Low DNA Concentrations Research Articles

Development of PCR-Based Molecular Marker for Detection of Xanthomonas campestris pv. campestris Race 6, the Causative Agent of Black Rot of Brassicas.

Xanthomonas campestris pv. campestris (Xcc), the pathogen of black rot which is the most destructive disease of Brassica vegetables throughout the world. Here, we reported two novel sequence-characterized amplified region (SCAR) markers (i.e., XccR6-60 and XccR6-67) for the detection of Xcc race 6 via re-alignment of the complete genome sequences of Xcc races/strains/pathovars. The specificity of SCAR primer sets was verified by mean of PCR amplification using the genomic DNA template of Xcc races/strains/pathovars and two other plant infecting bacterial strains. The PCR result revealed that the XccR6-60 and XccR6-67 primer sets amplified 692-bp and 917-bp DNA fragments, respectively, specifically from race 6, while no visible amplification was detected in other samples. In addition, the SCAR primers were highly sensitive and can detect from a very low concentration of genomic DNA of Xcc race 6. However, the complete genome sequence of Xcc race 6 is not yet publicly available. Therefore, the cloning and sequencing of XccR6-60 and XccR6-67 fragments from race 6 provide more evidence of the specificity of these markers. These results indicated that the newly developed SCAR markers can successfully, effectively and rapidly detect Xcc race 6 from other Xcc races/strains/pathovars as well as other plant pathogenic bacteria. This is the first report for race-specific molecular markers for Xcc race 6.

Efficacy of heated tourmaline in reducing biomass clogging within woodchip bioreactors

Woodchip bioreactors can effectively remove waterborne nitrates from subsurface agricultural drainage and prevent the eutrophication of receiving water, but rapid biofilm growth can severely reduce water flux and denitrification efficiency of this practice within a few years. Tourmaline minerals with thermal excitation could generate reactive oxygen species which would inhibit bacterial growth. In this study, laboratory scale woodchip bioreactors were set up to test the anti-clogging and denitrification efficiency of heated woodchips with tourmaline, heated woodchips without tourmaline, and unheated woodchips. The results showed that the heated tourmaline treatment could reduce the clogging and optimize the nitrate removal rate (47.6 g N/m3/day) under all three hydrologic retention times tested (1, 4, and 8 h). Dissolved oxygen and pH values fluctuated with the removal rate and temperature change, while temperature was identified as the key factor impacting the tourmaline treatment. The heated tourmaline treatment had the lowest biofilm growth (lowest DNA concentration), while the 16S rRNA and a higher abundance of nirS-, nirK-, and nosZ-encoding denitrifying bacteria (based on qPCR) confirmed the higher denitrification efficiency of the heated tourmaline treatment.

Study of the Effect of Material of Microfluid Chip on the Polymerase Chain Reaction

Effect of several polymer materials from different manufacturers on the polymerase chain reaction is tested. Absence of adsorption of reaction components at room temperature is demonstrated for a polymethylmethacrylate chip. However, a decrease in the reaction efficiency at low DNA concentrations is observed for the polymerase chain reaction in the microchip.

Low molecular weight serum cell-free DNA concentration is associated with clinicopathologic indices of poor prognosis in women with uterine cancer

BackgroundSerum cell-free DNA (cfDNA) holds promise as a non-invasive cancer biomarker. The objective of this study was to evaluate the association of cfDNA concentration with clinicopathologic variables of poor prognosis and overall survival among women with uterine cancer compared to benign cancer-free controls.MethodscfDNA was extracted from the serum of 91 women with multiple uterine cancer histologies and 22 post-menopausal controls without cancer. Low molecular weight (LMW) cfDNA was separated from contaminating genomic high molecular weight cfDNA using paramagnetic bead purification and its concentration was measured using fluorometric quantification. Clinicopathologic data was abstracted from the electronic medical record. The association between serum cfDNA concentration, clinicopathologic variables, and overall survival was assessed using linear regression modelling, Cox proportional hazards modelling, and the Kaplan–Meier method.ResultsMedian total serum cfDNA concentration for the cohort was 69.2 ng/mL (IQR 37.4, 132.3) and median LMW cfDNA concentration was 23.8 ng/mL (IQR 14.9, 44.4). There were no significant differences in total serum cfDNA concentration with any clinicopathologic variables. However, LMW cfDNA concentration was significantly higher in serum of women with cancer (25.8 ng/mL IQR 16.0, 49.6) compared to benign controls (15.5 ng/mL IQR 9.3, 25.8 ng/mL) (p < 0.01). It is also significantly higher among women with early stage cancer than benign controls (p < 0.01). There were also significant associations between LMW cfDNA concentration and stage of cancer (p = 0.01) and histology (p = 0.02). Patients with leiomyosarcoma and carcinosarcoma had higher cfDNA concentrations than those with endometrioid cancer. Over a median follow-up of 51.9 months, 75th percentile for overall survival for women with cancer was 24.0 months. Higher LMW cfDNA concentrations is associated with lower survival among women with cancer (p < 0.01).ConclusionsSerum LMW cfDNA concentration is associated with overall survival in women with uterine cancer, and it is higher among women with uterine cancer compared to those of controls.

