微滴式数字PCR技术用于生物样品种属鉴定和绝对定量

目的 运用微滴式数字PCR技术进行生物样品的种属鉴定和绝对定量.方法 选择人mtDNA两个编码基因ND4和16S rRNA,设计特异性引物与探针,用人来源及常见动物样本验证种属特异性,再用重组质粒和2组共16份人来源生物检材,倍比稀释.使用微滴式数字PCR技术进行种属检验和绝对定量,验证其灵敏度和稳定性.结果 人重组质粒FAM （ND4）可进行人来源样品的检测,其检测结果与各级稀释梯度基本吻合,微滴式数字PCR技术可以检测出反应体系中低至单拷贝的DNA.结论 微滴式数字PCR技术可以进行生物样品的种属鉴定和绝对定量,并具有很高的灵敏度和特异性,可应用于日常法医物证检验.     

Analysis of ancient human DNA and primer contamination: one step backward one step forward

The analysis of DNA from archaeological human remains is plagued by a unique set of methodological problems concerning contamination with modern exogenous DNA. Through an original approach, we propose complementary methods to identify all potential sources of contamination and complete guidelines for the validation of ancient human sequences. The study presented was conducted on non-European human samples (Polynesian and Amerindian) which were collected with all precautions during excavation. This permitted us to distinguish without ambiguity authentic and contaminant sequences. The samples' origins and histories were perfectly known, allowing us to trace all potential contamination sources and to determine the efficiency of precautions followed during all steps of the study. The data obtained confirm that precautions taken during sampling effectively prevent contamination. However, we demonstrate that human contamination can also be introduced during genetic analyses even if all precautions are strictly followed. Indeed, numerous human contaminations were detected in template-PCR products and negative controls, resulting in a striking diversity of contaminant mitochondrial DNA sequences. We argue that this contamination partly derives from the primers. To our knowledge, no previous experiment has been performed to investigate primers as a possible source of human contamination despite the fact that this specific type of contamination poses a real problem in terms of validating ancient human DNA studies. Finally, we confirm that the detection of contaminants in negative controls is clearly related to the number of PCR cycles used. This study enhances our understanding of contamination processes and confirms that, in reality, an absolutely contamination-free situation cannot be obtained. As a consequence, we propose improvements to the guidelines usually followed in the field in order to take the highly probable contamination of PCR reagents, including primers, into account.     

线粒体DNA短片段复合扩增系统鉴别种属的研究

目的建立线粒体DNA短片段复合扩增体系用于种属鉴定的方法。方法提取人、牛、猪、羊、鸡的DNA,用所选的3对引物复合扩增细胞色素b基因（cyt b）片段、16srRNA基因片段和ND4基因片段,扩增产物经琼脂糖凝胶电泳检测。结果人DNA扩增产物在358bp、157bp和110bp处各出现一条带;动物DNA扩增产物均只有358bp一条带。结论线粒体DNA短片段复合扩增鉴别种属的方法可区分人源性生物检材和其它动物样本,可应用于法庭科学实践。     

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

Are these food products fraudulent? Rapid and novel triplex-direct PCR assay for meat identification

Unintentional contamination and fraudulent labeling of food, especially in halal-certified products, breach both religious and international laws. DNA-based methods are widely employed to identify trace amount of contaminants for law enforcement. Direct PCR has proved successful in the DNA analysis from degraded samples and PCR-inhibited samples, but it has never been applied to meat identification from food products. In this study, we aimed to develop a multiplex direct PCR assay for simultaneous identification of three commonly consumed meats without the need to extract DNA. Species-specific primers were designed from the mitochondrial DNA using the alignment of sequences available on GenBank. The assay was validated for its specificity, sensitivity, and usefulness in market sample analysis. The results showed that a highly specific and sensitive multiplex direct PCR assay was developed and provided the expected PCR fragment of approximately 100, 119, and 133 bp for pork (Sus scrofa), mutton (Ovis aries), and chicken (Gallus gallus), respectively. Thirty-nine market samples were tested and a small number of fraudulent labeling was detected. In conclusion, we developed a rapid and inexpensive test for three meat species. This cost- and time-saving assay could be easily adopted in the world food hubs, which are mostly third-world countries.     

