Download presentation
Presentation is loading. Please wait.
Published byPaula Lipford Modified over 9 years ago
4
بسم الله الرحمن الرحيم (وإن لكم في الأنعام لعبرة نسقيكم مما في بطونه ، من بين فرث ودم لبناً خالصاً سائغاً للشاربين ) سورة النحل آيه (66)
5
Brucella spp. Brucella spp.( Coccobacilli )
6
Drinking Un- pasteurized camel, sheep, goat or cattle Milk Contact with Infected Animals
11
Surgeon David Bruce;1887 Micrococcus melitensis
12
Gram Negative coccobacilli Culture on Chocolate agar Microbiological Characteristics of Brucella spp
14
1 2 3 4
15
Portals of Entry
16
Fever, headache Sore throat Joint Pain Muscle Aches Ear Aches Fatigue Anorexia
17
1 Clinical 2 Blood Cultures 3 Serology 4 PCR
19
To compare 3 of the reported PCR techniques for Diagnosis of Brucellosis from Human blood and determine the most suitable technique for a Clinical Microbiology Lab in terms of Sensitivity, Specificity, Robustness and Ease of Implementation
21
Material 147 random samples from Brucellosis patients 50 control samplesQuestionnaire Brucella melitensis
22
Methods 3 PCR techniques using genus specific primers ElectrophoresisLimit of Detection DNA extraction
23
Primers (B 4 and B 5 ) Brucella abortus 223bp fragment B 4 =5 TGGCTCGGTTGCCAATATCAA3' B 5 =5' CGCGCTTGCCTTTCAGGTCTG-3' StepTempTime 1Initial Denaturation93 o C5 min 2Template Denaturation90 o C60 Sec 3Primer Annealing60 o C60 Sec 4Primer Extension72 o C60 Sec 5Final Extension72 o C7 min Cycling Profile Repeated 35 times
24
Primers (JPF and JPR) Brucella abortus encoding 193bp ) JPF=5' GCGCTCAGGCTGCCGACGCAA-3' JPR=5' ACCAGCCATTGCGGTCGGTA-3' StepTempTime 1Initial Denaturation94 o C4 min 2Template Denaturation94 o C60 Sec 3Primer Annealing60 o C60 Sec 4Primer Extension72 o C60 Sec 5Final Extension72 o C3 min Repeated 35 cycles Cycling Profile
25
Primers (F 4 and R 2 ) Brucella abortus 905bp fragment. (Romero et al.,1995 ). F4 = 5'TCGAGCGCCCGCAAAGGG-3'-63-79 R 2 = 5'AACCATAGTGTCTCCACTAA-3'947-966 Cycling Profile StepTempTime 1Initial Denaturation95 o C5 min 2Template Denaturation95 o C30 Sec 3Primer Annealing54 o C90 Sec 4Primer Extension72 o C90 Sec 5Final Extension72 o C6 min Repeated 30 cycles
26
(PCR Work Station - Plas Labs)
27
(MyCycler - Bio Rad)
28
STEPS INVOLVED Taq Polymerase Forward Primer Reverse Primer
31
P =0.05 Age Distribution of Brucella species Less than 25 ys25 to < 50 ysMore than 50 ys
32
Fever Anorexia Headache Vomiting Fatigue P=0.001 Symptoms of Brucellosis in the study population Body aches
33
Smudged bands DNA Extraction
34
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
35
Modifications
36
StepTempTime 1Initial Denaturation93 o C5 min 2Template Denaturation90 o C60 Sec 3Primer Annealing60 o C60 Sec 4Primer Extension72 o C60 Sec 5Final Extension72 o C7 min Repeated 40 times تكرر 40
37
Repeated 40 times StepTempTime 1Initial Denaturation94 o C4 min 2Template Denaturation94 o C60 Sec 3Primer Annealing60 o C60 Sec 4Primer Extension72 o C60 Sec 5Final Extension72 o C3 min تكرر 40
38
Repeated 35 times StepTempTime 1Initial Denaturation95 o C5 min 2Template Denaturation95 o C30 Sec 3Primer Annealing54 o C90 Sec 4Primer Extension72 o C90 Sec 5Final Extension72 o C6 min تكرر (35) تكرر 35
43
7x 10 2 cfu/ml
44
7 x 10 5 cfu/ml
45
7 x 10 7 cfu/ml
46
144 (98.0)* 130 (88.4)* 78 (53.1) * 69(147) 17 (147) 3 (147) (100) 50 P=0.0001 Results obtained using the 3 sets of primers
47
P = 0.05 Senitivity and Specificity of the 3 sets of primers
49
Egyptian Journal of Medical Microbiology, 2007
50
Canadian Journal of Microbiology, 2008
51
Thank you
Similar presentations
© 2024 SlidePlayer.com Inc.
All rights reserved.