Allopatric instead of parapatric divergence in an ectomycorrhizal fungus (Laccaria trichodermophora) in tropical sky-islands

Abstract In tropical sky-islands, cold-affinity populations tend to become isolated at highlands during the interglacial periods, and to expand into the lowlands where they become more connected during the glacial periods. Although this has been widely studied in trees, it is poorly understood how fungal symbionts can differentiate among mountains (allopatrically), or within a single mountain (parapatrically) due to climate fluctuations. Here, we conducted population genomic analyses on the ectomycorrhizal fungus Laccaria trichodermophora in three tropical sky-islands using Genotyping by Sequencing (GBS) at low DNA concentrations. There were no significant differences between altitudes within a single mountain, but we observed significant genetic differentiation among populations from different mountains, supporting the allopatric differentiation hypothesis. Our results indicate that L. trichodermophora populations are under a sky-island population dynamics that started during the Pleistocene climate fluctuations.

The Study of the Yield of DNA Extracted From Hairs of Postmortem Cases

Background: Problems frequently occur from hair extraction are low concentration and impurity of DNA.&#x0D; Methods: Hairs from 30 postmortem cases were collected. In each case, 5 and 10 hair roots were extracted DNA by Phenol/Chloroform/Isoamyl alcohol. The amount and purity of DNA (A260/280) were detected by NanoDrop spectrophotometer. The quality of DNA was examined using polymerase chain reaction (PCR). Amount of DNA was analyzed compared with gender, age, and manner of death.&#x0D; Results: The amount and purity of DNA extracted from 10 hair roots were slightly better than 5 hair roots (P &gt; .05). The mean (range) of DNA concentration was 200.4 (35.2 - 799.6) ng/µL from 10 hair roots vs 148.2 (21.7 - 571.5) ng/µL from 5 hair roots in 30 µL volume whereas the mean (range) of A260/280 was 1.61 (1.31 - 1.99) in 10 hair roots vs 1.54 (1.32 - 1.90) in 5 hair roots. The results from NanoDrop showed no difference among various gender, age, and manners of death.&#x0D; Conclusions: DNA can be extracted from hair by Phenol/Chloroform/Isoamyl alcohol. The quantity and quality of DNA were good enough for personal identification by PCR. DNA extracted from 10 hair roots is better than 5 hair roots.&#x0D;

DNA Photocleavage in the Near-Infrared Wavelength Range by 2-Quinolinium Dicarbocyanine Dyes.

Here, we report the syntheses of two pentamethine cyanine dyes containing quinolinium rings and substituted with either hydrogen (3) or bromine (4) at the meso carbon. The electron withdrawing bromine atom stabilizes dye 4 in aqueous buffer, allowing complex formation to occur between the dye and double-helical DNA. UV–visible, CD, and fluorescence spectra recorded at low DNA concentrations suggest that dye 4 initially binds to the DNA as a high-order aggregate. As the ratio of DNA to dye is increased, the aggregate is converted to monomeric and other low-order dye forms that interact with DNA in a non-intercalative fashion. The brominated dye 4 is relatively unreactive in the dark, but, under 707–759 nm illumination, generates hydroxyl radicals that cleave DNA in high yield (pH 7.0, 22 °C). Dye 4 is also taken up by ES2 ovarian carcinoma cells, where it is non-toxic under dark conditions. Upon irradiation of the ES2 cells at 694 nm, the brominated cyanine reduces cell viability from 100 ± 10% to 14 ± 1%. Our results suggest that 2-quinolinium-based carbocyanine dyes equipped with stabilizing electron withdrawing groups may have the potential to serve as sensitizing agents in long-wavelength phototherapeutic applications.

