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. RESULTS: A statistically significant increase of α-synuclein levels was found in serum, but not plasma, from GU-PD patients compared to healthy controls. A statistically significant decrease of α-synuclein levels was found in serum and plasma from symptomatic A53T mutation carriers compared to healthy controls. Plasma α-synuclein levels were modestly negatively correlated with UPDRS part III score and disease duration in A53T-PD patients. CONCLUSION: Increased α-synuclein levels in serum of GU-PD patients suggest a systemic deregulation of α-synuclein homeostasis in PD. The opposite results in A53T-PD highlight the complexity of α-synuclein homeostatic regulation in PD, and suggest the possibility of reduced expression of the mutant allele.

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. Detection and monitoring are accomplished either by naked eye or by scanning with a common flatbed scanner. The total assay time is ∼ 2 h. The method was evaluated by using 21 clinical samples of known genotypes. The results were fully concordant with the reference method (sequencing). The proposed method is accurate, simple, rapid, and cost-effective. Visual detection does not require specialized instrumentation or highly trained technical personnel. We anticipate that the proposed device will become a useful analytical tool for precision medicine of breast cancer.

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. Using PCR as a synthetic tool, a competitor was prepared for each target sequence containing the same primer binding sites as the target but differing in a short segment to enable discrimination by hybridization. The assay involves multiplex amplification of targets and competitors followed by a multiplex hybridization assay for the 16 amplification products. The assay was performed on optically encoded microspheres with oligonucleotide probes attached to their surface. The microspheres were analyzed rapidly (1 min) by flow cytometry. The signal ratio of the target and cognate competitor is a function of the target copy number in the sample prior to amplification. The multiplexing potential of the proposed method is much higher than real-time PCR and other end-point methods since there are 100 sets of commercially available microspheres.

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. The interrogated DNA sequence is amplified by polymerase chain reaction (PCR) followed by a primer extension reaction. The reaction products are captured through hybridization on the sensing areas (spots) of the strip. Streptavidin-horseradish peroxidase conjugate is used as a reporter along with a chemiluminogenic substrate. Detection of the emerging chemiluminescence from the sensing areas of the strip is achieved by digital camera or smartphone. For this purpose, we constructed a 3D-printed smartphone attachment that houses inexpensive lenses and converts the smartphone into a portable chemiluminescence imager. The device enables spatial discrimination of the two alleles of a SNP in a single shot by imaging of the strip, thus avoiding the need of dual labeling. The method was applied successfully to genotyping of real clinical samples. Graphical abstract Paper-based genotyping assays using digital camera and smartphone as detectors.

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. In this context, we report a novel, rapid and low-cost two-panel diagnostic platform for the simultaneous visual genotyping of the seven mutations (14 alleles). The proposed method comprises the following: (a) a multiplex PCR using genomic DNA isolated from peripheral blood, (b) a multiplex genotyping reaction based on allele-specific primer extension, and (c) visual detection of the genotyping reaction products by means of a multi-allele dipstick-type DNA biosensor, using gold nanoparticles as reporters. The method was applied to 40, previously characterized, and 15 blind clinical samples and the results were 100 % accurate. The proposed assay is simple to perform, requires no specialized and costly equipment, and eliminates multiple pipetting, incubation, and washing steps.

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. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.

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. RESULTS: All 13 "non-deletion" α-chain haemoglobin variants were successfully detected by high resolution melting analysis. All heterozygote samples and eight out of 10 available homozygotes were clearly differentiated from each other and from wild type in the same amplicon. Although not all homozygote samples were distinguishable from wild type samples, this should not present a problem in a clinical setting since all DNA results should be evaluated alongside the haematological and (if relevant) clinical findings in each case. CONCLUSIONS: The 13 "non-deletion" α-chain haemoglobin variants were successfully genotyped by high resolution melting analysis using LightScanner instrument and LCGreen Plus saturating dye. High resolution melting analysis is an accurate mutation scanning tool, advantageous as a closed-tube method, involving no post-PCR manipulations and requiring only around 5 min post-PCR analysis.

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. Here, we report a method that facilitates direct detection by naked eye of the 13 most common "nondeletion" alpha-globin gene mutations in populations around the Mediterranean and Middle East. METHODS AND RESULTS: The method comprises (i) PCR amplification of a single 1087 bp fragment for each HBA1 and HBA2 gene (separately); (ii) multiplex primer extension reaction of just 10 cycles, using unpurified amplification product as a template, to incorporate biotin into those allele-specific primers that extend and, finally, (iii) visual detection of the reaction products within minutes by the dipstick biosensor. The method was evaluated by analysing 105 samples of known genotypes and the results were found fully concordant with those obtained by the reference methods. CONCLUSIONS: The proposed assay is particularly suited for small molecular-diagnostic laboratories with a limited budget and a low-to-medium sample volume. In addition this platform represents a very simple and useful genotyping tool to support gene scanning methods whenever nucleotide variations have to be specified.

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. The 'diagnostic' membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3-8.2.

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. Up to 100 sets of microspheres, with unique "fluorescence signatures", are available. Allele discrimination was accomplished by primer extension reaction. The reaction products were captured via hybridization on the microspheres and analyzed, within seconds, by a flow cytometer. The "fluorescence signature" of each microsphere is assigned to a specific allele, whereas the signal from a reporter fluorophore denotes the presence of the allele. As a model, a panel of three SNPs was chosen that enabled identification of five common Greek olive cultivars (Adramytini, Chondrolia Chalkidikis, Kalamon, Koroneiki, and Valanolia).

