A New Multiplex Assay of 17 Autosomal STRs and Amelogenin for Forensic Application

This paper describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) systems for 17 autosomal loci (D1S1656, D2S441, D3S1358, D3S3045, D6S477, D7S3048, D8S1132, D10S1435, D10S1248, D11S2368, D13S325, D14S608, D15S659, D17S1290, D18S535, D19S253 and D22-GATA198B05) and Amelogenin. Primers for the loci were designed and optimized so that all of the amplicons were distributed from 50 base pairs (bp) to less than 500 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. Strategies were developed to overcome challenges that encountered in creating the final assay. The limits of the multiplex were tested, resulting in the successful amplification of genomic DNA range from 0.25–4 ng with 30 PCR cycles. A total of 681 individuals from the Chinese Han population were studied and forensic genetic data were present. No significant deviations from Hardy–Weinberg equilibrium were observed. A total of 180 alleles were detected for the 17 autosomal STRs. The cumulative mean exclusion chance in duos (CMEC_D) was 0.999967, and cumulative mean exclusion chance in trios (CMEC_T) was 0.99999995. We conclude that the present 17plex autosomal STRs assay provides an additional powerful tool for forensic applications.     

5个X-STR基因座荧光复合扩增体系的建立及法医学应用

为了发掘更多多态性高的X染色体短串联重复(X-STR)基因座, 以解决法医学实践中的特殊案例, 建立了一组荧光复合扩增体系, 同时检测DXS6803、DXS981、DXS6809、DXS6789和DXS7132 5个X-STR基因座, 并用ABI PRISM 3100作毛细管电泳和GeneMapper ID 3.1软件进行基因分型, 结果清晰, 灵敏度高, 重复性好。最低检出限为0.25 ng, 10~20 ng模板DNA能得到最佳结果, 在实际检案中能得到满意结果。实验表明, 本体系能为用X-STR基因座解决特殊的亲权鉴定案提供快速鉴定技术, 是常染色体STR、Y-STR等鉴定方法的良好补充, 在法医学实践中有较好的实用价值。     

Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR

Bacillus cereus group bacteria share a significant degree of genetic similarity. Thus, to differentiate and identify the Bacillus cereus group efficiently, a multiplex PCR method using the gyrB and groEL genes as diagnostic markers is suggested for simultaneous detection. The assay yielded a 400 bp amplicon for the groEL gene from all the B. cereus group bacteria, and a 253 bp amplicon from B. anthracis, 475 bp amplicon from B. cereus, 299 bp amplicon from B. thuringiensis, and 604 bp amplicon from B. mycoides for the gyrB gene. No nonspecific amplicons were observed with the DNA from 29 other pathogenic bacteria. The specificity and sensitivity of the B. cereus group identification using this multiplex PCR assay were evaluated with different kinds of food samples. In conclusion, the proposed multiplex PCR is a reliable, simple, rapid, and efficient method for the simultaneous identification of B. cereus group bacteria from food samples in a single tube.     

Human MECP2 gene at Q28 arm of X chromosome as a suitable target for monitoring PCR inhibition in a nested, multiplexed HIV-1 DNA detection protocol

Human MECP2 gene located at q28 arm of X chromosome was identified as target for thermal co-amplification with HIV-1 proviral DNA of infected individuals. The selected MECP2 gene-specific primers functioned at a wide range of annealing temperature, extension time and exhibited no significant interaction with pathogen specific primers. A 466 bp PCR amplicon originating from human MECP2 gene was found to be diagnostic for inhibition-free PCR reaction when co-amplified with the HIV-1 target gene in a multiplexed, nested PCR reaction. The 5' end of the MECP2 primers were engineered to position an EcoRI restriction endonuclease site to facilitate rapid cloning in various DNA vector molecules at the corresponding EcoRI sites. Cell mass of Escherichia coli (XL1Blue) harboring the recombinant plasmid when added to pleural fluid of HIV-1 infected individuals co-infected with Mycobacterium tuberculosis, generated the diagnostic 466 bp MECP2 PCR amplicon as well as the 194 bp PCR amplicon of target gene from M. tuberculosis. The experiment underlined potential of the region spanning nucleotide position 4118099 to 4118552 of human MECP2 gene (NCBI accession number NT_011726.13) as a reliable target for multiplex PCR to accommodate a wide range of thermal cycling and multiplex reaction conditions. In both cases of this study, electrophoresis-based separation of the 466 bp MECP2 fragment and the 232 bp and 194 bp HIV-1 and M. tuberculosis fragments respectively was distinct and unambiguous. The potential of this human MECP2 gene available from human genome or recombinant plasmid as a potent target to monitor PCR inhibition for a range of different PCR reactions is discussed.     

STRBase: a short tandem repeat DNA database for the human identity testing community

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/++ +strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes.     