Acoustic Methodology for Selecting Highly Dissipative Probes for Ultrasensitive DNA Detection.

The objective of this work is to present a methodology for the selection of nanoparticles such as liposomes to be used as acoustic probes for the detection of very low concentrations of DNA. Liposomes, applied in the past as mass amplifiers and detected through frequency measurement, are employed in the current work as probes for energy-dissipation enhancement. Because the dissipation signal is related to the structure of the sensed nanoentity, a systematic investigation of the geometrical features of the liposome/DNA complex was carried out. We introduce the parameter of dissipation capacity by which several sizes of liposome and DNA structures were compared with respect to their ability to dissipate acoustic energy at the level of a single molecule/particle. Optimized 200 nm liposomes anchored to a dsDNA chain led to an improvement of the limit of detection (LoD) by 3 orders of magnitude when compared to direct DNA detection, with the new LoD being 1.2 fmol (or 26 fg/μL or 2 pM). Dissipation monitoring was also shown to be 8 times more sensitive than the corresponding frequency response. The high versatility of this new methodology is demonstrated in the detection of genetic biomarkers down to 1-2 target copies in real samples such as blood. This study offers new prospects in acoustic detection with potential use in real-world diagnostics.

Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids

Multiplex amplification of DNA can be highly valuable in circulating tumor DNA (ctDNA) analysis due to the sheer number of potential mutations. However, commercial ctDNA extraction methods struggle to preconcentrate low concentrations of DNA and require multiple sample handling steps. Recently, magnetic ionic liquids (MILs) have been used to extract DNA and were integrated with a quantitative polymerase chain reaction (qPCR). However, in previous studies, DNA could not be preconcentrated from plasma and only one fragment could be amplified per reaction. In this study, MILs were utilized as DNA extraction solvents and directly integrated into a multiplex-qPCR buffer to simultaneously amplify wild-type KRAS, G12S KRAS, and wild-type BRAF, three clinically-relevant genes whose mutation status can affect the success of anti-EGFR therapy. DNA was desorbed from the MIL solvent during a multiplex-PCR without having a significant effect on the amplification efficiency, and allelic discrimination of single-nucleotide polymorphisms could still be achieved. Enrichment factors over 35 for all three sequences were achieved from Tris buffer using the [N8,8,8,Bz+][Ni(hfacac)3-]) and [P6,6,6,14+][Ni(Phtfacac)3-] MILs. DNA could still be preconcentrated from 2-fold diluted human plasma using the [N8,8,8,Bz+][Ni(hfacac)3-] MIL. Extractions from undiluted plasma were reproducible with the [P6,6,6,14+][Ni(Phtfacac)3-] MIL although DNA was not preconcentrated with enrichment factors around 0.6 for all three fragments. Compared to commercial DNA extraction methods (i.e., silica-based spin columns and magnetic beads), the MIL-based extraction achieved higher enrichment factors in Tris buffer and plasma. The ability of the MIL-based dispersive liquid-liquid microextraction (DLLME) direct-multiplex-qPCR method to simultaneously achieve high enrichment factors of multiple DNA fragments from human plasma is highly promising in the field of ctDNA detection.

Promotion and Inhibition of the Oxidase‐Mimicking Activity of Nanoceria by Phosphate, Polyphosphate, and DNA

Nanoceria (CeO2 nanoparticles) is an extensively studied nanozyme with interesting oxidase-mimicking activity. As they can work in the absence of toxic and unstable H2 O2 , CeO2 nanoparticles have been widely used in biosensing. CeO2 nanoparticles often encounter phosphate-containing molecules that can affect their catalytic activity, and various reports exist in the literature showing both promoted and inhibited activity. In this work, we systematically studied five types of phosphate: orthophosphate, pyrophosphate, triphosphate, trimetaphosphate, and a polyphosphate with 25 phosphate units (Pi25 ). In addition, DNA oligonucleotides of various length and sequence. DNA was included as they contain a phosphate backbone that can strongly adsorb on nanoceria. We observed that a high concentration of DNA in acetate buffer inhibited activity, whereas a low concentration of DNA in phosphate buffer increased activity. The change of activity was also related to the type of substrate and related to the aggregation of CeO2 . These discoveries provide an important understanding for the further use of CeO2 nanoparticles in biosensor development, materials science, and nanotechnology.

