The use of a random priming procedure to generate cDNA libraries of infectious bronchitis virus, a large RNA virus

An efficient method for the generation of gene banks from large RNA viruses is described using infectious bronchitis virus as an example. Randomly primed clones have been characterized and found to be representative of the viral genome including 5' leader sequences.     

A new h allele detected in Europe has a missense mutationin alpha(1,2)-fucosyltransferase motif II

BACKGROUND: The FUT1 gene encodes an alpha(1,2)-fucosyltransferase (H transferase), which determines the blood group H. Nonfunctional alleles of this gene, called h alleles and carrying loss-of-function mutations, are observed in the exceedingly rare Bombay phenotype. Twenty-three distinct h alleles have been characterized at the molecular level in various populations. The FUT2 (SE) gene is highly homologous to FUT1 (H:). STUDY DESIGN AND METHODS: The FUT1 gene of an Austrian proband with the Bombay phenotype was characterized by nucleotide sequencing of the full-length coding sequence. A PCR method using sequence-specific primers for FUT2 genotyping in whites was developed. The plasma alpha(1,2)-fucosyltransferase activity was determined. The distribution of the mutations underlying 24 h alleles and 7 se alleles was analyzed. RESULTS: The proband carried a new h allele. Two nucleotide changes, G785A and C786A, in codon 262 of the FUT1 gene resulted in the replacement of serine by lysine. No alpha(1,2)-fucosyltransferase activity was detected in the proband's plasma. The proband was homozygous for the seG428A allele. Six of 17 missense mutations in nonfunctional h and se alleles occurred in highly conserved fucosyltransferase motifs. No loss-of-function mutation was observed in the aminoterminal section encompassing the transmembraneous helix. CONCLUSION: The missense mutation S262K in the FUT1 gene caused the loss of H transferase activity. The analysis of the distribution of mutations in nonfunctional FUT1 and FUT2 genes can point to functionally important domains in the H transferase.     

青天葵ISSR-PCR体系优化及引物筛选

目的:建立适合青天葵遗传差异分析的简单重复序列区间(ISSR)-聚合酶链式(PCR)反应体系。方法:采用正交试验设计对影响青天葵ISSR-PCR反应体系的5种因素(dNTP、模板DNA、引物、Mg2+和Taq DNA聚合酶)4个水平进行优化,并结合新复极差法,对试验中各单因素进行分析。结果:确立了青天葵最佳反应体系,即在20μL的总反应体积中含有10×PCR buffer 2μL,dNTP 225μmol·L-1,Mg2+2.5 mmol·L-1,引物0.4μmol·L-1,Taq DNA聚合酶1.0 U,模板DNA60 ng;从100条引物中筛选出16条扩增稳定、多态性丰富的ISSR引物。结论:建立的青天葵ISSR-PCR反应体系,经过24份青天葵样品检验,得出该体系稳定可靠,可用于青天葵遗传差异分析。     

鹿鞭线粒体DNA指纹鉴定

目的探讨鹿鞭及牛鞭线粒体细胞色素b基因部分序列片段特征,建立中药材鹿鞭敏感、特异的DNA分子标记学鉴定方法。方法碱变性法提取新鲜鹿鞭及牛鞭,采用引物设计软件PrimerPremier5.0对鹿鞭及牛鞭的细胞色素b基因序列分析,用于鉴定正品鹿鞭的特异性引物,经聚合酶链式反应（PCR）扩增,对鹿鞭进行DNA指纹鉴定。结果对鹿鞭进行mtDNA鉴定图谱显示,真品鹿鞭有306bp条带,伪品没有。结论用此方法对鹿鞭进行鉴定,可准确辨别正品与伪品。重现性、稳定性好,简便准确而可行。鹿鞭mtDNA具有特异性指纹特征,所得鹿鞭DNA指纹特征图谱可用于鹿鞭的鉴定。     

三叶木通MSAP反应体系的优化及引物筛选

目的 研究三叶木通表观遗传多样性,建立并优化三叶木通甲基化敏感扩增多态性（methylation sensitive amplified polymorphism,MSAP）反应体系。方法 以三叶木通叶片为材料,利用正交试验建立三叶木通MSAP的预扩增和选择性扩增的最佳反应体系。结果 建立的MSAP最佳反应体系为酶切反应：HpaII/MspI 10 U,EcoR I 10 U;预扩增反应：总体积20 μL,连接产物2 μL,E00、HM00各125 ng,dNTPs 0.312 5 mmol/L,Mg^2＋ 1.875 mmol/L,Taq DNA聚合酶2 U;选择性扩增反应：总体积20 μL,稀释200倍的预扩增产物5 μL,引物E-ACT、HM-CAT各50 ng,dNTPs 0.250 mmol/L,Mg^2＋ 1.250 mmol/L,Taq DNA酶1 U。利用优化的体系,最终从MSAP的80对引物中筛选出有效引物6对。结论 建立的MSAP反应体系可用于后续三叶木通DNA甲基化的相关研究。     

