Detect NHS by PCR Technique .

Detect
Streptococcuspneumoniae, Haemophilusinfluenzae
and
Neisseriameningitidis
Causes meningitis
by
PCR Techniques
S.B .Modhafar Qadir Sabeer
Director of Epidemiological Unit / CPHL
Baghdad

PCR Technique
• Polymerase Chain Reaction )PCR )is
increasingly being used in clinical microbiology
laboratories for the detection of agents of
infectious disease, since the conventional
diagnostic methods, such as microscopy,
culture, and serology, fail to identify the
responsible pathogen in many occasions.

PCR Technique
PCR is a technique that takes a specific sequence of
DNA of small amounts and amplifies it to be used
for further testing .
It is called “polymerase” because the only enzyme
used in this reaction is DNA polymerase.

PCR Technique
• It is called “chain” because the products of the
first reaction become substrates of the
following one, and so on.

Why We use PCR to detect Streptococcus
pneumoniae, Haemophilus influenzae and
Neisseria meningitidis Causes meningitis ?
• Streptococcus pneumoniae and Haemophilus influenzae are major
causes of community-acquired pneumonia and as Neisseria
meningitidis they are important agents of meningitis.
• Identification of the microbiological cause of meningitis is
important, as it enables pathogen-directed antibiotic therapy.
Conventional detection of bacteria is based on culture and
phenotypic characterization. However, culture methods are time-
consuming and have relatively low sensitivity, especially when
antibiotics have been given to the patient prior to sampling. The
use of nucleic acid amplification tests, such as polymerase chain
reaction (PCR), have enabled more sensitive and rapid detection of
pathogens in many samples such as blood ,respiratory secretions
and cerebrospinal fluid (CSF) etc…..

Why We use PCR to detect Streptococcus
pneumoniae, Haemophilus influenzae and
Neisseria meningitidis Causes meningitis ?
• The major agent of causes meningitis are :
1- 9802 gene fragment in Streptococcus pneumoniae.
2- omp P6 gene in Haemophilus influenzae .
3- ctrA gene in Neisseria meningitidis .
We can not detect these agents by Conventional lab.
methods , and therefore we use PCR Techniques to
ensure detect these agents .

What we need to begin PCR ?
(The “Reaction” Components )
1-Target DNA - contains the sequence to be amplified .
2- Pair of Primers - oligonucleotides that define the sequence to
be amplified .
3-dNTPs - deoxynucleotidetriphosphates: DNA building blocks .
4- Thermostable DNA Polymerase - enzyme that catalyzes the
reaction .
5- Mg++ ions - cofactor of the enzyme .
6- Buffer solution – maintains pH and ionic strength of the
reaction solution suitable for the activity of the enzyme .

PCR STEPS
Adenine
Thymine
Guanine
Cytosine

Extraction & purification
of
DNA
• To obtain of pure DNA for Streptococcus pneumoniae,
Haemophilus influenzae and Neisseria meningitidis
,we use a DNA / RNA Prep kit ( Sacace biotechnologies /
Italy ) for nucleic acid extraction and purification of total
DNA / RNA from clinical materials , we use a suspension
of bacteria as a specimen by take a loop full from growth
bacteria and inoculated in 2ml of ( BHI ) and incubated
for 24h/ 37c with Co2 condition, 100µl of this suspension
was taken and according to the steps of extraction kit we
obtained a pure DNA ready to use for PCR Process .

PCR Process to detect
NHS ( Master Mix )
• To make a master mix for amplification of NHS DNA we must
prepare these materials :
• 1. Template DNA (NHS DNA).
• 2. Forward and reverse PCR primers .
• 3. MgCl2 (25 mM).
• 4. dNTPs (a mixture of 2.5 mM dATP, dCTP, dGTP, and dTTP).
• 5. 10× PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 25°C.
• 6. Thermal stable DNA polymerase (e.g., Taq DNA
polymerase).

PCR Process to detect
NHS ( Master Mix )
Method
According of NHS meningitidis Kit (Sacace biotechnologies / Italy )
Follow these steps :
1- Prepare required quantity of tubes PCR-mix-1.
2- Pipeete 10µl of PCR-mix -2 into each PCR-mix-1.
3- Add to approprative tube 10µl of DNA sample obtained after sample
preparation .
4- Add 10µl of DNA – buffer to the tube for Negative Control of
Amplification.
5- Add 10µl of Streptococcus pneumoniae C+, Haemophilus
influenzae C+ and Neisseria meningitidis C+ to the tubes for
Positive Control of Amplification.
6- Close PCR-mix-1 tubes , transfer theme into thermalcycler and start
following program :

Thermalcycler Programe
• 1- 95°C for 5 min ( 1 cycle ).
• 2- 95°C for 20 sec, 65°C for 20 sec , 72°C for 40 sec
( 40 cycle ).
3- 72°C for 1min ( 1 cycle ).
4- Storage at 10°C forever.

Analysis PCR Amplification
• To measure the success of a PCR amplification,
5 to 10 μL of the final PCR product is run on
2% agarose gel and visualized by staining with
ethidium bromide. The Length of specific DNA
Band fragments is :
• 1- Streptococcus pneumoniae ( 792 bp ).
• 2- Haemophilus influenzae ( 516 bp ).
• 3- Neisseria meningitidis ( 341 bp ).

• Gel electrophoresis of positive NHS controls & samples :
• 1- DNA size marker ( 100 bp ).
• 2- Str.pnemoniae pos.c. 792 bp.
• 3- Str.pnemoniae neg sample.
• 4- Haemophilus influenzae pos.c. 516 bp.
• 5- Haemophilus influenzae pos. sample .
• 6- Neisseria meningitis pos.c.341 bp.
• 7- Neisseria meningitis pos. sample .
• 8- Negative Controle ( No band ).

PCR - Major Advantages
1- Rapid and easy to perform.
2- High Sensitivity (theoretically one DNA copy can
be multiplied and detected but practically we
need certain numbers of DNA copies in the
specimen ).
3- All samples can be used as : sputum ,
urine , blood , CSF …etc .
4- Qualitative.
5- Quantitative: follow up of viral load before and
after therapy.
6- Genotyping .
7- Drug Resistance.
8- Result with in 2-3 h.

Major Disadvantages
1- Expensive .
2- high technology
3- Special lab design (Must have separated
rooms ).
4- Contamination .

Detect NHS by PCR Technique .

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