Sequencing Analysis of Plasma Epstein-Barr Virus DNA Reveals Nasopharyngeal Carcinoma-Associated Single Nucleotide Variant Profiles.

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. Plasma EBV DNA is a validated screening tool for NPC. In screening, there are some individuals who do not have NPC but carry EBV DNA in plasma. Currently it is not known from screening if there may be any genotypic differences in EBV isolates from NPC and non-NPC subjects. Also, low concentrations of EBV DNA in plasma could pose challenge to such EBV genotypic analysis through plasma DNA sequencing. In a training dataset comprised of plasma DNA sequencing data of NPC and non-NPC subjects, we studied the difference in the EBV single nucleotide variant (SNV) profiles between the two groups. The most differentiating SNVs across the EBV genome were identified. We proposed an NPC risk score to be derived from the genotypic patterns over these SNV sites. We subsequently analyzed the NPC risk scores in a testing set. A total of 661 significant SNVs across the EBV genome were identified from the training set. In the testing set, NPC plasma samples were shown to have high NPC risk scores, which suggested the presence of NPC-associated EBV SNV profiles. Among the non-NPC samples, there was a wide range of NPC risk scores. These results support the presence of diverse SNV profiles of EBV isolates from non-NPC subjects. EBV genotypic analysis is feasible through plasma DNA sequencing. The NPC risk score may be used to inform the cancer risk based on the EBV genome-wide SNV profile.

Effects of sample age on data quality from targeted sequencing of museum specimens: what are we capturing in time?

BackgroundNext generation sequencing (NGS) can recover DNA data from valuable extant and extinct museum specimens. However, archived or preserved DNA is difficult to sequence because of its fragmented, damaged nature, such that the most successful NGS methods for preserved specimens remain sub-optimal. Improving wet-lab protocols and comprehensively determining the effects of sample age on NGS library quality are therefore of vital importance. Here, I examine the relationship between sample age and several indicators of library quality following targeted NGS sequencing of ~ 1300 loci using 271 samples of pinned moth specimens (Helicoverpa armigera) ranging in age from 5 to 117 years.ResultsI find that older samples have lower DNA concentrations following extraction and thus require a higher number of indexing PCR cycles during library preparation. When sequenced reads are aligned to a reference genome or to only the targeted region, older samples have a lower number of sequenced and mapped reads, lower mean coverage, and lower estimated library sizes, while the percentage of adapters in sequenced reads increases significantly as samples become older. Older samples also show the poorest capture success, with lower enrichment and a higher improved coverage anticipated from further sequencing.ConclusionsSample age has significant, measurable impacts on the quality of NGS data following targeted enrichment. However, incorporating a uracil-removing enzyme into the blunt end-repair step during library preparation could help to repair DNA damage, and using a method that prevents adapter-dimer formation may result in improved data yields.

Probe-based multiplex qPCR identifies blood-meal hosts in Anopheles mosquitoes from Papua New Guinea

BackgroundDetermination of blood-meal hosts in blood-fed female Anopheles mosquitoes is important for evaluating vectorial capacity of vector populations and assessing effectiveness of vector control measures. Sensitive molecular methods are needed to detect traces of host blood in mosquito samples, to differentiate hosts, and to detect mixed host blood meals. This paper describes a molecular probe-based quantitative PCR for identifying blood-meal hosts in Anopheles malaria vectors from Papua New Guinea.MethodsTaqMan oligonucleotide probes targeting specific regions of mitochondrial or nuclear DNA of the three primary Anopheles blood-meal hosts, humans, pigs and dogs, were incorporated into a multiplex, quantitative PCR which was optimized for sensitivity and specificity.ResultsAmplification of serially diluted DNA showed that the quantitative PCR detected as low as 10−5 ng/μl of host DNA. Application to field-collected, blood-fed Anopheles showed that the quantitative PCR identified the vertebrate hosts for 89% (335/375) of mosquitoes whereas only 55% (104/188) of blood-meal samples tested in a conventional PCR were identified. Of the 104 blood-fed Anopheles that were positive in both PCR methods, 16 (15.4%) were identified as mixed blood meals by the quantitative PCR whereas only 3 (2.9%) were mixed blood meals by the conventional PCR.ConclusionsThe multiplex quantitative PCR described here is sensitive at detecting low DNA concentration and mixed host DNA in samples and useful for blood-meal analysis of field mosquitoes, in particular mixed-host blood meals.

