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1.
Advances in molecular techniques for the detection and quantification of genetically modified organisms 总被引:2,自引:1,他引:2
Elenis DS Kalogianni DP Glynou K Ioannou PC Christopoulos TK 《Analytical and bioanalytical chemistry》2008,392(3):347-354
Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been
altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries
require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical
methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction
(PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed
by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification
and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE)
systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization
assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening
as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including
surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent
dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower
cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have
been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput
multiplex competitive quantitative PCR have been reported. 相似文献
2.
In this work, it was demonstrated that on-line pre-concentration and separation of DNA fragments within bared silica column by dynamic coating capillary electrophoresis and UV detection. The DNA fragments were pre-concentrated with long electrokinetic injecting time (99 s), peak height increased dramatically as a function of injection time, especially for shorter length DNA. The concentration sensitivity of DNA fragments can be improved from 20- to 100-fold relative to a normal injection (5 s). The electro-osmotic flow (EOF) and DNA-wall interactions within the capillary were eliminated effectively by dynamic coating method. Employing 0.5% poly(ethylene oxide) (PEO) in Tris-phosphate-EDTA (TBE) buffer as sieving matrix, DNA fragments, ranging from 11 to 657 bp, were separated within 20 min. The linear coefficient of linear relation between the migration and DNA length is 0.999. The DNA fragments amplified from transgenic oilseed rape by polymerase chain reaction (PCR) were separated and detected by this method, demonstrating the potential use of this method for effective DNA analysis and detection of genetically modified organisms (GMO). 相似文献
3.
A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs). The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms. The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model. Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA. The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure. Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates. The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm. The glass slide was etched to a depth of 30 microm for both the injection and separation channel. The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication. The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s. As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material. This corresponds to only 20 genome copies of genetically modified soybean. 相似文献
4.
Marko Stojkovic Narasimha R. Uda Peter Brodmann Milica Popovic Peter C. Hauser 《Journal of separation science》2012,35(24):3509-3513
The use of CE with contactless conductivity detection for the determination of PCR products is demonstrated for the first time. The separation of specific length PCR products according to their size could be achieved using 5% PVP as a sieving medium in a separation buffer consisting of 20 mM Tris and 20 mM 2‐(cyclohexylamino)ethansulphonic acid (pH 8.5). A fused silica capillary of 60 cm length and 50 μm id and an applied separation voltage of –15 kV were employed and separations could be completed within 20–50 min. PCR amplified DNA fragments of different sizes obtained from different bacterial plasmid templates as well as a fragment from genomic DNA of genetically modified soybeans could be successfully identified. 相似文献
5.
New trends in bioanalytical tools for the detection of genetically modified organisms: an update 总被引:2,自引:1,他引:2
Michelini E Simoni P Cevenini L Mezzanotte L Roda A 《Analytical and bioanalytical chemistry》2008,392(3):355-367
Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially
in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources,
the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary
principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations
necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow
the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil
some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently
been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including
its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability
to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection
are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance
sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques
has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released
onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous
GMOs, including unknown GMOs. 相似文献
6.
Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize 总被引:2,自引:0,他引:2
Roda A Mirasoli M Guardigli M Michelini E Simoni P Magliulo M 《Analytical and bioanalytical chemistry》2006,384(6):1269-1275
Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to
control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein
from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on
96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by
employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the
enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy
and precision and exhibited limits of detection of a few pg mL−1 Cry1Ab (3 or 5 pg mL−1, depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection
of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput
analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption
of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay
is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically
modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified
material according to the actual EU regulation. 相似文献
7.
Valdemar I. Esteves Susana S.F. Lima Diana L.D. Lima Armando C. Duarte 《Analytica chimica acta》2004,513(1):163-167
The simultaneous separation and determination of organic acids in several samples of white and red Port wines was performed by capillary zone electrophoresis using indirect UV detection with 2,6-pyridinedicarboxylic acid as a background electrolyte buffer. Operational parameters like migration time, temperature, voltage and capillary length were optimized. Sixteen samples of red wine and four samples of white wine were used to analyze for tartaric, malic, lactic, succinic and acetic acids using glyoxylic acid as the internal standard. The method is rapid, sensitive and quantitative, and time-consuming sample preparation, such as solid-phase extraction or liquid-liquid extraction procedure, is not required. 相似文献
8.
转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究 总被引:1,自引:0,他引:1
采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1%). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求. 相似文献
9.
