). Thus the presence of a gene in the DNA isolated from clinical specimen is the indicator of the Methicillin-resistant Staphylococcus aureus  infection. Polymerase chain reaction is used widely to identify a specific DNA sequence within a sample. Since, polymerase chain reaction involves binding of primers complimentary to specific sequences of DNA and amplifying a specific segment of DNA, successful polymerase chain reaction proves the presence of a specific DNA sequence. MecA gene is conserved in Staphylococcus species, so detecting MecA by PCR is considered as standard method for identifying Methicillin-resistant Staphylococcus aureus (MRSA) (Maes N 2002), (Bignardi GE 1996) (Murakami K 1991).

Actually, the resistant bacteria acquires a transposable element named staphylococcal cassette chromosome (SCCmec) in which the MecA gene is contained (C. Cuny 2005). Sccmec contains incomplete inverted repeat at both ends and integrated at the 3′-end of orfXregion, within a site named attBSCC (Hiramatsu K 2002). SCCmec element is classified in five classes according to the mec gene, insertion complex, site-specific recombinase genes and transposon tn554 (Ma X 2002). Identification of MecA gene alone can give false positive result since it is present both in Methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci. So polymerse chain identification of the region covering theorfX and SCCmec can be used to specifically identify Methicillin-resistant Staphylococcus aureus. For these purpose, five forward primers for five types of SCCmec elements and one reverse primer fororfX region can be used (Huletsky A 2004).

Coagulase-negative staphylococci (CoNS) is another vital cause of infection (von Eiff C 2001) and commonly isolated in laboratories (CC. 1990). Coagulase-negative staphylococci (CoNS) also contain multi-drug resistance. So MRSA and CoNS must be distinguished (Zhang K 2004) for rapid detection and proper treatment ofMRSA. MRSA and CoNs are not distinguishable in PCR detection method of MecA gene since MecA genes of both species have 99% sequence similarity (Ryffel C 1990). For this purpose presence coag gene (S Rallapalli1 2008)is analysed besides MecA gene in multiplex PCR systems. In MRSA both MecA and coag genes are detected by PCR where in CoNS, only MecA gene is detected by PCR (S Rallapalli 2008). Another method of distinguishing MRSA and CoNS is multiplex PCR to co-amplify both mecA gene responsible for methcillin resistance and femA gene responsible for coagulase activity (Kohner P 1999) (Sakoulas G 2001) (Vannuffel P 1995) (Kobayashi N 1994) since mecA gene is present both in MSRA and CoNS where femA is present only is MSRA.

PCR methodologies


Forward Primer

5’-3’ direction

Reverse Primer

 5’-3’ direction

Reaction Mix

Cycles

Length (bp)

Reference

SCCmec

elements

rjmec

57641–57656

TATGATATGCTTCTCC

ORFX1r

58042–58022

AACGTTTAGG
CCCATACACCA

Total Volume= 25 µL


-Taq DNA polymerase (1.3 U)

-0.5 µL DNA (10 ng)

-primer(2.5 pmol)

-dNTP(200 µM) each in Tris-HCl (10 mM)

-KCl(500 mM)

-MgCl2(1.5 mM)















Total 30 cycles


Initial Denaturation

94°C 

3 min


Denaruration-

94°C

30 s


Annealing-

55°C

30 s


Extension-

72°C

30 s


Final extension-

72°C  4 min.

400

(C. Cuny 2005)

mecA

mecA1

318-338

GTAGAAATGACT
GAACGTCCG

mecA2

603-627

CCAATTCCACATT
GTTTCGGTCTAA

Total volume=50ml


-Taq DNApolymerase(2U)

-DNA(30 ng)

-each primer(50 pmol)

-dNTP(200 mM)

-10X reaction buffer[(NH4)2SO4((160 mM), Tris HCl (670 mM ) of pH8.8, Tween 80( o.1%)]

-MgCl2(2.5 mM)




Total 30 cycles

Initial denaturation

94o C

five minutes,

denaturation a

94oC

30 seconds

annealing

55oC

30 seconds

Extension

72oC

30 seconds

final extension

72oC

10 minutes.

310

(Riza Adaleti 2008)

mecA

primer 1

37-57

GTTGTAGTT
GTCGGGTTlTGG

Primer 2

1827-1854 CCACCCAATlTTGTCT
GCCAGTTTCTCC

Total Volume=25 μL


-Taq polymerase (2.5 U)

-3 μL of template DNA

-Primers (100 pmol of each)

-dNTPs

-PCR Buffer

Mg (2.5 mm)

Total 40 cycles


Initial denaturation

94o C

3 minutes


Denaturation

94 °C

30 sec


Annealing

60 °C

45 sec


Extension

72 °C

1.5 minutes


Final Extension

72 °C

3.5 minutes.

1800

(S Ercis 2010)

Multiplex PCR to Distinguish Between MRSA and CoNS

MecA







coag

1282 to 1303 AAAATCGATG
GTAAAGGTTGGC




CGAGACCAA
GATTCA ACAAG

1793 to 1814 AGTTCT
GCAGTACCGGATTTGC





AAA GAA AAC
CACTCA CAT CAG T.

Total Volume=20 µL


- 1µL  DNA (50 ng)

-Buffer (500mM KCl, 100mM Tris-HCl, pH 8·3)

- dNTP(0.5 mM each)

- MgCl 2 (2.5 mM) 

-coag primer (0.15 µM of each )

-mecA primer(0.1 µM)

- Taq DNA polymerase(2U)



Total 40 cycles


Initial denaturation

95o C

five minutes,


Denaturation a

94oC

30 seconds


Annealing

55oC

30 seconds


Extension

72oC

90 seconds


Final extension

72oC

5 minutes.


533






810

(S Rallapalli 2008)

mecA






femA

MecAl

1282-1303

AAAATCGATGG
TAAAGGTTGGC




FemAl

595-614 AGACAAATA
GGAGTAATGAT 

MecA2

1739-1814

AGTTCTGCA
GTACCGGATTTGC




FemA2

1084-1103

AAATCTAA
CACTGAGTGATA

Total Volume= 100 µL

- Taq DNApolymerase (1U)

-10 µL DNA

-Primer (100 pM each)

-dNTP (200 µM each)

 -Tris-HCl (10 mM, pH 8 3),

-KCI(50 mM)

-MgCl2(1-5 mM)

Total 25 cycles


Denaturation

94oC

1 minutes


Annealing

57oC

1 minutes


Extension

72oC

2 minutes


Final extension

72oC

5 minutes.


533








509

(Kobayashi N 1994)

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