Forensic validation of a SNP and INDEL panel for individualisation of timber from bigleaf maple (Acer macrophyllum Pursch)

Illegal logging is one of the largest illicit trades in the world, with high profits and generally low risks of detection and prosecution. Timber identification presents problems for law enforcement as traditionally used forensic methods such as wood anatomy and dendrochronology are often unable to confidently match wood evidence to the remains of illegally felled trees. Here we have developed and validated a set of genetic markers for individualisation in bigleaf maple (Acer macrophyllum), a high value timber species often felled illegally in the USA. Using 128 single nucleotide polymorphisms and three insertion/deletion markers developed through massively parallel sequencing, 394 individuals were genotyped on the MassARRAY® iPLEX™ platform (Agena Bio-science™, San Diego, USA) to produce a population reference database for the species. We demonstrate that the resulting DNA assay is reliable, species specific, effective at low DNA concentrations (<1 ng/μL) and suitable for application to timber samples. The PID for the most common profile, calculated using an overall dataset level FST-correction factor, was 1.785 × 10−25 and PID-SIB across all individuals (treated as a single population) was 2.496 × 10-22. The further development of forensic identification assays for timber species has the potential to deliver robust tools for improved detection and prosecution of illegal logging crimes as well as for the verification of legality in reputable supply chains.

Microbiome applications for pathology: challenges of low microbial biomass samples during diagnostic testing.

The human microbiome can play key roles in disease, and diagnostic testing will soon have the ability to examine these roles in the context of clinical applications. Currently, most diagnostic testing in pathology applications focuses on a small number of disease‐causing microbes and dismisses the whole microbial community that causes or is modulated by disease. Microbiome modifications have already provided clinically relevant insights in gut and oral diseases, such as irritable bowel disease, but there are currently limitations when clinically examining microbiomes outside of these body sites. This is critical, as the majority of microbial samples used in pathology originate from body sites that contain low concentrations of microbial DNA, including skin, tissue, blood, and urine. These samples, also known as low microbial biomass samples, are difficult to examine without careful consideration and precautions to mitigate contamination and biases. Here, we present the limitations when analysing low microbial biomass samples using current protocols and techniques and highlight the advantages that microbiome testing can offer diagnostics in the future, if the proper precautions are implemented. Specifically, we discuss the sources of contamination and biases that may result in false assessments for these sample types. Finally, we provide recommendations to mitigate contamination and biases from low microbial biomass samples during diagnostic testing, which will be especially important to effectively diagnose and treat patients using microbiome analyses.

Application and Evaluation of the Loop-Mediated Isothermal Amplification assay for the Detection of Aflatoxigenic Fungi Contaminants of Rice Grains in Kenya

Aflatoxigenic fungi are most common filamentous fungi that synthesis aflatoxins and represent the major fungal pathogens to agricultural products. Aflatoxins remain a major threat to global food security, these molecules could be resisted into food during processing and in additional may remain within the food chain. Aflatoxins are carcinogenic, hepatotoxic, mutagenic, teratogenic, can inhibit numerous metabolic systems and immunosuppressive properties. Studies of aflatoxigenic strains can help to enhance strategies control and prevent aflatoxigenic fungi contamination and aflatoxins production in foodstuffs. In this study, isolation of Aspergillus species was based on morphological characteristics including the mycelium growth pattern, color, and properties of fruiting bodies of the fungi. The innovated technique loop-mediated isothermal amplification assay was applied to amplify Norsolorinic Acid gene. The loop-mediated isothermal amplification have been optimized by combination of the rapidity, simplicity and specificity to detect the target genomic DNA in the reactions. The amplification curves monitored by Loopamp realtime Turbidimeter were analyzed in order to distinguish aflatoxigenic and non-aflatoxigenic strains. Overall, the results showed that the loop-mediated isothermal amplification method was effective in detecting aflatoxigenic strains with high specificity of 71.5% and sensitivity under lower concentrations of DNA. In additional, it was faster than the conventional polymerase chain reaction. The loop-mediated isothermal amplification assay described in this study might be a promising tool for prediction potential threats by aflatoxigenic fungi and aflatoxins risk in food and commodities.