Accreditation of GMO detection laboratories: Improving the reliability of GMO detection 总被引:4,自引:0,他引:4
Jana Žel Katarina Cankar Maja Ravnikar Marjana Camloh Kristina Gruden 《Accreditation and quality assurance》2006,10(10):531-536
Reliable and efficient methods for detecting genetically modified organisms (GMOs) are essential for establishing an effective system for traceability all along the supply chain from seed producers to final consumers. The latter is especially meaningful in European Union and other countries where strict legislations on GMOs were set up. Performance of the methods used in laboratories around the world should be uniform, in order to obtain reliable and comparable results. Accreditation is a suitable system for harmonising procedures in each testing laboratory. In this paper, key elements for the accreditation of molecular biology methods for GMO detection according to ISO/IEC 17025 are described. The procedures described are also valuable for the accreditation of molecular methods for all laboratory diagnostics where qualitative and quantitative characterisation of nucleic acids is needed. 相似文献
10.
Jerman S Podgornik A Cankar K Cadet N Skrt M Zel J Raspor P 《Journal of chromatography. A》2005,1065(1):107-113
The availability of sufficient quantities of DNA of adequate quality is crucial in polymerase chain reaction (PCR)-based methods for genetically modified food detection. In this work, the suitability of anion-exchange CIM (Convective Interaction Media; BIA Separations, Ljubljana, Slovenia) monolithic columns for isolation of DNA from food was studied. Maize and its derivates corn meal and thermally pretreated corn meal were chosen as model food. Two commercially available CIM disk columns were tested: DEAE (diethylaminoethyl) and QA (quaternary amine). Preliminary separations were performed with standard solution of salmon DNA at different pH values and different NaCl concentrations in mobile phase. DEAE groups and pH 8 were chosen for further isolations of DNA from a complex matrix-food extract. The quality and quantity of isolated DNA were tested on agarose gel electrophoresis, with UV-scanning spectrophotometry, and by amplification with real-time PCR. DNA isolated in this way was of suitable quality for further PCR analyses. The described method is also applicable for DNA isolation from processed foods with decreased DNA content. Furthermore, it is more effective and less time-consuming in comparison with the existing proposed methods for isolation of DNA from plant-derived foods. 相似文献
11.
N-Methylimidazolium modified magnetic particles (MIm-MPs) were prepared and applied in the solid phase extraction of genomic deoxyribonucleic acid (DNA) from genetically modified soybeans. The adsorption of MIm-MPs for DNA mainly resulted from the strong electrostatic interaction between the positively charged MPs and the negatively charged DNA. The elution of DNA from MPs–DNA conjugates using phosphate buffer resulted from the stronger electrostatic interaction of phosphate ions with MPs than DNA. In the extraction procedure, no harmful reagents (e.g. phenol, chloroform and isopropanol, etc.) used, high yield (10.4 μg DNA per 30 mg sample) and high quality (A260/A280 = 1.82) of DNA can be realized. The as-prepared DNA was used as template for duplex-polymerase chain reaction (PCR) and the PCR products were analyzed by a sieving capillary electrophoresis method. Quick and high quality extraction of DNA template, and fast and high resolution detection of duplex PCR products can be realized using the developed method. No toxic reagents are used throughout the method. 相似文献
12.
Dinelli G Bonetti A Marotti I Minelli M Navarrete-Casas M Segura-Carretero A Fernández-Gutiérrez A 《Electrophoresis》2006,27(20):4029-4038
The aim of the present study was to develop a quantitative-competitive PCR (QC-PCR) method to detect DNA from transgenic herbicide-resistant (roundup ready, RR) soybean and maize. Since no QC-PCR system for the quantification of RR maize had been published at the time of writing, a specific competitor DNA for transgenic event was developed. For the QC-PCR of RR-soybean, a commercially available competitor was employed. These internal standards were calibrated by coamplifying with mixtures containing RR-soybean and maize DNAs. The calibrated QC-PCR systems were applied to certified RR-soybean and maize flour mixtures in order to demonstrate their suitability not only for the quantification of the glyphosate resistance traits in DNA matrices, but also in practically relevant samples. In addition, a special focus of the present work was to compare the detection of QC-PCR products by slab gel and CGE with UV detection. CGE permitted the precise detection of transgenic events also below the equivalence points; while in slab gel electrophoresis, due to the low sensitivity the quantification of genetically modified DNA was allowed only at the equivalence point. 相似文献
13.
