口蹄疫病毒3D基因的真核表达及其表达产物的功能分析

利用RT-PCR技术扩增口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,经测序确认后将其克隆到真核表达质粒载体pcDNA3.1( )中,重组质粒pcDNA3.1( )-3D经HindⅢ、XbaⅠ双酶切后转染至BHK-21细胞,用纯化的目的蛋白进行Western-blotting鉴定,并预测该3D基因表达产物的三级结构。结果显示,获得分子质量约55 ku的单一3D基因表达产物,其三级结构较为保守。利用RNA细胞内复制体系和荧光定量RT-PCR技术,证明3D基因表达产物在细胞内可促进口蹄疫病毒基因组的复制。     

Simplified Direct PCR Method for Reference Buccal Samples Using a Non-FTA Card by Omitting the Pretreatment Step

When using non-FTA cards in commercial multiplex STR kits for direct PCR, pretreatment steps with specific buffers are recommended. Here, we designed a rapid direct PCR method utilizing a non-FTA card, Oral Cell Sampling Kit, by omitting the pretreatment step involving Prep-n-Go™ Buffer, and it showed compatibility with the GlobalFiler™ Express PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit, and PowerPlex^® Fusion system. To optimize the PCR conditions, we tested the method with different final PCR volumes and cycles. Finally, we conducted a performance test using 50 Korean buccal samples and confirmed the high performance of the method, detecting more than 90% of the samples with full profiles when using GlobalFiler™ PCR Amplification Kit and PowerPlex^® Fusion system at 29 cycles in a 10 μL final PCR volume. Thus, we report a simple direct PCR set-up to analyze reference samples collected using a non-FTA card manufactured in Korea.     

病毒性出血败血症病毒实时荧光RT-RPA恒温检测方法的建立

为建立一种简单、快速的病毒性出血败血症病毒(VHSV)检测方法,本研究通过比较分析VHSV N基因的保守序列,设计了多对引物和一条探针。经过筛选优化,建立了实时荧光RT-RPA检测方法。该方法在39℃恒温反应20 min即可快速、特异性地检测出VHSV,与其他水生动物病毒不发生交叉反应,该方法检测限为1.83×10^3PFU/mL,利用本研究方法对30份鲑鳟鱼临床样品以及9份鱼类疫病病原样品进行检测,检测结果与荧光定量PCR方法结果一致。上述结果表明,本研究建立的检测方法操作简单,反应灵敏,结果可靠,仪器依赖性低,适用于现场对VHSV快速检测。     

荧光标记复合扩增检测4个STR基因座多态性

目的建立荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座检测分型方法,并对成都汉族群体4个基因座的遗传多态性进行调查。方法用6-FAM标记D1S2142和D15S659引物,HEX、TMR分别标记D14S306和D13S1492引物,PCR复合扩增,310基因分析仪电泳自动收集电泳结果数据,GeneScan Analysis Software3.7NT软件计算扩增产物片段相对大小,Genotyper(3.7NT软件进行样本基因型分型,建立了荧光标记复合扩增检测4个STR基因座基因型的方法,对145名成都汉族无关个体样本进行分型。结果荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,每个STR基因座都获得了清晰的基因型分型结果。145份样本,4个STR基因座分别检出10,14,7,12个等位基因和22,54,21,39种基因型,其基因型分布均符合Hardy-W e inberg平衡。4个基因座在成都汉族群体的杂合度分别依次为0.7793,0.8345,0.7793和0.8345;多态信息含量分别依次为:0.7656,0.8730,0.7470和0.8312。累计非父排除率为0.9783,累计个人识别机率为0.9999 917。结论荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,可实现对每个基因座准确分型;成都汉族群体该4个基因座的遗传学数据,可为群体遗传学和法医学研究与应用提供基础资料。     

