Peripheral alpha-synuclein levels in patients with genetic and non-genetic forms of Parkinson's disease.

Background: Variations of α-synuclein levels have been reported in serum and plasma in Parkinson's Disease (PD) Patients.

Methods: Serum and plasma were obtained from PD patients without known mutations (GU-PD, n = 124)), carriers of the A53T/G209A point mutation in the α-synuclein gene (SNCA) (n = 29), and respective age-/sex-matched controls. Levels of total α-synuclein were assessed using an in-house ELISA assay.

Paper-based device providing visual genetic signatures for precision medicine: application to breast cancer.

Genome-wide association studies have demonstrated that combinations of single nucleotide polymorphisms (SNPs), rather than individual SNPs, represent genetic signatures that correlate with heterogeneous and complex diseases. In this context, we developed a paper-based device that provides visual detection of a 10-SNP panel as a genetic signature associated with the risk for breast cancer. The method involves multiplex PCR amplification, multiplex extension reaction of allele-specific primers, without prior purification of the amplified sequences, and, finally, capture and visualization of the extension products within minutes on the device.

Multianalyte quantitative competitive PCR on optically encoded microspheres for an eight-gene panel related to prostate cancer.

Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene.

Digital camera and smartphone as detectors in paper-based chemiluminometric genotyping of single nucleotide polymorphisms.

Chemi(bio)luminometric assays have contributed greatly to various areas of nucleic acid analysis due to their simplicity and detectability. In this work, we present the development of chemiluminometric genotyping methods in which (a) detection is performed by using either a conventional digital camera (at ambient temperature) or a smartphone and (b) a lateral flow assay configuration is employed for even higher simplicity and suitability for point of care or field testing. The genotyping of the C677T single nucleotide polymorphism (SNP) of methylenetetrahydropholate reductase (MTHFR) gene is chosen as a model.

Τwo-panel molecular testing for genetic predisposition for thrombosis using multi-allele visual biosensors.

Thrombosis is considered as the most typical example of multigenic/multifactorial disorder. The three most common genetic risk factors for thrombotic disorders are the G1691A mutation in factor V gene (FV Leiden), the G20210Α mutation in prothrombin gene (FII), and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. An additional panel of biomarkers predisposing for thrombotic events includes the H1299R variant in factor V gene (HR2), A1298C variant in MTHFR gene, the V34L mutation in fibrinogen stabilizing factor XIII (FXIII) gene as well as the 4G/5G polymorphism in plasminogen activator inhibitor type-1 (PAI-1) gene.

Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene.

Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date.

Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis.

Background: Screening for "non-deletion" α-chain haemoglobin variants resulting from point mutations or short deletions/insertions has attracted an increased interest during recent years, especially in areas where α-thalassaemia is prevalent. We describe a method utilising high resolution melting analysis for detecting the 13 most common "non-deletion" α-thalassaemia mutations in populations around the Mediterranean and Middle East.

Methods: The method comprises: (1) amplification of a 1087 bp fragment for each of the duplicated α-globin genes (HBA1 and HBA2) flanking all 13 mutations using a common forward primer and different reverse primers specific for HBA1 and HBA2, respectively; (2) nested amplification of three fragments in HBA2 flanking 10 mutations and two fragments in HBA1 flanking 5 mutations; (3) High resolution melting analysis of the amplicons using a LightScanner Instrument and LC Green.

Multi-allele DNA biosensor for the rapid genotyping of 'nondeletion' alpha thalassaemia mutations in HBA1 and HBA2 genes by means of multiplex primer extension reaction.

Background: Alpha-thalassaemia is an autosomal recessive disorder characterized by defective production of the alpha chain of haemoglobin. It is caused mainly by deletions of one or both of the duplicated alpha-globin genes on chromosome 16, and/or by nucleotide variations, known as "nondeletion" mutations. Definition of the alpha globin genotype in carriers supports genetic counselling, and in patients with Hb H disease is useful to predict prognosis and management options.

Olive oil DNA fingerprinting by multiplex SNP genotyping on fluorescent microspheres.

Olive oil cultivar verification is of primary importance for the competitiveness of the product and the protection of consumers and producers from fraudulence. Single-nucleotide polymorphisms (SNPs) have emerged as excellent DNA markers for authenticity testing. This paper reports the first multiplex SNP genotyping assay for olive oil cultivar identification that is performed on a suspension of fluorescence-encoded microspheres.

Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters.

There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp.

A simplified approach for FSHD molecular testing.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is characterized by complex genetics linked to DNA rearrangements in a polymorphic genomic region of tandemly repeated D4Z4 segments. A panel of FSHD biomarkers including contracted D4Z4 array repeat combined with the 4qA(159/161/168)PAS haplotype has been proposed as molecular signature for defining alleles causally related to FSHD. The aim of the present study was to develop a simple approach for FSHD molecular testing in order to extend studies related to the applicability of FSHD molecular signature in Greek population.

Lateral flow dipstick test for genotyping of 15 beta-globin gene (HBB) mutations with naked-eye detection.

For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896 bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers.

Dipstick test for DNA-based food authentication. Application to coffee authenticity assessment.

This paper reports DNA-based food authenticity assays, in which species identification is accomplished by the naked eye without the need of specialized instruments. Strongly colored nanoparticles (gold nanoparticles) are employed as reporters that enable visual detection. Furthermore, detection is performed in a low-cost, disposable, dipstick-type device that incorporates the required reagents in dry form, thereby avoiding multiple pipetting and incubation steps.

Quantitative bioluminometric method for DNA-based species/varietal identification in food authenticity assessment.

