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Volume 6 Number 5, September/October 1996, pp.301-303 Reverse-Hybridisation Detection of CF Mutations - A Line- Probe Assay Karen Behm, Murex Diagnostics Australia Pty Ltd, PO Box 351, North Ryde, NSW 2113
Code Number: AU96017
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[ALL TABLES AND FIGURES AT END OF TEXT] A DNA probe-based test that is suitable for simultaneous detection of the eight most common cystic fibrosis mutations is described. The test involves a membrane immobilised probe with chromogenic signal visualisation Cystic Fibrosis Cystic fibrosis (CF) is an inherited condition which results in the disordered secretion of all exocrine glands. Viscous secretions accumulate in bronchi and intestines and consequentially lead to respiratory and digestive problems. Additional symptoms include: fibrosis of the pancreas, reduced fertility or sterility, regular infections of the lungs, vitamin deficiency and sweat with increased levels of sodium and chloride ions. The gene involved in CF is located on the long arm of human chromosome 7 (region q31-q32). The locus spans approximately 27 exons. It encodes the cystic fibrosis transmembrane-conductance regulator (CFTR), a protein of 1480 amino acids. It is mutations within this gene which leads to CF. At present, more than 250 different CF-causing mutations have been identified including single amino acid substitutions, non-sense mutations and mutations affecting splicing sites. CF is the most common autosomal recessive genetic disorder in caucasian populations, with a carrier rate of 1 in 22 individuals. Its incidence rate ranges from 1 in 1600 to 1 in 2500, depending on the ethnic group considered. Early postnatal diagnosis of CF patients can improve efficiency of the treatment. In addition, a patient's genotype at the CFTR locus may be a helpful diagnostic indicator. There is thus a need for a screening test able to distinguish between the variety of cystic fibrosis allelles present in an affected population. Using the INNO-LiPA CF2 strips, carriers, diseased and healthy individuals can be distinguished from one another.
Principle of the INNO-LiPA Tests The INNO-LiPA CF2 is a fast, easy and highly specific DNA hybridisation test based on the "reverse hybridisation" principle (Fig.1). Amplified biotinylated DNA material, from the multiplex amplification of the four exons of the CFTR gene, is hybridised with oligonucleotide probes specifically designed to hybridise with the wild-type sequence and probes designed to correspond with the mutant sequence. These probes are immobilised as parallel lines on membrane-based strips. After hybridisation, streptavidin labelled with alkaline phosphatase is added and bound to any biotinylated hybrid previously formed. Incubation with BCIP/NBT (bromochloroindoylphosphate/ nitroblue tetrazolium) chromogen results in a purple/brown precipitate. The test can easily be performed in 3.5 hours, after sample preparation and amplification (Table I). The hybridisation step, the stringent wash step and the colorimetric detection of the strips are all performed in handy troughs, included in the kits. The strips stay in the same troughs throughout these three steps. Example of Results The eight mutations screened by the INNO-LiPA CF2 strips are: DF508, G542X, N1303K, 1717-1(G to A), W1282X, G551D, R553X and DI507. A black line is drawn on the top of the strips for orientation. The conjugate control line provides an internal control for colour development (Fig. 2). A positive signal with a mutant probe indicates that this particular mutation is present in the genome of the individual. If a negative signal is obtained with the wild-type probe, while the mutant probe is positive, the individual is homozygous for that particular mutation. Features of the INNO-LiPA CF Test The INNO-LiPA CF2 is a reliable reverse hybridisation test with the following advantageous characteristics:
-differentiation between normal individuals and healthy carriers; -highly specific: 100%; -highly sensitive: depending on the ethnical group under study; -fast: results within three hours after amplification; -many samples can be processed simultaneously; -easy interpretation of results; -safe: no toxic components in the hybridisation or stringent wash buffers; non-radioactive; -test completely automated; a walk-away system when used in conjunction with Auto-LiPA instrumentation; -no stress to patients; -no expensive equipment needed.
From several LiPA assays performed where amplification of DNA was variable in degree of success, it was concluded that even low quality DNA (low signals in the gel) yield good interpretable results in the INNO-LiPA. Applications The reliability and ease-of-use allows the INNO-LiPA CF test to be used in a variety of applications. These include:
-general population screening; -postnatal CF diagnosis; -screening of newborns; -CF mutation analysis for disease prognosis and research studies for mutation frequencies.
Tsui L.C. and Buchwals, M. in "Advances in Human Genetics"; Harris and Hirschhorn Ed., Vol 20, Plenum Press, NY p. 153 (1992) Tsui L.C., Trends in Genetics; 11:392 (1992) Super M. Brit. Med. Bull.; 4:717 (1992) Cuppens H. et al.; Mol. Cell Probes; 6:33 (1992) n.b. The polymerase chain reaction (PCR) is covered by patents to Hoffman-LaRoche. If you are performing PCR, you may therefore wish to contact Hoffman-LaRoche for information. Nothing in this product information should be considered as an authorisation or an implicit license to practice PCR under any patents held by Hoffman-LaRoche.
Background History of Murex Diagnostics and Innogenetics Murex Diagnostics is a fully integrated diagnostics company with extensive world-wide distribution and product sales predominantly in the infectious disease areas of blood viral screening, bacteriology and DNA probes. Murex, as an entity, was formed four years ago when International Murex Trading Corp., IMTC, acquired Wellcome Diagnostics, a division of Burroughs Wellcome pharmaceuticals. Innogenetics NV was founded in Belgium in 1985 and is widely regarded as an innovative biotechnology company by the scientific community in Europe. Key technological areas include genetic engineering, line probe assay (LiPA) with multiple DNA probe assays on a single strip, line immunoassay (LIA) with multiple immunoassays on a single strip and DNA spacer probe methods to identify specific infectious species. These technologies are supported by some 200 scientists working with strong ties with various academic and medical institutions. As part of continued advancement and development, due for launch in 1997 is the INNO-LiPA CF15 kit. Based on all the same principles as its predecessor, the INNO-LiPA CF15 is for the simultaneous detection of 22 CF-causing mutations.
---------------------------------------------------------------- Table 1. The three major steps in the INNO-LiPA CF test ---------------------------------------------------------------- Major steps and total assay time amplification of target DNA 120-180 min 1 - hybridisation 90 min 2 - stringent wash 30 min 3 - colorimetric detection 80 min Total assay time (after amplification) \< 3.5 hrs ----------------------------------------------------------------- Copyright 1996 Australian Biotechnology Association Ltd. The following images related to this document are available:Photo images[au96017b.jpg]Line drawing images[au96017a.gif] |
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