Real time PCR and it's functions in diagnosis

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Nowadays HIV is strikingly spreading out whole the world. so in order to diminish its distribution , it is necessary to detect it as soon as possible & for this purpose, Real time PCR is recommended by scientist.

In this method ,’ pol’’ gen of the virus, is amplified in thermocycler.

26 patient have been studied. infection in these patients was confirmed by ELISA & western blot.

Then what was done?

Sampling & RNA extracting from patients.

Cloning of target segment by using Xba I & Hind III. And 180 bp primers.

Standard virus mRNA was extracted.

Quantitative analysis of HIV virus by SYBR-green Real Time RT-PCR.(3,4,5)

Introduction

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

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0.09

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180 bp

marker

cycle

Flu

- control

product

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 24

Detection of unknown deleted genes in Thalassemia

Thalassemia is the most commonplace hemoglobin disorder which is caused by deletion of one or both globin genes.

By the way deletion or any other change can be detected quantitatively by real time PCR in a real time, we can determine the sort of deletion.

Alpha thalassemia is caused mostly by whole gene deletion but beta is by piont mutation.

60 patients were under study.(6,7,8)

Primer for deleted genes were designed using Primer Express soft ware & its sequence confirmed in NCBI/BLAST data bank. after PCR any changes was detected.

Introduction

Advantage disadvantages

Product Detection

Conclusion

Reference

Real Time PCR

in medicine

Peripheral blood findings and molecular evidence for loss of the telomeric region of chromosome 16p in a patient with acquired hemoglobin H and myelodysplastic syndrome.

Wright-Giemsa–stained peripheral blood smear demonstrates severe anisopoikilocytosis.

(B) Brilliant cresyl blue stain reveals with classical HbH inclusions .

(C) Metaphase spread and 2 interphase nuclei.

(D) Southern blot of the 3 hypervariable region in the alpha-globin cluster

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 25

Cystic fibrosis (CF) is the most common inherited disease among Caucasian populations with an incidence of ~1 in 2500 births.(9)

Couples in which both individuals carry a mutant copy of the CF gene have a one in four chance of having an affected child.The conditions caused by these mutations range from mild to lifethreatening.

A3 base pair (bp) deletion, designated DF508, accounts for nearly 70% of CF cases and causes severe manifestations of the disease. It results in the absence of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and this error prevents normal processing and translocation of the polypeptide chain to apical membranes of epithelial cells.This deletion can be detected by molecular beacons in real time PCR.