DIAGNOSIS OF CYSTIC FIBROSIS

USING MULTIPLEX PCR

CYSTIC FIBROSIS

A chronic, progressive, and frequently fatal genetic

disease of the bodys mucus glands.
Affects the respiratory and digestive systems in
children and young adults.
An average person has a lifespan of 40 years with
the right treatment.
The name cystic fibrosis refers to the characteristic
scarring (fibrosis) and cyst formation within the
pancreas, first recognized in the 1930s.

DEFECTIVE CFTR GENE

Cystic Fibrosis is caused by a defective CFTR [cystic

fibrosis transmembrane conductance regulator] gene

which codes for a Na+ and Cl- transporter found on the
surface of epithelial cells of lungs and other organs.
This results in thickened secretions in the airways,
pancreatic ducts, biliary tree, intestines and reproductive
tract leading to the clinical manifestations of the disease.
As many as 1300 mutations in the CFTR gene have been
observed worldwide.
The severity of the disease is directly related to the
characteristic effects of the particular mutations that have
been inherited by the individual sufferer.

CHRONIC INFECTIONS

The lungs of individuals with cystic fibrosis are colonized and

infected by bacteria from an early age. These bacteria, which often
spread among individuals with CF, thrive in the altered mucus, which
collects in the small airways of the lungs. This mucus leads to the
formation of bacterial microenvironments known asbiofilmsthat are
difficult for immune cells and antibiotics to penetrate.

A variety of organisms then colonize the infected lungs. In the early

stages organisms like Staphylococcus aureus& Hemophilus
influenzaecolonize and infect the lungs.

With the advancement in infection, organisms likePseudomonas

aeruginosa & Burkholderia cepacia dominates.

Once within the lungs, these bacteria adapt to the environment and
developresistanceto commonly used antibiotics.

SIGNS AND SYMPTOMS

The hallmark signs and symptoms of cystic
fibrosis are:
salty tasting skin
poor growth
poor weight gain despite a normal food intake
accumulation of thick, sticky mucus
frequent chest infections
coughing or shortness of breath.

TEST FOR CYSTIC FIBROSIS

Sweat Test If a person shows symptoms of CF or if a baby has a positive newborn

screen for CF, a doctor may order a sweat test. This simple, painless test is the best
way to diagnose CF. It measures the concentration of salt in a persons sweat. A high
salt level indicates CF.
Sweat tests should be done at a Cystic Fibrosis Foundation-accredited care center
where strict guidelines help ensure accurate results.
Newborn Screening Newborns screened for cystic fibrosis can benefit from early
diagnosis and treatment, which can:
Improve growth;
Help keep lungs healthy;
Reduce hospital stays; and
Add years to life.
While newborn screening is not a definitive diagnostic test for cystic fibrosis, it may
lead to tests that can rule out or confirm a CF diagnosis. All states in the U.S. screen
newborns for cystic fibrosis.

Genetic Carrier Testing More than 10 million Americans are

symptomless carriers of the defective CF gene. This blood test
can help detect carriers, who could pass CF onto their children.
To have cystic fibrosis, a child must inherit one copy of the
defective CF gene from each parent.

Each time two carriers of the CF gene have a child, the chances
are:

25% (1 in 4) the child will have CF;

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

Multiplex PCR,is a modification of PCRin order

to rapidly detect deletionsorduplicationsin a
largegene.
This processamplifies genomicDNAsamples
using multiple primersand a temperaturemediatedDNA polymerasein athermal cycler.

PRIMER DESIGN PARAMETERS FOR MULTIPLEX PCR

Primer Length
Melting Temperature
Specificity
Avoid Primer Dimer Formation

TYPES OF MULTIPLEX PCR

Multiplexing reactions can be broadly divided in 2
categories:
1.
Single
Template
PCR
Reaction
This technique uses a single template which can be
a genomic DNA along with several pairs of forward
and reverse primers to amplify specific regions
within a template.
2.
Multiple
Template
PCR
Reaction
It uses multiple templates and several primer sets
in the same reaction tube. Presence of multiple
primers may lead to cross hybridization with each
other and the possibility of mis-priming with other
templates.

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

Prior to PCR the buffers and the PCR mix without

primers were decontaminated under UV radiations.
Cells were lysed using alkaline phosphate buffer and
multiplex PCR was performed with taq polymerase
and gene amp PCR system.
Primers were fluorescently labelled and the process
began with denaturation (95C; 5 mins)followed by
addition of primers and annealing (55oc; 60 sec) &
finally elongation (68c).

The PCR master mix is prepared just prior to use and

contains the following final concentrations:
10 mM Tris-Cl, 50 mM KCl,
0.12 mM 5'-biotinylated primer mix,
0.3 mM dATP, 0.3 mM dCTP,
0.3 mM dGTP,
0.6 mM dUTP,
3 U uracil N-glycosylase,
15 U FastStart Taq DNA polymerase,
and 8 mM MgCl2

Multiplex PCR amplification is performed using a

GeneAmp PCR System 9600 thermal cycler.

Samples are amplified as follows:

hold for 10 min at 42C (to activate uracil N-glycosylase);
hold for 6 min at 93C (to inactivate uracil N-glycosylase);

And then cycled (32 cycles) with denaturation, annealing &elongation.

Hold upto 2hr at 15oc

Following amplification, they are analysed immediately or stored

at -10C.

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

Tris-borate-EDTA PAGE is performed.

The multiplex PCR samples are mixed with 2 l of 6X
sample buffer containing bromophenol blue and Ficolltype 400.
Samples are electrophoresed on 4% mini-polyacrylamide
gels in 1X TBE at 75 V for 1 hr at RT.
Gels are stained with ethidium bromide for 10 min and
photographed.
Electrophoretic migration of multiplex amplicons is
compared to DNA molecular weight 100 bp ladders

It consists of multiple primer sets within a single

PCR mixture to produceampliconsof varying
sizes that are specific to different DNA
sequences.
By targeting multiple genes at once, additional
information may be gained from a single test-run
that otherwise would require several times the
reagents and more time to perform.

TESTING AFTER DIAGNOSIS

Imaging tests.
Lung function tests.
Sputum culture
Organ functioning test

TREATMENT AND DRUGS

There is no cure for cystic fibrosis, but treatment can
ease symptoms and reduce complications.
The goals of treatment include:
- Preventing and controlling lung infections,
-Loosening and removing mucus from the lungs,
- Preventing and treating intestinal blockage,
- Providing adequate nutrition.

MEDICATIONS

The options include:

Antibioticsto treat and prevent lung infections
Mucus-thinning drugsto help you cough up the
mucus, which improves lung function
Bronchodilatorsto help keep your airways open
by relaxing the muscles around your bronchial
tubes
Oral pancreatic enzymesto help your digestive
tract absorb nutrients

THANK YOU!!