牛疱疹病毒Ⅰ型二重LUX荧光PCR鉴别检测方法的建立

采用LUX新型荧光PCR技术原理,建立了快速检测牛疱疹病毒Ⅰ型(BHV-1)以及鉴别野毒感染和基因缺失疫苗免疫动物的二重实时荧光PCR方法。结果显示,该方法对多株BHV-1病毒均呈典型gE、gC双基因阳性反应,对其他常见动物疱疹病毒以及健康牛基因组DNA等均呈阴性反应;对细胞增殖病毒液的gE、gC双基因鉴别的检测敏感性可达0.4~0.04 TCID50,比常规PCR方法高100倍以上;对带毒牛血清、抗凝全血、牛新鲜精液和冷冻精液基因鉴别的检测敏感性分别达0.04 TCID50、0.4 TCID500、.4 TCID50和4 TCID50。采用该方法从临床牛血样、鼻拭子样品中检出IBRV阳性样品,检测全程仅需约2 h。对单基因克隆质粒的检测进一步证实该方法能特异地鉴定gE、gC基因,检测灵敏度分别达90、30拷贝。结果表明,该方法可应用于临床快速诊断BHV-1病毒感染,鉴别BHV-1病毒感染与对应的基因缺失疫苗免疫动物。     

猪胸膜肺炎放线杆菌PCR检测方法的建立及临床应用

为建立直接检测可疑病料中胸膜肺炎放线杆菌(APP)的PCR诊断方法,对用以扩增apxⅣA毒力基因3′端长约442 bp的核苷酸片段的引物、模板、TaqDNA聚合酶、dNTPs的最适剂量及退火温度进行了优化筛选,并进行了特异性及重复性试验;以筛选出的优化条件,对临床可疑病料进行了PCR检测。结果,在25μL体系中,以5 pmol/μL引物、0.25μLTaqDNA聚合酶、2 mmol/L dNTPs、56℃退火温度对APP标准菌株apxⅣA毒力基因的扩增效果最好,重复性和稳定性高;而对类症菌株的扩增结果则为阴性,表明其特异性强。对分离到APP的病料PCR阳性率为100%(5/5);未分离到APP的纤维渗出性病料中,新鲜与陈旧病料PCR阳性率分别为70.59%(12/17)、50.00%(4/8),而非纤维渗出性病料的PCR结果均为阴性(0/45、0/9)。结果表明,建立的PCR方法特异、快速、稳定、敏感,优于细菌学方法,它可作为APP可疑病料的理想诊断方法。     

用多重PCR鉴别猪伪狂犬病野毒与疫苗毒的研究

根据基因库中的猪伪狂犬病病毒(PRV)各基因的序列,设计了与PRV的 gB、gD、gE基因序列互补的3对引物。对样品中的PRV DNA模板进行了多重PCR扩增及反应条件的优化,结果同时得到与设计相符合的3条特异性条带,分别为549 bp(gB)、429 bp(gD)、366 bp(gE)。用这3对引物对三基因缺失疫苗毒的样品DNA模板进行多次多重 PCR扩增,均能稳定得到与设计相符合的2条特异性条带。敏感性试验结果表明,多重 PCR可以检测到 106 pg三基因缺失疫苗毒或756 pg PRV野毒的核酸模板量。特异性试验结果表明,以正常对照细胞及猪圆环病毒和猪细小病毒DNA为模板进行多重PCR扩增,均无任何条带。     

江西汉族人群D6S1043等9个STR基因座的遗传多态性研究

目的研究D6S1043、D8S1132、D11S2368、D7S3048、D18S1364、D20S478、D22-GATA198B05、D2S1772、D13S325共9个STR基因座遗传多态性。方法应用PCR扩增,聚丙稀酰胺垂直电泳及银染技术,对200例中国江西汉族人群上述9个STR基因座首次进行了检验分析。结果获得了中国江西汉族人群基因频率分布资料,基因频率分布符合Hardy-Weinberg平衡,杂合度(H)0.7830～0.8768,个体识别率(DP)0.9166～0.9680,非父排除率(PE)0.5795～0.7468,多态信息总量(PIC)0.7493～0.8616。结论D6S1043等9个STR基因座是一组高多态性的遗传标记系统,在法医学及人类遗传学研究中具有重要意义。     

基于TaqMan探针的猪圆环病毒1型实时荧光定量 PCR检测方法的建立

采用PCR方法克隆了猪圆环病毒1型(PCV1)ORF2基因,构建了重组质粒pMD-ORF2并将其作为标准阳性模板.通过对反应条件进行优化,以定量的10倍系列稀释的质粒pMD-ORF2为标准品进行荧光定量PCR扩增,建立了检测PCV1的荧光定量PCR方法.结果显示,该方法的检测灵敏度可达1.0×101拷贝/μL;对起始浓度为1.0×107、1.0×106、1.0×105拷贝/μL标准品的最终实际测得值分别为1.001×107、0.869×106和0.988×105拷贝/μL,变异系数分别为4.758%、1.865%和3.836%.表明,此方法具有良好的准确性和重现性.     

