甜瓜黄斑病毒SYBR Green Ⅰ实时荧光定量PCR方法

为建立检测甜瓜黄斑病毒(melon yellow spot virus, MYSV)的SYBR Green Ⅰ实时荧光定量PCR(qPCR)方法。基于MYSV核衣壳蛋白基因保守序列设计qPCR特异性引物对,针对引物退火温度、引物浓度、特异性和敏感性进行系列优化。结果显示,优化后的qPCR方法最适退火温度为61.3℃,最适引物浓度为0.65μmol·L^-1,特异性强,灵敏度高,比PCR高100倍。以携带目的基因片段的重组质粒为标准品,构建的qPCR标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.999 7。实验样品验证表明建立的qPCR方法可用于MYSV的定量检测。     

薄壳山核桃疮痂病菌TaqMan荧光定量PCR检测方法的建立及应用

疮痂病是薄壳山核桃上最具毁灭性的病害,带菌植物材料是传播疮痂病的重要来源。准确、灵敏、快速的检测方法可为该病害流行规律调查和防控提供有力的依据。本文通过比较薄壳山核桃疮痂病菌Venturia effusa及其近似种之间的ITS序列差异,设计了特异性引物和TaqMan探针,建立了薄壳山核桃疮痂病菌的荧光定量PCR检测方法。特异性检测结果表明,该方法可以检测不同地区的薄壳山核桃疮痂病菌菌株,而对其近似种以及薄壳山核桃上的其他真菌均没有信号。本研究建立的检测方法对薄壳山核桃疮痂病菌DNA的最低检测限可达0.5 pg/μL。该方法用于田间样品检测时,检测时间仅需1 h,远快于常规的分离培养法。本研究建立的基于TaqMan探针的荧光定量PCR检测方法为薄壳山核桃疮痂病菌的快速检测和监测提供了有力工具。     

荧光定量PCR法检测点带石斑鱼神经坏死病毒研究

根据己公布的神经坏死病毒全基因序列,应用引物设计软件依据Gene Bank所收录的各种石斑鱼神经坏死病毒RNA2基因组全序列选择保守区域,根据Taqman探针引物设计原则,设计Taq Man探针及其引物。采用Taq Man探针技术,并且将假病毒技术构建的含有神经坏死病毒RNA2基因组的MS2噬菌体假病毒作为阳性质控品,建立Taq Man探针检测点带石斑鱼神经坏死病毒的荧光定量PCR方法,拟合标准曲线,能够在病毒检测中进行绝对定量,能检测到10个病毒拷贝模板数,在临床检测中得到很好运用。本研究建立实时荧光定量RT-PCR,有助于日常检验工作,为鱼类神经坏死病毒的筛选和监测提供基础,有助于控制神经坏死病的疫情传播。     

某牛场粪源大肠杆菌0157∶H7的分离鉴定

为了解重庆市某牛场牛群大肠杆菌O157∶H7的感染情况,我们以牛场犊牛为研究对象,采集其新鲜粪便,经LB培养基增菌培养、免疫磁珠富集后,在山梨醇麦康凯琼脂上划线培养,最后PCR扩增eaeA基因目的片段,以此分离鉴定粪源大肠杆菌O157∶H7。结果显示：在所采集的39份犊牛粪便样品中,分离得到一株符合大肠杆菌O157∶H7生长特点且含有eaeA基因的菌株。     

Investigation of biofilm production and icaA and icaD genes in Staphylococcus aureus isolated from heifers and cows with mastitis

Biofilm formation and antimicrobial resistance of Staphylococcus aureus are important virulence factors in cases of mastitis in dairy cows. However, few studies have investigated mastitis strains isolated from heifers. Within this context, the objective of the present study was to investigate biofilm formation on Congo red agar, the presence of the icaA and icaD genes by polymerase chain reaction (PCR), and the percentage of in vitro antimicrobial resistance of 110 S. aureus isolates from mammary gland secretions of heifers and cows with mastitis. PCR detected the icaA and icaD genes in 98% and 100% of isolates, respectively. However, only 55.5% of all isolates produced a biofilm on Congo red agar. Antimicrobial susceptibility testing revealed that 47.0% of isolates from heifers and 70.4% of isolates from cows were resistant to at least one of the antimicrobial agents tested. Resistance to penicillin and/or ampicillin was the most frequent (44.5%). These results indicate the need to implement prophylactic and control measures of mastitis for heifers. Heifers and cows can carry resistant strains with the capacity of biofilm production, a fact representing a threat to public health and animal well‐being and generating losses to dairy farmers.     

关岭牛MyoD Ⅰ基因在不同组织和时期中的表达分析

为研究MyoDⅠ基因在关岭牛不同组织表达量的差异及在肌肉发育过程中的表达情况,试验采用实时荧光定量PCR技术对关岭牛背最长肌、大腿肌、心脏、肝脏、脂肪、小肠6个组织的MyoDⅠ基因相对表达量进行检测。同时分析关岭牛胎儿、18月龄、30月龄时期背最长肌组织中MyoDⅠ基因的表达规律。结果显示,MyoDⅠ基因在背最长肌中表达量最高,在出生后的关岭牛肌肉组织中也具有较高的表达量,且随着出生时间的推移呈上升趋势。推测可能由于背最长肌是肌肉富集地方,因此基因表达量较高,随着年龄增大,肌肉丰满度增加,基因表达量也随着增加。     

The use of COLD‐PCR,DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours

The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different ‘driver’ somatic mutations of c‐KIT, together with the wild‐type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild‐type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50–20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5–1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.     

桤木群体遗传分化研究I.DNA提取和PCR条件的建立

桤木(ＡｌｎｕｓｃｒｅｍａｓｔｏｇｙｎｅＢｕｒｋ)为桦木科(Ｂｅｔｕｌａｃｅａｅ)植物,主要分布于四川盆地及其周边地区。桤木喜光、喜湿,根系发达,对土壤要求不严,适应性强,在长江中下游地区防护林建设中具有重要作用。国外对桤木属(Ａｌｎｕｓ)植物的研究起步较早,主要集中于生理生化方面包括生长、固Ｎ、抗涝性以及种间杂交研究等[1　3]。虽有学者利用ＲＦＬＰ研究其核糖体ＤＮＡ的基因结构、以同工酶技术研究桤木属的群体遗传变异[4　6],但未见应用ＲＡＰＤ技术的研究报道。随着现代生物技术的发展,生物多样性的检测技术日趋成熟和多…     

枫香DNA提取方法与PCR扩增程序的优化

从SDS、CTAB、高盐低pH值3种方法中选择了CTAB法作为枫香适合的DNA提取方法,同时构建了枫香DNA的PCR扩增程序,并从Mg^ 浓度、Taq酶用量、dNTP浓度、引物浓度、DNA模板用量等方面优化了PCR扩增程序。