实时荧光定量PCR对兔戊型肝炎病毒(HEV)的检测及评估

目的 兔戊型肝炎病毒(HEV)是近年来新发现的,根据其分子生物学特征可能成为HEV的新基因型。为提高对兔HEV的检出率,本研究设计了针对兔HEV RNA的特异性引物和TaqMan探针并对其进行考评。方法 1)从GenBank中下载14条兔HEV全长序列,分别在其ORF1、ORF3片段的保守区域设计一条TaqMan探针和一对上、下游特异性引物,其中ORF1片段的探针及引物为C组,ORF3片段的为B组;A组的探针及引物为Jothikumar N等所报道的通用引物;2)制备相应的质粒标准品来构建定量标准曲线,并利用上述3套引物、探针对49份兔粪便及44份兔血液样品进行检测,将B、C组检测结果与A组的检测结果进行比较。结果 新建立了两套兔HEV定量PCR的TaqMan探针及引物(B、C组),其标准曲线的斜率分别为-3.455和-3.469;使用上述3套引物及探针(A、B、C组)对49份粪便标本进行荧光定量PCR检测,其HEV RNA阳性率分别为67.35%(33/49)、67.35%(33/49)、57.14%(28/49),平均HEV RNA(log copies/g)拷贝数为6.18、5.90、6.11;44份血液标本的HEV RNA阳性检出率分别为65.90%(29/44 )、56.82%(25/44)、50.00%(22/44),平均HEV RNA(log copies/mL)拷贝数为3.62、3.43、3.03;结果显示以上3组探针及引物均可用于粪便和血液标本的定量检测。结论 兔HEV虽然有其独特的基因结构,但使用HEV通用引物检测不影响检出率。     

基因组简并寡核苷酸引物聚合酶链反应的影响因素

目的:对影响现有微量基因组扩增方法--简并寡核苷酸引物聚合酶链反应(degenerate oligonucleotideprimed PCR,DOP-PCR)的因素进行探讨.方法:针对现有DOP-PCR扩增过程中存在的影响产物产量和特异性的因素进行分析,分别从不同的DNA模板来源、模板梯度稀释与否以及DOP-PCR扩增产物是否采用低熔点胶回收去除干扰分析的小片段等方面进行研究,最后以特异性基因(FTCD和CBS)PCR扩增的结果作为评价指标,了解上述因素对DOP-PCR扩增的影响.结果:以小鼠单个卵细胞基因组为模板与以肝基因组DNA为模板相比,在产量上前者明显低于后者,特异性上两者相当.小鼠肝基因组DNA梯度稀释作为模板进行扩增的结果没有明显差异.与未经低熔点胶回收的DOP-PCR产物相比,经低熔点胶回收的DOP-PCR产物在常规PCR扩增反应中得到的产物特异性更好.结论:应用DOP-PCR方法进行扩增时,小鼠单个卵细胞可以用来进行DOP-PCR的扩增从而用于对特异性基因的检测,但是在扩增产量方面需要进一步的改进或优化.对现有DOP-PCR反应进行产物低熔点胶回收纯化,能够增加产物的特异性,效果满意.     

Molecular diagnosis of leishmaniosis in the Paraná state of southern Brazil

The objective of the present study was to establish a polymerase chain reaction (PCR) technique for the diagnosis of cutaneous and mucocutaneous leishmaniosis from autochthonous cases in the state of Paraná in southern Brazil as well as imported cases. We sought to determine its utility and accuracy compared with smears and present culture methods. To standardize PCR samples, skin and mucosal punch biopsies from human lesions were performed on patients living in different regions of the Paraná state (76 cases) and other endemic areas of Brazil and Argentina (7 cases). For PCR standardization, two pairs of primers (MP1L/MP3H and B1/B2) were utilized for amplification of the conserved sequences in the minicircle of kinetoplast DNA (kDNA) for the Leishmania braziliensis complex. Two other primer pairs (b1/b2 and a1/a2) were species-specific for L. (V.) braziliensis and L. (V.) amazonensis, respectively. After differential diagnosis, eight patients had clinical diagnosis of the cutaneous ulcer changed to others pathologies such as syphilis, baso-cellular carcinoma, varicose ulcer, ecthyma and paracoccidioidomycosis. Of the 75 patients with cutaneous (CL) and mucocutaneous (MCL) lesions who provided samples, 47 (46 CL + 1 MCL) were diagnosed with leishmaniosis by smear and 57 (52 LC + 5 MCL) were diagnosed by culture methods. In contrast, our PCR technique presented higher accuracy when compared to the direct examination and culture of parasites. PCR is applicable both for CL where all 61 lesions were diagnosed, and MCL where 12 of 14 lesions were diagnosed. This molecular biology technique is also a faster and more specific diagnostic method compared with present parasitological procedures.     

通过引物设计和提高退火温度提高实时定量PCR检测microRNA的特异性

目的：了解因某些microRNA (miRNA) 家族成员序列相似,以及同一种成熟miRNA的长度不均一对PCR定量检测结果的影响,寻找区分相似miRNA成员和准确定量的方法和条件。方法: 采用RNA加尾和引物延伸实时定量RT-PCR法,设计不同位置错配的引物,以克隆到质粒中的全长成熟miRNA为模板,比较在不同退火温度下各种错配引物与完全匹配引物在扩增miRNA上的效率差异,并根据由此得出的经验设计let-7家族成员引物,以克隆的成熟miRNA为模板,检测其在不同退火温度下区分相似家族成员的能力。此外,设计了10组3′末端碱基位置不同的特异引物,比较了它们在检测小鼠大脑miRNA表达水平上的差异。结果：提高退火温度12 ℃~14 ℃将最大限度增加特异性而不降低敏感性,将引物的3′ 末端置于差异碱基或其附近可以有效区分相似miRNA序列,将引物3′ 末端置于miRNA第18~20个碱基,可避免漏检较短的成熟miRNA,使定量更真实准确。结论：精心设计引物并提高退火温度可提高实时定量PCR检测microRNA的特异性和准确性。