Development of a real-time PCR assay to control the illegal trade of meat from protected capercaillie species (Tetrao urogallus)

A rapid and highly species-specific real-time polymerase chain reaction (PCR) assay has been developed for the detection of capercaillie DNA (Tetrao urogallus) in meat and meat mixtures. The method combines the use of capercaillie-specific primers, that amplify a 142bp fragment of the mitochondrial 12S rRNA gene, and a positive control primer pair that amplifies a 141bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. SYBR(?) Green dye or TaqMan(?) fluorogenic probes were used to monitor the amplification of the target genes. Results obtained with the use of TaqMan(?) probes as detection platform increased the specificity of the real-time PCR assay in comparison with the results obtained using SYBR(?) Green. The proposed real-time PCR assay represents a rapid and straightforward method for the accurate identification of capercaillie that could be used by law enforcement agencies as a tool for the control of poaching and illegal trade of meat from this protected species.     

从指甲中提取DNA进行PCR-STR初步研究

本文成功的提取了指甲中的细胞核DNA,并进行多个PCR-STR位点扩增,具有实际应用价值.     

Identification of mammal species using species-specific DNA pyrosequencing

In forensic casework it is highly relevant to be able to deduce the species origin of an unknown biological sample. For such a purpose we have designed and developed an assay for species identification based on DNA sequencing of two short mitochondrial DNA amplicons. In short, partial 12S rRNA and partial 16S rRNA fragments (approximately 100bp) are amplified by PCR followed by direct sequencing using pyrosequencing technique. Due to properties of the chosen targets, the same PCR conditions and primers were used irrespective of the true species of an unknown sample. A total of 28 different mammals present in the European fauna were sequenced both for the partial 12S rRNA and the partial 16S rRNA sequences for accuracy verification. Together the two sequences showed to have a high divergence factor, discriminating almost all mammals. Furthermore, the human reference nucleotide sequences were always at least nine nucleotides different compared to the other sequenced species both at the partial 12S rRNA and the partial 16S rRNA sequences.     

Simple and Sensitive Method for Identification of Human DNA by Allele-Specific Polymerase Chain Reaction of FOXP2

Abstract:  The forkhead box P2 ( FOXP2 ) gene is specifically involved in speech and language development in humans. The sequence is well conserved among many vertebrate species but has accumulated amino acid changes in the human lineage. The aim of this study was to develop a simple method to discriminate between human and nonhuman vertebrate DNA in forensic specimens by amplification of a human-specific genomic region. In the present study, we designed an allele-specific polymerase chain reaction (PCR) using primers to amplify smaller than 70-bp regions of FOXP2 to identify DNA as being of human or nonhuman, including ape, origin. PCR amplification was also successfully performed using fluorescence-labeled primers, and this method allows a single PCR reaction with a genomic DNA sample as small as 0.01 ng. This system also identified the presence of human DNA in two blood stains stored for 20 and 38 years. The results suggested the potential usefulness of FOXP2 as an identifier of human DNA in forensic samples.     

Discrimination between human and animal DNA: application of a duplex polymerase chain reaction to forensic identification

Identification of a report's species is one of the basic analyses in forensic laboratories. The authors report the case of 6 bone fragments recovered in a wooded area, which were not attributable to 1 animal species on the basis of morphologic examination. The aim of this study was to develop a duplex polymerase chain reaction (PCR) to discriminate human and animal origin of bone fragments. The method is based on the PCR amplification of cytochrome b and a 16S ribosomal mitochondrial DNA fragment, which has never been tested up to now. Our protocol combines a single-round PCR with direct visualization of amplicons in agarose gel, without sequencing analysis of the PCR products. The presence of a single band (359 bp) indicates a nonhuman origin of the sample, whereas 2 bands (157 and 359 bp) indicate a human biologic sample.This method revealed to be useful for forensic purposes because the 16S ribosomal mitochondrial DNA is a small human-specific fragment that is easily amplifiable even with degraded DNA from biologic materials such as old bones.     

