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Method of detection of SP-A2 gene variants useful for prediction of predisposition to aspergillosis

a technology of aspergillosis and gene variants, applied in the field of detection of spa2 gene variants, can solve problems such as morbidity and mortality

Inactive Publication Date: 2007-02-08
SARMA PURANAM USHA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pulmonary aspergillosis is a serious threat to those immunocompromised as a result of disease or therapy, and has been identified as a major cause of morbidity and mortality in asthmatic and cystic fibrosis patients.

Method used

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  • Method of detection of SP-A2 gene variants useful for prediction of predisposition to aspergillosis
  • Method of detection of SP-A2 gene variants useful for prediction of predisposition to aspergillosis

Examples

Experimental program
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Effect test

example 1

Identification of Allelic Variants of SP-A2 Gene

[0056] This example describes the identification of allelic variant of human surfactant protein A 2 gene by PCR and sequencing using certain oligonucleotide primers. According to the invention DNA was extracted from human peripheral blood leukocytes using a modification of salting out procedure. The concentration of the DNA was determined by measuring the optical density of the sample, at a wavelength of 260 nm. The DNA was then amplified by PCR by using the oligonucleotide primers:

(SEQ. ID. No. 1)5′ TGC CTG GAG CCC CTG GTG TCC CTG GAG AGC 3′(Forward)(SEQ. ID. No.2)5′ TGC CTC GTC CGC ATT CAC CCT TCA GAC TGC 3′(Reverse).

[0057] The sample were denatured at 95° C. for 5 minutes followed by 28 cycles of denaturation (95° C., 1 minutes), annealing (70° C., 1 minute), extension (72° C., 1 minute) and a final extension of 7 minutes at 72° C. in a PE GeneAmp PCR System 9600. This reaction produced a DNA fragment of 459 bp when analysed by ...

example 2

Nucleotide Sequence of the Allelic Variant of SP-A2 Gene

[0058] The nucleotide seq. of the allelic variant of SP-A 2 gene derived using the method as described in example 1.

[0059] In the above sequence the 2 SNP's as given in table II are at nucleotide position 1649 and 1660.

example 3

Patients with A Allele at 1660 Position are at Nearly Zero Risk for the ABPA Disease

[0060] A method as described in example 1 is applied to a series of DNA samples extracted from ABPA positive individuals and normal controls. There is observed a statistically significant difference (At position 1649 p=0.1193 and at position 1660 p=0.0156) in the frequency distributions of the SNP haplotypes generated using SNP in normal and ABPA patient SP-A2 chromosome. The results obtained are summarized in table below

TABLE IVSNP (G vs C) atSNP (G vs A) at1649 position1660 positionODDS RATIO (ABPA2.12644.7778patient vs Normal)Chi-square2.4265.846p-value0.11930.0156

[0061] A strong association of G (at 1649 position) and G (at 1660 position) haplotypes with ABPA disease chromosome indicated that SP-A2 alleles with the G (at 1649 position) and G (at 1660 position) haplotypes are predisposed to the disease. Therefore, these SNP haplotypes in the human SP-A2 gene could be used as a method of establ...

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Abstract

Allele specific primers and probes suitable for detecting allelic variants of human SP-A2 gene for applications such as molecular diagnosis, prediction of an individual's susceptibility, and / or the genetic analysis of SP-A2 gene in a population.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation-in-part of U.S. application Ser. No. 10 / 102,731, filed Mar. 22, 2002 and which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION [0002] The present invention relates to a method of detection of SP-A2 gene variants useful for prediction of predisposition to aspergillosis. The utility of the invention is in applications such as molecular diagnosis, prediction of an individual's disease susceptibility and genetic analysis of SP-A2 gene in a population. The invention also provides primer sequences useful in detecting these polymorphic variations in SP-A2 gene and their use in diagnosis and prediction of an individual's susceptibility to Allergic bronchopulmonary aspergillosis (ABPA). BACKGROUND OF THE INVENTION About the Disease [0003] Aspergillosis is a group of fungal diseases which include allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, chronic necrotising aspergil...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q2600/156C12Q1/6883
Inventor SARMA, PURANAM USHAMADAN, TARUNASAXENA, SHWETA
Owner SARMA PURANAM USHA