A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SNPs). These single-base changes in DNA are particularly useful because they enable discrimination of closely related species and/or varieties. As a model, quantitative authentication studies were performed on coffee.

Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected.

Quadruple-allele dipstick test for simultaneous visual genotyping of A896G (Asp299Gly) and C1196T (Thr399Ile) polymorphisms in the toll-like receptor-4 gene.

Background: Toll-like receptor-4 (TLR4) is a central regulators of innate immune response as it interacts with bacterial lipopolysaccharide (LPS) and also with endogenous molecules, such as heat-shock proteins and fibrinogen. Two common single nucleotide polymorphisms, A896G (Asp299Gly) and C1196T (Thr399Ile), have been found in the exon 3 of human TLR4 gene, which lead to structure alteration of the extracellular domain of TLR4 thereby influencing the receptor ability for recognition and ligand binding.

Methods: We propose a simple, rapid and reliable method for the simultaneous detection of the two SNPs in TLR4 gene that involves: (a) exponential amplification of the genomic region that spans the two SNPs, (b) quadruple primer extension (PEXT) reaction using two allele-specific primers per SNP, and (c) a simple-to-perform dipstick test that allows visual and simultaneous detection of the four alleles within minutes without the need for specialized instrumentation.

Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization.

Presently, there is a growing interest in the development of lateral flow devices for nucleic acid analysis that enable visual detection of the target sequence (analyte) while eliminating several steps required for pipetting, incubation, and washing out the excess of reactants. In this paper, we present, for the first time, lateral flow tests exploiting oligonucleotide-functionalized and antibody-functionalized carbon nanoparticles (carbon nano-strings, CBNS) as reporters that enable confirmation of the target DNA sequence by hybridization. The CBNS reporters were applied to (a) the detection of PCR products and (b) visual genotyping of single nucleotide polymorphisms in human genomic DNA.

A nanoparticle-based sensor for visual detection of multiple mutations.

Disposable dipstick-type DNA biosensors in the form of lateral flow strips are particularly useful for genotyping in a small laboratory or for field testing due to their simplicity, low cost and portability. Their unique advantage is that they enable visual detection in minutes without the use of instruments. In addition, the dry-reagent format minimizes the pipetting, incubation and washing steps.

Development of a three-biosensor panel for the visual detection of thrombophilia-associated mutations.

We developed a rapid and low-cost panel of three assays for visual genotyping of the three most common genetic risk factors in thrombophilia, namely, the single-point mutations in the FV (Leiden factor), FII and MTHFR genes. A triplex PCR was developed for simultaneous amplification of three fragments spanning the interrogated loci. Allele discrimination was accomplished by a 5-min primer extension reaction on each locus.

Visual screening for JAK2V617F mutation by a disposable dipstick.

During the last 5 years, it was discovered that the JAK2V617F somatic mutation is present in virtually all patients with polycythemia vera and a large proportion of patients with essential thrombocythemia, primary myelofibrosis, and refractory anemia with ring sideroblasts and thrombocytosis. As a result, JAK2V617F was incorporated as a new clonal marker in the 2008 revision of the WHO diagnostic criteria. Current methods for JAK2 genotyping include direct sequencing, pyrosequencing, allele-specific PCR with electrophoresis, restriction fragment length polymorphism, real-time PCR, DNA-melting curve analysis, and denaturing HPLC.

Association of TLR4 single-nucleotide polymorphisms and sarcoidosis in Greek patients.

The present study investigates the potential role of Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile single-nucleotide polymorphisms (SNPs) as risk factors in the development of sarcoidosis using a novel high-throughput microtiter well-based bioluminometric genotyping assay. One hundred and nineteen Greek patients with sarcoidosis and 209 control subjects were genotyped for the two SNPs of the TLR4 gene. The genotypes observed were in Hardy-Weinberg equilibrium.

Bioluminometric assay for relative quantification of mutant allele burden: application to the oncogenic somatic point mutation JAK2 V617F.

Unlike the inherited mutations, which are present in all cells, somatic (acquired) mutations occur only in certain cells of the body and, quite often, are oncogenic. Quantification of mutant allele burden (percentage of the mutant allele) is critical for diagnosis, monitoring of therapy, and detection of minimal residual disease. With point mutations, the challenge is to quantify the mutant allele while discriminating from a large excess of the normal allele that differs in a single base-pair.

Multianalyte, dipstick-type, nanoparticle-based DNA biosensor for visual genotyping of single-nucleotide polymorphisms.

DNA biosensors involve molecular recognition of the target sequence by hybridization with specific probes and detection by electrochemical, optical or gravimetric transduction. Disposable, dipstick-type biosensors have been developed recently, which enable visual detection of DNA without using instruments. In this context, we report a multianalyte DNA biosensor for visual genotyping of two single-nucleotide polymorphisms (SNPs).

Quadruple-allele chemiluminometric assay for simultaneous genotyping of two single-nucleotide polymorphisms.

We developed a rapid, simple, cost-effective and high sample-throughput method for the simultaneous detection of four alleles in single-nucleotide polymorphisms (SNPs). The method was applied to the simultaneous genotyping of two common SNPs within the TLR4 gene, the A896G and C1196T polymorphisms. The method consists of a single PCR of the region spanning the A896G and C1196T polymorphic sites, followed by a quadruple primer extension (PEXT) reaction in a single tube.

Visual genotyping of SNPs of drug-metabolizing enzymes by tetra-primer PCR coupled with a dry-reagent DNA biosensor.

Background: SNP-typing strategies involve an exponential amplification step, an allele discrimination reaction and detection of the products. Usually, allele discrimination is performed after amplification. Tetra-primer PCR allows allele discrimination during the amplification step, thereby avoiding additional genotyping reactions.