This paper demonstrates the diagnosis of -thalassemia by capillary electrophoresis in conjunction with laser-induced fluorescence using poly(ethylene oxide) (PEO) solutions in the presence of electroosmotic flow (EOF). During the electrophoretic separation, PEO solution entered a capillary from the anodic vial by EOF. The separation of a mixture of the polymerase chain reaction (PCR) products (330 and 334 base pairs) from a healthy person and a -thalassemia patient was accomplished within 15 min at 15 kV using 1.5% PEO containing 2 M urea at 30 °C. The electropherogram patterns instead of migration times were used to diagnose -thalassemia, with an accuracy of 100% for the analyses of 11 blood samples from suspected patients. After injecting a large volume of the mixture to the capillary filled with 800 mM Tris-borate buffer (pH 10.0), the DNA fragments stacked due to increases in viscosity and sieving when migrating into 1.5% PEO solution. As a result of improved sensitivity, only 15 PCR cycles were required when using 500 ng of DNA templates. The results shown in this study indicate the potential of this simple, rapid, and cost-effective method for the diagnosis of -thalassemia.Abbreviations CE
Capillary electrophoresis - EOF
Electroosmotic flow - EtBr
Ethidium bromide - LIF
Laser-induced fluorescence - PCR
Polymerase chain reaction - PEO
Poly(ethylene oxide) - TRIS
Tris(hydroxymethyl)aminomethane - TB
TRIS-borate 相似文献
14.
Cheng Xiao Quyi Xu Huan Li Xiaolin Zhu Zhonghao Yu Jian Zhao Yue Li Hong Liu He Shi Chao Liu 《Electrophoresis》2021,42(11):1270-1278
In recent years, the DNA detection of drowning-related diatoms, cyanobacteria, and aeromonas has gradually attracted interest from forensic scientists. In this study, we described the validation and application of a novel multiplex PCR system. This system integrated 12 fluorescently labelled primers designed to amplify specific genes of diatoms, cyanobacteria, and aeromonas. The specificity studies demonstrated that this multiplex PCR system could detect nine species of diatom, seven species of cyanobacteria, and five species of aeromonas, all of which were drowning-related and widely distributed in various water circumstance of southern China. The sensitivity studies indicated that the limit concentration of template DNA was 0.0125 ng. Besides, this multiplex PCR system had good performance in sizing precision and stability, but it is not suitable for degraded DNA samples. The application into forensic casework showed that all the tissue samples from ten nondrowning cases showed negative results, and the positive rates of lung, liver, kidney, and water samples from 30 drowning bodies were 100, 86.7, 90, and 100%, respectively. Combined with results of diatom tests of MD-VF-Auto SEM method, this multiplex PCR system could help rule out nondrowning bodies and provide extra evidences to support drowning diagnosis, especially for those cases with few diatoms observed. It is expected that this multiplex PCR system has great potential for forensic drowning diagnosis. 相似文献
15.
This paper proposes a novel strategy to enhance selectivity and sensitivity in CE, using supported liquid membrane (SLM) and off-line SPE simultaneously. The determination of ochratoxin A (OA) in wine has been used to demonstrate the potential of this methodology. In the SLM step, the donor phase (either a 20 mL volume of a standard solution at pH 1 or a wine sample at pH 8) was placed in a vial, where a micromembrane extraction unit accommodating the acceptor phase (1 mL water, pH 11) in its lumen was immersed. The SLM was constructed by impregnating a porous Fluoropore Teflon (PTFE) membrane with a water-immiscible organic solvent (octanol). In the off-line SPE step, the nonpolar sorbent (C-18, 4 mg) selectively retained the target ochratoxin, enabling small volumes of acceptor phase (1 mL) to be introduced. The captured analytes were eluted in a small volume of methanol (0.1 mL). This procedure resulted in sample cleanup and concentration enhancement. The method was evaluated for accuracy and precision, and its RSD found to be 5%. The LODs for OA in the standard solutions and wine samples were 0.5 and 30 microg/L, respectively. The results obtained demonstrate that SLM combined with off-line is a good alternative to the use of immunoaffinity columns prior to CE analysis. 相似文献
16.
Hsiao-Wei Liao I-Lin Tsai Guan-Yuan Chen Chun-Ting Kuo Ming-Feng Wei Tzung-Jeng Hwang Wei J. Chen Li-Jiuan Shen Ching-Hua Kuo 《Analytica chimica acta》2013
CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes. 相似文献
17.
Aleksandra Karczmarczyk Ciril Reiner-Rozman Simone Hageneder Monika Dubiak-Szepietowska Jakub Dostálek Karl-Heinz Feller 《Analytica chimica acta》2016
Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL−1 and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL−1 by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied. 相似文献
18.