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.     

Quantification of human mitochondrial DNA using synthesized DNA standards

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.     

用ESI-TOF-MS分型技术检测线粒体DNA高变区碱基组成的多态性

目的 用ESI-TOF-MS分型技术检测线粒体DNA的D环高变区,通过碱基组成分析其多态性.方法 在PLEX-ID技术平台上,分别对mtDNA高变区1(HVⅠ,15924-16428nt)和mtDNA高变区Ⅱ(HVⅡ,31-576 nt)进行碱基组成分析,考察mtDNA在华东汉族人群的多态性,并将该技术应用于一例特殊的亲子鉴定案件.结果 用ESI-TOF-MS分型技术检测线粒体DNA,在高变区Ⅰ的8个区段检见碱基组成的多态性,在mtDNA高变区Ⅱ的10个区段检见多态性.在所应用的亲子鉴定案例中,线粒体DNA标记成了常染色体STR基因座的重要补充,经高变区Ⅰ和高变区Ⅱ的碱基组成检测,最后排除了非母.结论 ESI-TOF-MS检测mtDNA的技术具有良好的应用前景,在一些特殊的案件中,该法可为最终获得可靠鉴定结论提供技术支撑.     

PCR-based identification of postmortem microbial contaminants--a preliminary study.

Investigation of postmortem blood can reveal the presence of significant ethanol levels. However, in some instances it cannot easily be determined if the source of ethanol is from ingestion or from postmortem endogenous fermentation by contaminating microbes. Described here is a robust polymerase chain reaction (PCR)-based method for detecting the presence of common ethanol producing microbial contaminants in human blood. A set of DNA primers were designed for use in PCR to amplify and detect the genomic DNA from humans and three test microorganisms Escherichia coli, Proteus vulgaris, and Candida albicans. A rapid and reproducible protocol was developed for isolating genomic DNA from mixed human blood-microorganism samples that yields a suitable template for PCR. The organism-specific primer pairs can detect the presence of the target microorganisms in human blood at concentrations as low as 10 colony forming units/mL. The PCR products readily can be detected after agarose gel electrophoresis. This method provides an additional means of rapidly identifying microbial contaminants in postmortem blood samples.     

High-performance LAMP-based method for human sex identification using Y chromosome-specific genetic markers

Finding fast and cheap strategies for DNA typing and human sample identification is of interest in forensic science. We report a new versatile alternative for molecular sex determination using Y-specific targets (TSPY, TTTY, alphoid regions, and Y-Amelogenin). This system uses an isothermal loop-mediated DNA amplification (LAMP) with a set of 6 primers for each target, designed to improve sensibility and specificity, and reducing detection time to only 45 min. Furthermore, detecting the different targets on the Y chromosome either individually or in combination revealed accurate results. Assay sensitivity was determined with a mixture of human female and male DNA at different concentrations to mimic forensic samples. Single primer sets showed high sensitivity at DNA concentrations ranging from 58.6 to 3.7 pg/µL. When a combined primers set was used, sensitivity yielded a detection as low as 0.1 pg/µL of male DNA, making it 10 times more sensitive than qPCR-DNA quantification kits. Finally, high specificity was observed when tested against 6 domestic species.     

A method for quantifying mixed goat cashmere and sheep wool

Cashmere is a high-priced commodity in the world market. For financial gains, various interested parties often adulterate cashmere with cheap sheep wool. Here, we describe a method that can quickly extract mitochondrial DNA from natural or processed animal hair. We further designed two sets of TaqMan polymerase chain reaction (PCR) primers and probes that can react specifically to goat and sheep mitochondrial 12S ribosomal RNA (rRNA) genes. Using TaqMan PCR, we can not only distinguish between cashmere and wool but also quantify their contents in a cashmere/wool mixture. The method can be applied directly to examine the quality of cashmere products in the world markets.     

Multiplex PCR and minisequencing of SNPs--a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.     

Quantification of mitochondrial DNA in human blood cells using an automated detection system

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.