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. METHODS AND RESULTS: The method comprises: (i) visual genotyping of the common 4qA and 10qA subtelomeric haplotypes by a multiplex assay in a dipstick format. (ii) Detection of 4qA161 haplotype in D4Z4 contracted alleles by tri-primer PCR. (iii) Detection of PAS SNP in PLAM region and G>C SNP in the first proximal D4Z4 unit by tri-primer PCR. The method was evaluated by analysing DNA from monoallelic sources representing common 4q and 10q haplotypes, samples from 3 FSHD families, 36 unrelated probands and 38 control individuals of Greek origin. CONCLUSIONS: The proposed method could be a very useful tool for FSHD testing making it more accessible to clinical diagnostic laboratories and the wider FSHD community.

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. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ~2 h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood.

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. These involved the determination of the percentage of Arabica and Robusta species based on a SNP in the chloroplastic trnL(UAA)-trnF(GAA) intraspacer region. Following polymerase chain reaction (PCR), the Robusta-specific and Arabica-specific fragments were subjected to 15 min extension reactions by DNA polymerase using species-specific primers carrying oligo(dA) tags. Biotin was incorporated into the extended strands. The products were captured in streptavidin-coated microtiter wells and quantified by using oligo(dT)-conjugated photoprotein aequorin. Aequorin was measured within 3 s via its characteristic flash-type bioluminescent reaction that was triggered by the addition of Ca(2+). Because of the close resemblance between the two DNA fragments, during PCR one species serves as an internal standard for the other. The percentage of the total luminescence signal obtained from a certain species was linearly related to the percent content of the sample with respect to this species. The method is accurate and reproducible. The microtiter well-based assay configuration allows high sample throughput and facilitates greatly the automation.

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. Due to its simplicity, the method does not require highly qualified personnel. The procedure comprises the following steps: (i) PCR amplification of the DNA segment that flanks the unique SNP (species marker); (ii) a 15 min extension reaction in which DNA polymerase extends an allele-specific primer only if it is perfectly complementary with the target sequence; (iii) detection of the products of the extension reaction within a few minutes by the naked eye employing the dipstick. No purification is required prior to application of the extension products to the dipstick. The method is general and requires only a unique DNA sequence for species discrimination. The only instrument needed is a conventional thermocycler for PCR, which is common equipment in every DNA laboratory. As a model, the method was applied to the discrimination of Coffea robusta and arabica species in coffee authenticity assessment. As low as 5% of Robusta coffee can be detected in the presence of Arabica 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. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

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. RESULTS: The method was applied to the simultaneous detection of the two SNPs in 90 samples of general Greek population and the results showed 100% concordance with those obtained by direct sequencing. The entire assay, starting from genomic DNA, can be run in less than 1.5h. CONCLUSIONS: The dipstick test eliminates multiple incubation and washing steps that are common in microtiter well-based assays and does not require highly trained personnel. Because of these advantages, it is suitable for the routine clinical laboratory or even for point-of-care testing.

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. In this work, we significantly enhance the multiplexing capabilities of lateral flow strip biosensors without compromising their simplicity. Multiplex genotyping is carried out by polymerase chain reaction (PCR) followed by a single primer extension reaction for all target alleles, in which a primer is extended and biotin is incorporated only if it is perfectly complementary to the target. Multiallele detection is achieved by multiple test spots on the membrane of the sensor, each comprising a suspension of polystyrene microspheres functionalized with capture probes. The products of the primer extension reaction hybridize, through specific sequence tags, to the capture probes and are visualized by using antibiotin-conjugated gold nanoparticles. This design enables accommodation of multiple spots in a small area because the microspheres are trapped in the fibres of the membrane and remain fixed in site without any diffusion. Furthermore, the detectability is improved because the hybrids are exposed on the surface of the trapped microspheres rather than inside the pores of the membrane. We demonstrate the specificity and performance of the biosensor for multiallele genotyping.

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. Biotinylated PCR product was hybridized with a dA-tailed probe. In one assay configuration, the hybrid is captured at the test zone of the strip by immobilized streptavidin and detected by (dT)(30)-CBNS. In a second configuration, the hybrids are captured from immobilized (dA) strands and detected by antibiotin-CBNS. As low as 2.5 fmol of amplified DNA can be detected. For visual genotyping, allele-specific primers with a 5' oligo(dA) segment are extended by DNA polymerase with a concomitant incorporation of biotin moieties. Extension products are detected either by (dT)(30)-CBNS or by antibiotin-CBNS. Only three cycles of extension reaction are sufficient for detection. No purification of the PCR products or the extension product is required.

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. Detection of the extension products was performed by means of a dual-allele dipstick-type DNA biosensor. The biosensor is disposable and allows visual detection and confirmation of the genotyping products within 15 min by hybridization without the need for specialized instruments or expensive reagents. The proposed method was evaluated by genotyping 40 samples with a variety of genotypes for all three mutations. The genotyping results were in full concordance with those obtained by restriction fragment length polymorphism analysis and sequencing.

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. Some of these methods are labor intensive and time consuming, while the others require specialized costly equipment and reagents. We report a method for direct detection of the JAK2V617F allele by the naked eye using a dipstick test in a dry-reagent format. The method comprises a triprimer PCR combined with visual detection of the products within minutes by the dipstick test. Specialized instrumentation is not involved. The requirements for highly qualified technical personnel are minimized. Because the detection reagents exist in dry form on the dipstick, there is no need for multiple pipetting and incubation steps.