定量PCR技术在法医学中应用的研究

目的研究荧光定量PCR技术在法医学中的应用。方法应用Taqman技术对法医各种生物检材进行DNA定量。结果该定量PCR技术对各种法医生物检材进行了准确定量,并判断检材中是否存在抑制物,从而指导了后续STR的检验。结论定量PCR技术是法医DNA检验中一项不可缺少的辅助技术。     

猪链球菌2型和9型广东分离株的病原特性

2005年夏秋季从广东地区9例病猪中分离到2株猪源链球菌,均呈链球菌的特征形态,α溶血,生化试验结果相似,且都不与A~G链球菌群特异性血清发生凝集。经PCR及测序鉴定,一株为猪链球菌2型,另一株为猪链球菌9型。2型分离株的3个毒力基因均为阳性(sly+epf+mrp+),但对小鼠的毒力较低,2/10小鼠在接种细菌后第6~15 d发病致死,细菌在小鼠中的带菌时间至少15 d;9型分离株3个毒力因子均阴性(sly-epf-mrp-),但对小鼠有较强毒力,9/10小鼠在接种细菌后5 d内发病死亡,对小鼠的半数致死量为1.70×106CFU。表明广东地区致病性猪链球菌不仅存在2型,而且还有9型。     

Assessing the Risk of Secondary Transfer Via Fingerprint Brush Contamination Using Enhanced Sensitivity DNA Analysis Methods

Experiments were performed to determine the extent of cross‐contamination of DNA resulting from secondary transfer due to fingerprint brushes used on multiple items of evidence. Analysis of both standard and low copy number (LCN) STR was performed. Two different procedures were used to enhance sensitivity, post‐PCR cleanup and increased cycle number. Under standard STR typing procedures, some additional alleles were produced that were not present in the controls or blanks; however, there was insufficient data to include the contaminant donor as a contributor. Inclusion of the contaminant donor did occur for one sample using post‐PCR cleanup. Detection of the contaminant donor occurred for every replicate of the 31 cycle amplifications; however, using LCN interpretation recommendations for consensus profiles, only one sample would include the contaminant donor. Our results indicate that detection of secondary transfer of DNA can occur through fingerprint brush contamination and is enhanced using LCN‐DNA methods.     

Effectiveness of Coupled Application of AmpFℓSTR Yfiler Kit and Reduced Size Y‐chromosomal Short Tandem Repeat Analysis for Archeological Human Bones

The AmpF?STR Yfiler PCR Amplification (Yfiler) kit continues to be improved for a better analytical efficiency in cases of highly degraded DNA. The authors endeavored to determine whether coupling of the Yfiler kit with supplemental multiplex amplification of some Y‐STR loci is a more efficient analytical mode for poorly preserved human femurs (n = 15) discovered at Korean archeological sites. To reveal locus profiles not easily obtained by Yfiler analysis, custom‐designed primers were adopted for the DYS390, DYS391, DYS392, DYS438, DYS439, and DYS635 loci. The success rate for 16 Y‐STR locus profiles obtained from the 15 femurs was improved from 18.33% (in the use of Yfiler kit only) to 49.17% (the coupled use of Yfiler and custom‐designed primers). In this study, the authors established that the custom‐designed primers offer a markedly improved success rate for obtainment of Y‐STR profiles from degraded aDNA not easily identified by sole use of the Yfiler assay.     

Development of a Real‐Time PCR‐Based Method for Analyzing Semen‐Specific Unmethylated DNA Regions and Methylation Status in Aged Body Fluid Stains

The detection of semen in forensic investigation is considered important evidence of sexual assault. In this study, we report the development of a real‐time polymerase chain reaction‐based method for identifying semen that can simply and rapidly analyze the semen‐specific unmethylated region of the DACT1 gene. Using two fluorescent probes designed for the methylated or unmethylated status, this method could perform quantitative analysis of the methylation status in this region. Furthermore, this method was used to analyze various body fluid samples, including 29‐year‐old semen and blood stains. The results showed that this method can detect almost exclusively semen or nonsemen signals even in highly decomposed samples, while a few semen or nonsemen samples showed slight signals of the other fluorescence probe. Although there is still a need for further analysis such as setting thresholds to analyze unknown samples, this method could be a useful supplementary tool for identifying semen, especially in old stains such as those in cold‐case investigations.