牛布氏杆菌和副结核分枝杆菌双重PCR检测方法的建立

为快速检测牛布氏杆菌和副结核分枝杆菌,根据GenBank中的布氏杆菌OMP31基因序列(JF918757)及副结核分枝杆菌ISMav2基因序列(AF286339)设计、合成2对特异性引物,通过对PCR条件的优化,建立了快速鉴别检测牛布氏杆菌和副结核分枝杆菌的双重PCR方法。经对建立的方法进行特异性和敏感性试验,结果表明,分别扩增出602、246bp的特异性牛布氏杆菌和副结核分枝杆菌DNA目的条带,作为对照的双芽巴贝斯虫、大肠杆菌、沙门氏菌、弓形虫、链球菌、牛放线菌的DNA及其混合物均未扩增出任何条带。牛布氏杆菌与副结核分枝杆菌的最低检测限分别为1.92和2.51pg;牛肉样品中人工污染的牛布氏杆菌和副结核分枝杆菌的检测敏感性分别为6×104和7×104 CFU/mL。该双病原检测体系的成功建立为牛布氏杆菌病及副结核病的检测、鉴定和流行病学调查提供了有力的技术支持。     

鸭源大肠杆菌ZYD_2与多杀性巴氏杆菌YB_2原生质体融合菌株的鉴定分析

以新霉素标记的ZYD2(NEOs)和YB2(NEOr)作为亲本菌株,培育具有双亲本菌株免疫原性的融合菌株。在溶菌酶作用下,制备两亲本菌株原生质体,聚乙二醇4000诱导原生质体发生融合,利用NEO标记及亲本菌株培养条件不同筛选阳性融合子。ZYD2和YB2可分别得到95.32%和95.25%的原生质体制备率以及32.19%和33.52%的再生率。在含新霉素的麦康凯平板上传代培养获得5株稳定遗传的融合菌株。5株融合菌株可同时凝集双亲本菌株的鸭抗阳性血清。染色镜检观察融合菌株为革兰氏阴性中等大小杆菌。利用双重PCR检测,2株检测到双亲本菌株部分外膜蛋白A(OmpA)基因和部分外膜蛋白H(OmpH)基因,2株只检测到部分OmpA基因,另1株检测不到任何条带。测序结果表明检测到的部分基因片段与亲本菌株的相关基因片段同源性为100%。微量凝集反应显示,外周血可以检测到双亲本菌株抗体,抗体效价在二次免疫后可以维持较稳定水平。结果表明获得5株具有双亲本菌株免疫原性且可稳定遗传的融合菌株。     

海南汉族人群19个STR基因座遗传多态性及其应用

目的建立海南地区汉族人群19个常染色体STR基因座的遗传多态性数据资料,并探讨此19-STR基因座系统在亲子鉴定中的应用。方法对海南汉族462例无血缘关系个体,采用Goldeneye~(TM) 20A系统复合扩增并检测,得到19个STR基因座的遗传数据信息;在283例亲子鉴定案例中,评价19-STR基因座系统的应用。结果 19个STR基因座的基因频率分布均符合Hardy-Weinberg平衡(P0.05),杂合度在0.603~0.914之间,累积个体识别率大于0.999 999 999 999 999,累积三联体非父排除率为0.999 999 994。283例亲子鉴定中,三联体170例,二联体113例;认定案例247例(87.3%),排除案例36例(12.7%);发生等位基因突变案例14例(4.9%),均为一步突变。结论 19个STR基因座中的14个基因座具有高度遗传多态性,19-STR基因座复合扩增分型系统具有较高的非父排除效能,可满足海南地区亲子鉴定的需要,同时应注意亲子鉴定中的基因突变现象。     

