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CYSTIC FIBROSIS
CHRONIC INFECTIONS
Once within the lungs, these bacteria adapt to the environment and
developresistanceto commonly used antibiotics.
Each time two carriers of the CF gene have a child, the chances
are:
50% (1 in 2) the child will carry the CF gene but not have CF; and
25% (1 in 4) the child will not carry the gene and not have CF
MULTIPLEX PCR
Primer Length
Melting Temperature
Specificity
Avoid Primer Dimer Formation
DIAGNOSIS
Sample
preparation
Electrophoretic
separation
Multiplex PCR
Amplification
Data analysis
DNA EXTRACTION
1. Collection of the sample
Whole blood samples are usually collected from neonates
however in some cases (PGD) leukocytes and blastomers
can also be used.
The samples were collected in calcium and magnesium
free phosphate buffered saline solution (pbs) with
0.1mg/ml phenol red.
After transferring the cells to 0.2ml reaction tube, cellular
DNAse heat inactivation was done by incubating the
tubes at 65c for 10 minutes.
The cells were then stored at -20 c until PCR was
performed.
MULTIPLEX PCR
QUALITY CONTROL
Three quality control materials negative, normal, and abnormal
are routinely amplified.
The negative control consists of 25 l of water to the
amplification mix. No visible amplification products should
be detected on the hybridization strip.
The normal control consists of a bloodspot collected from a
normal individual. Only normal alleles should be detected on
the hybridization strip.
The abnormal control consists of a bloodspot collected from
a individual with homozygous delta F508 cystic fibrosis.
The deltaF508 mutation should be the only mutation detected
on the hybridization strip; all other alleles should be normal.
ELECTROPHORESIS
Imaging tests.
Lung function tests.
Sputum culture
Organ functioning test
MEDICATIONS
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