Исследование влияния материала микрофлюидного чипа на полимеразную цепную реакцию

The effect of a number of polymer materials of domestic and foreign manufacturers on the polymerase chain reaction has been verified. It was found that in the microchip from polymethylmethacrylate there was no adsorption of the components of the reaction mixture at room temperature, however, during the polymerase reaction in the microchip, the reaction efficiency decreases at low DNA concentrations.

EIS Biosensor for Detection of Low Concentration DNA Molecules

The results of the theoretical simulation of an electrolyte-insulator-semiconductor (EIS) biosensor made on a silicon nanowire for the detection of low concentration DNA molecules are presented. The equivalent electrical scheme for the EIS structure is constructed. The phenomenon of charge distribution in a depletion layer of semiconductor is taken into account in computations. The behavior and dependence of the total capacitance for the EIS sensor and its capacitive sensitivity on the DNA concentration in aqueous solution are investigated and analyzed. It is shown that the capacitance of the EIS sensor is very sensitive to the presence of the DNA molecules and the sensitivity has comparatively high value at the low concentration of the DNA molecules. The increase of the DNA concentration results in the decrease of the EIS total capacity. It is shown that one can register and determine the number of charged DNA molecules in aqueous solution by measuring the changes in the EIS capacity. The sensitivity increases with the growth of the gate voltage. It is shown that the threshold of sensitivity can be very low and the signal-to-noise ratio can reach high values for the EIS biosensor.

Analysis of Microsatellites (SSRs) in Processed Olives as a Means of Cultivar Traceability and Authentication.

Select cultivars of table olives have more desirable traits and a higher economic value. There are suspected issues with cultivar mislabeling and traceability in the supply chain. Here, we describe a method to identify cultivars by genotyping of processed olives. DNA was extracted from leaves and California-style olives of seven commonly packed cultivars. Processed olive fruits yielded relatively low DNA concentrations (0.04-0.86 μg/g), and extracts had more impurities compared with leaves. From 15 candidate SSRs, five markers showing the highest number of unique allele combinations and discriminatory power were selected. These SSRs were successfully amplified and analyzed in all cultivars of olives except one. When directly comparing any two cultivars, different allele combinations were typically present for at least four of the five SSRs. Microsatellite analysis shows potential as a simple yet robust diagnostic tool. The method can be expanded to include other cultivars, styles of table olives, and potentially other processed plant-based foods.

The screening of rare blood donors in a highly admixed population: A new approach for Holley and Diego genotyping and impact of genomic and self-reported ancestry.

The present study aimed to develop strategies for genotyping DO*HY (Dombrock system) and DI*A/DI*B (Diego system) alleles and to evaluate the impact of genomic and self-declared ancestry on rare donor screening in admixed populations. The antigens Hy and Dib demonstrate clinical importance. The lack of antisera for the serological evaluation of these antigens makes it necessary to develop molecular methods. In addition, considering that some rare red blood cell phenotypes present differences in frequency between ethnic groups, it is important to assess the applicability of self-declared ancestry in the search for rare donors in admixed populations. DO*HY and DI*A/DI*B genotyping based on real-time polymerase chain reaction (PCR) was standardised. A total of 457 blood donors clustered by self-defined skin colour/race categories were genotyped. Furthermore, individual genomic ancestry was used in the analyses. The assays developed are reproducible and provide satisfactory results even at low concentrations of DNA, which make them useful in situations where the DNA is scarce, such as dried blood spots on filter paper, or when screening for pooled samples. No significant difference was observed in the frequencies of the DI*A, DI*B and DO*HY, comparing the self-declared White (branco) donors with those who are Black (preto) and Brown (pardo). Real-time PCR, especially using pooled samples, is a promising strategy to screen rare blood donors. Although both self-reported race/colour and some blood group phenotypes are associated with ancestry, the results point to a greater complexity in the application of self-declared race/colour in the screening of rare donors in admixed populations.