鸭圆环病毒与鸭Ⅰ型肝炎病毒二重 PCR 检测方法的建立

目的：建立一种同时检测鸭圆环病毒（DuCV）和鸭I型肝炎病毒（DHV）病原体的二重PCR技术。方法：根据DuCV和DHV的基因文库,分别设计了2对与DuCV和DHV某段基因序列互补的引物,用这2对引物对同一样品中DuCV和DHV模板进行二重PCR扩增。结果与结论：用建立的方法均同时得到了2条特异性的大小与实验设计相符（DuCV：245bp;DHV：569bp）的二重PCR扩增带,而且对其他禽病病原的PCR扩增结果均为阴性,能同时检出56Pg的DHVRNA模板和6Pg的DuCVDNA模板。     

X染色体STR基因座的法医学研究进展

对X染色体的结构特征、遗传特征等相关基础知识及其短串联重复序列(STR)基因座在法医学领域的研究历史、现状, 复合扩增的研究动态和应用等方面进行了综述。同时收集了国内外X染色体STR 基因座在法医DNA工作者实践中的研究和应用资料, 分析了X染色体STR基因座在法医学应用中的利弊, 以促进X染色体STR基因座在法医学实践中的应用。     

Fragment size difference between multiplex and singleplex PCR products and their practical implications

By simultaneously amplifying several loci in the same reaction, multiplex PCR has been used in gene mapping and DNA typing with polymorphic short tandem repeat loci. Previous studies have discussed in detail the various parameters and conditions that influence the quantity of individual products generated by multiplex PCR. In practice, when a primer pair fails to amplify in a multiplex PCR for some individuals, singleplex PCR is often employed as a supplement to amplify the primer pair. However, the reliability of this procedure is unknown. In this study, we used six primer pairs from ABI PRISM Linkage Mapping Set version 2 to perform multiplex and singleplex reactions. The fluorescence-labeled amplification products were separated and detected on ABI PRISM 310 Genetic Analyzer. We found that for the marker D1S468, multiplex and singleplex reactions for the majority of individuals yielded reactions of different sizes. Therefore, the potential size difference between multiplex and singleplex reactions needs to be investigated. This investigation is essential to employ multiplex PCR supplemented with singleplex PCR in gene mapping and DNA typing.     

Amplification of Y-chromosomal STRs from ancient skeletal material

The adaptation to ancient DNA analysis of a Y-chromosomal STR (short tandem repeat) multiplex comprising the four STR systems DYS19, DYS390, and DYS389I/II shows the suitability of Y-chromosomal STR typing on ancient human remains. A new primer site for the system, DYS389I/II, resulting in products shortened by 94 bp, was chosen to serve the special needs of amplification of ancient DNA. For the first time, it was possible to amplify STR loci of the Y chromosome from historical and prehistorical bones of up to 3000 years old. Received: 8 September 1998 / Accepted: 7 December 1998     

Extraction and purification of microbial DNA from petroleum-contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis

We investigated the use of multiplex polymerase chain reaction (FCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWWO), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWWO, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10* to 10* cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 μL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradarJve cells, at a lower detection limit of = > 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.     

Multiplex PCR to detect four different tomato-infecting pathogens

This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200–800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.     

A novel real-time quantitative PCR method using attached universal template probe

A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5′ end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real-time PCR set-up. In addition, this method could be used for simultaneous detection using a 6-carboxy-fluorescein-labeled UT probe for the target gene and a 5-hexachloro-fluorescein-labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT–PCR analysis is relatively flexible, and it could be as short as 56 bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT–PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT–PCR technique is efficient, reliable, inexpensive and less labor-intensive for quantitative PCR analysis.     

Short tandem repeat typing technologies used in human identity testing

Short tandem repeat (STR) typing methods are widely used today for human identity testing applications including forensic DNA analysis. Following multiplex PCR amplification, DNA samples containing the length-variant STR alleles are typically separated by capillary electrophoresis and genotyped by comparison to an allelic ladder supplied with a commercial kit. This article offers a brief perspective on the technologies and issues involved in STR typing.     

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.     

Comparison of proteases in DNA extraction via quantitative polymerase chain reaction

We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.     

Genetic analysis of the 10 ChrX STRs loci in Chinese Han nationality from Guangdong province

This study is to explore the polymorphic nature of X-Chromosome short tandem repeats (ChrX STRs) loci, and to determine its application in kinship tests for forensic cases. A new fluorescent multiplex PCR that simultaneously amplifies the 10 ChX STRs loci in the same PCR reaction had been set up. DXS7132, DXS981, DXS6801, DXS6809, DXS6789, DXS7424, DXS101, DXS7133, GATA165B12 and GATA31E08 were analyzed in a sample of 511 (399 males and 112 females) unrelated individuals from Guangdong Han nationality in China. One hundred and one alleles were observed in all the loci. Here, we investigated the allele frequencies and mutation rates of the ten loci, and then made the comparison of allele frequencies distribution among different populations. The results show the ten loci in the multiplex systems may provide high polymorphism information for kinship testing and relationship investigations, and it is necessary to gain allele frequency and mutation rate of different population for forensic application.     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

Background

Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.

Results

The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.

Conclusions

A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.     

Highly accurate SNP genotyping from historical and low‐quality samples

Historical and other poor‐quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error‐prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of the DNA into small fragments. Single nucleotide polymorphisms offer potential advantages for assaying nuclear variation in historical and poor‐quality samples, because the amplified fragments can be very small, varying little or not at all in size between alleles, and can be amplified efficiently by polymerase chain reaction (PCR). We present a method for highly multiplexed PCR of SNP loci, followed by dual‐fluorescence genotyping that is very effective for genotyping poor‐quality samples, and can potentially use very little template DNA, regardless of the number of loci to be genotyped. We genotyped 19 SNP loci from DNA extracted from modern and historical bowhead whale tissue, bone and baleen samples. The PCR failure rate was < 1.5%, and the genotyping error rate was 0.1% when DNA samples contained > 10 copies/µL of a 51‐bp nuclear sequence. Among samples with ≤ 10 copies/µL DNA, samples could still be genotyped confidently with appropriate levels of replication from independent multiplex PCRs.