Performance characteristics of commercial Y-STR multiplex systems

In this work, a number of performance checks were carried out to evaluate the efficacy of commercial Y-short tandem repeats (Y-STR) kits for casework applications. The study evaluated the sensitivity, specificity and stability of the Y-STR markers used and the ability to obtain a male profile from postcoital samples taken at various time points after intercourse. All systems performed well with 1-3 ng of male DNA as recommended by the manufacturers. All systems gave full profiles at 100 pg of input DNA, which is within the realm of low copy number DNA analysis. Moreover all, except Y-Plex12, gave full profiles with 30-50 pg of male DNA. No increased performance was obtained with any of the systems by increasing the cycle number beyond that recommended by the various manufacturers. When up to 1 microg of female DNA was used (in the absence of male DNA) no female DNA cross reactivity was observed with the Y-Plex 12 and Y-Filer systems. PowerPlex Y produced female DNA derived products near the DYS438 and within the DYS392 loci at a rare allele position with high input DNA levels (300 ng and 1 microg, respectively). Male/female DNA admixture experiments indicated the particularly high specificity of the Y-Filer and PowerPlex Y systems under conditions of several thousand fold female DNA excess. All systems were able to detect the minor alleles in male/male DNA admixtures at a 1:5 dilution with the PowerPlex Y and Y-Filer being able to detect some minor alleles at 1:20. Species testing indicated some limited, minor cross reactivity of the commercial systems with some domestic male mammals although it is easily recognizable and would not pose any problems in casework analysis. As expected a significant number of cross-reacting products were obtained with nonhuman primate species. All Y-STR multiplex systems tested were able to produce complete Y-STR profiles from bloodstains and semen stains exposed up to 6 weeks when the samples were protected against precipitation and sunlight. However, exposure of the samples to precipitation either in the presence or absence of sunlight resulted in Y-STR profile loss over time, with total profile loss occurring with all systems after 3 weeks or more. Complete Y-STR profiles of the male donors up to 72 h postcoitus were obtained with all of the multiplex systems tested, except for Y-Plex12, which gave partial profiles.     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

Molecular Assay for Screening and Quantifying DNA in Biological Evidence: The Modified Q‐TAT Assay*

Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing.     

Fast PCR amplification using AmpFlSTR Identifiler: Second report

At ISFG2007, an earlier conference, we presented reports on two fast PCR cycling methods using AmpFlSTR Identifiler. In our current study, which involved PCR amplification using AmpliTaq Gold Fast PCR Master Mix, UP, and a standard PCR thermal cycler rather than the Fast PCR thermal cycler, we succeeded in allele typing while reducing PCR running times by half (to approximately 90 min).     

Rapid amplification of commercial STR typing kits

Forensic DNA typing is currently conducted in approximately 8–10 h. The process includes DNA extraction, quantitation, multiplex PCR amplification, and fragment length detection. Today's commercial multiplex short tandem repeat (STR) typing kits are not optimized for rapid PCR thermal cycling. Current protocols require approximately 3 h for amplifying a multiplex containing 15 STR loci plus amelogenin. With the continuing development of miniaturization technologies such as microfluidic and micro-capillary devices, there is a desire to reduce the overall time required to type DNA samples. Such miniature devices could be used for initial screening at a crime scene, at a border, and at airports. There is also the benefit of reducing the required PCR amplification time for labs typing single-source reference samples. Surveys of fast processing polymerases working in combination with rapid cycling protocols have resulted in the development of a ‘rapid’ PCR amplification protocol. Results are obtained in less than 36 min run on a standard peltier-based thermal cycler employing a heating rate of 4 °C/s. Capillary electrophoresis characterization of the PCR products indicates good peak balance between loci, strong signal intensity and minor adenylation artifacts. Genotyping results are concordant with standard amplification conditions utilizing a standard 3 h (non-rapid) thermal cycling procedure. The rapid assay conditions are robust enough to routinely amplify 0.5 ng of template DNA (with 28 cycles).     

Automated DNA extraction using the QIAsymphony platform: Estimation of DNA recovery from simulated forensic stains

The aim of this study was to evaluate the forensic protocol recently developed by Qiagen for the QIAsymphony automated DNA extraction platform. Samples containing low amounts of DNA were specifically considered, since they represent the majority of samples processed in our laboratory. The analysis of simulated blood and saliva traces showed that the highest DNA yields were obtained with the maximal elution volume available for the forensic protocol, that is 200 μl. Resulting DNA extracts were too diluted for successful DNA profiling and required a concentration. This additional step is time consuming and potentially increases inversion and contamination risks. The 200 μl DNA extracts were concentrated to 25 μl, and the DNA recovery estimated with real-time PCR as well as with the percentage of SGM Plus alleles detected. Results using our manual protocol, based on the QIAamp DNA mini kit, and the automated protocol were comparable. Further tests will be conducted to determine more precisely DNA recovery, contamination risk and PCR inhibitors removal, once a definitive procedure, allowing the concentration of DNA extracts from low yield samples, will be available for the QIAsymphony.     

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR^® Idenitfiler^® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.