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Dr. M. H. Shahhosseiny
Rapid detection of
Staphylococcus aureus
and Streptococcus
pneumonia sinusitis by
PCR
• - Sinusitis or rhinosinusitis is
inflammation of the paranasal
sinuses, which may be due to
infection, allergy, or autoimmune
issues.
Sinusitis
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• - Sinusitis affects 1 in 7
• adults in the U.S. - 31 -35 million diagnosed
per year
• - 500 000 sinus surgeries performed
• - 73 million days restricted activity/year
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• 4 paranasal sinuses
• Frontal
• Maxillary
• Ethmoid
• Sphenoid
Infectious or noninfectious inflammation of 1 or more sinuses
•Sinusitis
• Classification
Sinusitis
• Acute:
– Symptoms up to 4wks
• Subacute:
– Symptoms from 4-12wks
• Chronic:
– Symptoms >12wks
• Recurrent acute:
– 4+ episodes in one year, each lasting >7days
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Signs and symptoms
Sinusitis
5
.
Tthe most common Bacterial causative agents : Sinusitis
Streptococcus pneumoniae
Staphylococcus aureus
Haemophilus influenza
Moraxella catarrhalis
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• Clinical
• X-rays photography of the sinuses
• CT scan of the paranasal sinuses
• Culture fluids obtained from the sinuses
Diagnosis
Sinusitis
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√Cultural properties
√Morphological //
√ Physiological //
√Biochemical //
√Antigenic //
*Biotyping
* Phagetyping
* Morphotyping
* Bacteriocintyping
* Serotyping(FIA-RIA-EIA)
†API
† Entrotube
† Micro ID
† RAP ID N/H
† R/B System
† MicroScan
† Minitek System
† UNI-N/F-Tek Plate
† Vitek system
† Bac-T-Screen system
TRADITIONAL METHODS (Culture)
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TRADITIONAL METHODS have many drawbacks
1.Time consuming
2. Some microorganisms are unculturable at present & (routine culture)
3. Labor-intensive(extremely fastidious)
4. Prohibitively expensive & danger
5. Low sensitivity
6. VBNC
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1. Broadrange (B. V. P. F. …)
2. Reproducible, Long periods, Different centers
3. Ease and Fast
4. Don’t cross reactivity with related microbes
5. Able to detection in different sample(Clinical &
Environmental)
6. Low dosage (High sensitivity)
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Modern & Molecular Methods 1) LPS, Fatty Acids
2) Proteins
3) Polynucleotide
A:In-Vitro Nucleic Acid Amplification (NAT)
B:Real Time C:DNA Microarray & GeneChip
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1) Thermostable DNA Polymerase
2) Accessibility to reagents to kit format
3) Accessibility to sequence information
4) Cheap oligonucleotide synthesis
5) PCR derivative
*6) Primer modification
*7) Mixing Hybridization techniques with amplification
protocol
*8) Innovation in Thermocycler
*9) Nanobiotechnology & Bioinformatic
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PCR
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• Target Amplification
Methods
PCR
TAS
3SR
SDA
LAMP
Probe Amplification
Methods
1)LAR=LCR
2) QB-
replicase
based
amplificati
on
Signal Amplification Methods
1) Compound
Probes
2) bDNA Probes
NAT
Comparison between conventional & NAT
Speed Sensitivity Specificity
-Culture + +++ +++
-IF +++ ++ ++
-ELISA +++ ++ ++
-Non-Amplification ++ ++ +++
Probes
-Gene ++++ ++++ ++++
Amplification
Methods
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•EXTRACTION
•AMPLIFICATION
•DETECTION
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Molecular Diagnostic Methods
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This study has been performed on
40 samples which obtained in the
hospital during the surgery from patients with sinusitis.
