Detect Streptococcus pneumoniae,

Haemophilus influenzae and

Neisseria meningitidisCauses meningitis

byPCR Techniques

S.B .Modhafar Qadir SabeerDirector 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 .

Reaction Mixture

PCR STEPS

Adenine

Thymine

Guanine

Cytosine

PCR STEPS

pcr.exe

PCR STEPS

Extraction & purificationof

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 Advantages1- 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 .

THE END

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