应用反转录-聚合酶链反应检测动物组织中的口蹄疫病毒

应用反转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术检测了猪和牛的扁桃体、淋巴结、脊髓、肌肉和蹄冠皮肤中的口蹄疫病毒（ＦＭＤＶ）。与接种乳鼠测毒法比较，该方法的灵敏度达０．１６ＬＤ５０～０．３２ＬＤ５０病毒量。检测人工感染后７～３５ｄ的猪组织样品７７份，２５份为阳性；接种乳鼠并盲传３代，乳鼠－间接血凝试验（ＩＨＡ）鉴定６份为阳性。检测人工感染后４～７ｄ的牛组织样品５２份，２０份为阳性；接种乳鼠－ＩＨＡ鉴定１２份为阳性。检测野外鲜肉样品４７份，１２份为阳性；任选其中８份样品接种细胞单层，盲传至第６～７代，先后有５份样品产生ＣＰＥ；其中６份样品同时接种乳鼠，盲传至第６～７代，３组乳鼠出现ＦＭＤＶ致病症状，ＩＨＡ鉴定为阳性。检测冻猪肉样品３７份，３份为阳性；冻牛肉样品９份、冻羊肉样品１０份，均为阴性     

H9N2亚型禽流感病毒聚合酶基因的克隆及序列分析

采用RT-PCR方法对1株H9N2亚型禽流感病毒Ck/GS/2/99株RNA聚合酶基因PB2、PB1和PA分别进行了扩增,将其克隆到pGEM-T easy载体后进行序列测定与分析。结果表明,该株病毒的PB2、PB1和PA基因开放阅读框(ORF)分别由2280、2274和2 151个碱基组成,分别编码759、757和716个氨基酸。经同源性与系统发生树分析,该毒株RNA聚合酶基因PB2、PB1和PA与Ck/NX/4/99株的核苷酸和氨基酸同源性均在98%以上,另外这3个基因与Dk/HK/Y280/97、Sw/HK/9/98和Qu/HK/NT/28/99株的关系密切。     

5个STR基因座的遗传多态性及其法医学应用

目的获得D11S4951、D11S4957、GATA193H05、D2S2951、D6S2421基因座的群体遗传学数据,并分析其在法医学中的应用价值。方法随机抽取成都地区汉族群体无血缘关系个体的静脉血,EDTA抗凝,用Chelex-100法提取DNA,应用PCR技术,扩增上述5个短串联重复序列基因座,聚丙烯酰胺凝胶垂直板电泳分型。结果5个基因座在中国成都汉族人群中分别发现了7、10、8、6、8个等位基因,5个基因座的基因型分布符合Hardy-Weinberg平衡(P>0.05)。各基因座的杂合度分别为0.743、0.772、0.833、0.650和0.800;非父排除概率分别为0.497、0.549、0.662、0.356和0.599;个人识别几率分别为0.863、0.912、0.947、0.829和0.931。结论5个基因座在中国汉族群体中具有法医学应用价值。     

An innovative transfer DNA experimental design and qPCR assay to identify primary,secondary, and tertiary DNA transfer

“Touch DNA” is a form of trace DNA that is presumed to be deposited when an individual touches something and leaves behind DNA-containing skin cells, sweat, or other fluids. While touch DNA is often the result of direct contact (i.e., primary transfer), it can also be indirectly transferred between surfaces or individuals (e.g., secondary or tertiary transfer). Even experts cannot distinguish between different types of transfer and do not fully understand which variables affect direct versus indirect transfer or how often each type of transfer occurs. In this study, we utilize an innovative protocol that combines a paired male and female transfer DNA experimental design with an Amelogenin qPCR assay to generate data on primary, secondary, and tertiary DNA transfer. We report frequencies of indirect DNA transfer and also investigate the potential effects of participant age, self-identified ethnicity, and skin conditions on DNA transfer. Out of 22 experimental trials, we detected primary transfer (male + female) in 71% of trials, secondary DNA transfer in 50% of trials, and tertiary DNA transfer in 27% of trials. No significant associations were found between primary DNA transfer and age, self-identified ancestry, or skin conditions, however, all individuals with sloughing skin conditions demonstrated primary DNA transfer and we suggest this variable be explored in larger samples. These results contribute to a better understanding of the conditions under which secondary and tertiary DNA transfer occurs and can be used to propose realistic DNA transfer scenarios in court cases.