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•Extraction
•DNG-Plus
• Boiling + DNG-Plus
Target Gene
S.Pneu S.aur
Nuclease
Gene SPN
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Staphylococcus aureus Optimization
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M C+ C-
1500
850
400
200
50
279 bp
M : Low range DNA Ladder1103 (Fermentas)
C+ : Amplicon (279 bp)
C- : negative control
Staphylococcus aureus Sensitivity
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M C+ 1 2 3 4 5 6 7 C-
1500
850
400
200
50
1: 1 million CFU 2: 100/000 CFU 3: 10/000 CFU 4: 1000 CFU 5: 100 CFU 6: 10 CFU 7: 1 CFU
M : Low range DNA Ladder 1103 (Fermentas)
C+ : Amplicon (279 bp)
C- : negative control
Staphylococcus aureus +ve & -ve samples
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From the 40 samples, 50% of them were positive for Staphylococcus aureus
1500
850
400
200
50
M C+ 1 2 3 4 C-
M : Low range DNA Ladder 1103 (Fermentas)
C+ : Amplicon (279 bp)
C- : negative control
1: Negative sample 2-4: Positive samples
Streptococcus pneumoniae Optimization
M C+ C-
M : 1 Kb DNA Ladder (Fermentas)
C+ mplicon (227 bp)
C-
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M C+ C-
M : Low range DNA Ladder1103 (Fermentas)
C+ mplicon (227 bp) C-
227
bp
1500
850
400
200
50
227 bp
Streptococcus pneumoniae Sensitivity
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M C+ 1 2 3 4 5 6 7 C-
1500
850
400
200
50
M : Low range DNA Ladder1103 (Fermentas)
C+ mplicon (227 bp) C-
1: 1 million CFU 2: 100/000 CFU 3: 10/000 CFU 4: 1000 CFU 5: 100 CFU 6: 10 CFU
Streptococcus pneumoniae Specificity
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M C+ 1 2 3 4 5 6 7 C-
1500
850
400
200
50
M : Low range DNA Ladder1103 (Fermentas)
C+ mplicon (227 bp) C- 1: Staphylococcus aureus 2: Bacillus spp. 3: Streptococcus pyogenes 4: Lactobacillus plantarum 5: Heamophilus influenzae 6: Moraxella catarrhalis 7: Mycoplasma pneumoniae
Streptococcus pneumoniae +ve & -ve samples
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From the 40 samples, 37% of them were positive for Streptococcus pneumonia.
M C+ 1 2 3 4 C-
M : Low range DNA Ladder1103 (Fermentas) C+: Positive control mplicon 227 bp C- : Negative control 1 : negative sample 2-5: Positive samples
Staphylococcus aureus & Streptococcus pneumoniae
PCR Product Cloning
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Blue
White
Plasmid Extraction
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Staphylococcus aureus & Streptococcus pneumoniae
PCR Product Cloning
M : Low range DNA Ladder1103 (Fermentas)
C- : Negative control 1: Cloned PCR Product C+: Positive control Amplicon 227 bp
C- 1 M C+ C- 1 M 2 C-
M : Low range DNA Ladder1103 (Fermentas)
C- : Negative control 1: Cloned PCR Product 2: Positive control Amplicon 279 bp
279 bp
227 bp
50%نمونه مورد بررسی 40از نمونه ها از جهت استافیلوکوکوس
نمونه ها برای 37%اورئوس و استرپتوکوکوس پنومونیه مثبت
نمونه هر دو مثبت 13گردید و در .بود
:نتیجه گیری
وسااااا ه PCRنتاااااشان نداااااشندو ساااااه ااااار ااااا مدی جه دایی
اشفته، به شدت اژه وبزور سشرو
وساااااااااااااتشفی وسوسو و ر ساااااااااااارا ساااااااااااای و اتهشی .وسترپتوسوسو پ ومونیه وس
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Conclusion: The results showed that
the two newly developed PCR
methods are a highly specific and
efficient tool for the rapid detection of
S. aureus and S. pneumonia sinusitis.
This technique can be safely used for
early diagnosis of sinusitis with high
accuracy.
From the 40 samples, 50% of them
were positive for Staphylococcus
aureus and 37% of them were
positive for Streptococcus
pneumonia.
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Tuesday, May 28, 2013
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