H. M. Liebich G. Xu C. Di Stefano R. Lehmann H. U. Häring P. Lu Y. Zhang 《Chromatographia》1997,45(1):396-401
Summary Modified nucleosides excreted in urine have been studied as potential diagnostic markers for cancer and AIDS, and as indicators
for the whole-body turnover of RNA. Until now, reversed-phase (RP) HPLC and, to some extent, immunoassays are the preferred
analytical methods for urinary nucleosides. A new capillary electrophoretic method for the analysis of normal and modified
nucleosides in urine has been developed and optimized in our laboratory. The separation of nucleosides extracted from normal
human urine on phenyl boronic acid affinity chromatography columns was performed in uncoated 565 mm (500 mm to detection window)
× 50 μm i.d. capillary tubing using a 300 mM SDS—25 mM borate—50 mM phosphate buffer (pH 6.7), a 45-s load, a voltage of 7.5
kV (41 μA) and UV detection at 260 and 210 nm. The average recovery of the nucleosides was 91 %. The calibration curves were
linear over all physiological and pathophysiological concentration ranges and the limits of detection were at micromolar levels.
Reproducibility of migration times were better than 1 % (coefficient of variation,CV), and the reproducibilities of the determined concentrations were better than 5 % for standards and 6–15 % for extracted
urine. The developed method was used to quantify 15 normal and modified nucleosides in 25 normal urines to establish reference
ranges. The analysis time was less than 45 min.
Dedicated to Professor E. Bayer on the occasion of his 70th birthday.
Presented at the 21st ISC held in Stuttgart, Germany, 15th–20th September, 1996. 相似文献
19.
BRAF mutations are found in many human tumors, namely melanomas ( approximately 70%) and colon carcinomas ( approximately 15%). This paper presents a method for identification of exon 15 BRAF mutations by denaturant capillary electrophoresis (CE), an analysis method that is sensitive, cost-effective (involving only polymerase chain reaction (PCR) and electrophoresis) and capable of high-throughput screening. In total, we found 21 (70%) out of 30 melanoma cell lines with BRAF mutations in exon 15: two of which were the p.Val600Asp (c.1799-800TG>AT) mutation, one cell line contained the p.Val600Arg (c.1798-99GT>AG) mutation, and 18 cell lines contained the p.Val600Glu (c.1799T>A) mutation. Of the nine cell lines that did not contain a BRAF mutation, five contained an NRAS mutation at exon 2, and no mutations were detected in NRAS exon 1. There was no overlap of NRAS and BRAF mutations in the same cell line. In addition, we looked at 221 colon biopsy samples and identified one further BRAF mutation, the p.Asp594Gly (c.1781A>G) mutation, in seven samples. The p.Val600Glu mutation was identified in 11 of the colon biopsy samples. Using the four mutations of BRAF exon 15, we then constructed a denaturing CE standard capable of distinguishing between each of the mutations; therefore, sequencing does not need to be performed to confirm the mutation. In conclusion, this sensitive, cost-effective mutation assay for BRAF (and RAS) will provide the opportunity to detect and determine mutations without the need to purify samples for sequencing. Future large-scale studies will provide the clinical usefulness of such mutations. 相似文献
20.
Yiling Qu RuiYang Tao Huan Yu Qi Yang Ziwei Wang Rui Tan Xiaochun Zhang Ruocheng Xia Lei Xiong Shihan Xi Jun Wu Yuzhen Gao Suhua Zhang Chengtao Li 《Electrophoresis》2021,42(14-15):1419-1430
This paper describes the development and validation of a novel 31-locus, six-dye STR multiplex system, which is designed to meet the needs of the rapidly growing Chinese forensic database. This new assay combines 20 extended-CODIS core loci (D3S1358, D5S818, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D8S1179, D18S51, D16S539, D13S317, FGA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045), nine highly polymorphic loci in Chinese Han population (D3S3045, D6S1043, D6S477, D8S1132, D10S1435, D15S659, D19S253, Penta D, and Penta E), and two gender determining markers, amelogenin and Y-Indel, which could amplify DNA from extracts, as well as direct amplification from substrates. To demonstrate the suitability for forensic applications, this system was validated by precision and accuracy evaluation, concordance tests, case sample tests, sensitivity, species specificity, stability, stutter calculation, and DNA mixtures, according to the guidelines described by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and regulations published by the China Ministry of Public Security. The validation results indicate the robustness and reliability of this new system, and it could be a potentially helpful tool for human identification and paternity testing in the Chinese population, as well as facilitating global forensic DNA data sharing. 相似文献