犬复孔绦虫ITS及5．8 SrDNA的PCR扩增、克隆及序列分析

目的以从我国广东广州和湛江犬小肠中采集的2条犬复孔绦虫作为研究对象。以保守引物NC5及NC2扩增犬复孔绦虫的ITS-1，5．8S及ITS-2rDNA片段并进行序列分析。方法将PCR扩增出的片段纯化后克隆至pGEM-TEasy载体，重组质粒通过菌落PCR和酶切鉴定后，对阳性菌落进行序列测定。结果来自广州和湛江的2条犬复孔绦虫ITS及5．8 S rDNA序列总长分别为1536bp、1385bp，2条犬复孔绦虫的ITS-1、ITS-2序列相差较大，分别为20．80％、27．17％，而5．8S序列相差较小（1．49％）。结论由于犬复孔绦虫ITS序列复杂，种内存在的差异大，故不适于作为犬复孔绦虫种的遗传标记。     

人神经营养素—4基因的扩增及序列分析

从人基因组DNA获取神经营养素－4（neurotrophin-4,NT-4)基因，并对该目的基因进行序列测定，本实验直接从人脑组织中提取基因组DNA，根据人神经营养素－4的cDNA序列，设计一对寡核苷酸引物，采用PCR，获得人NT－4基因，用DNA序列分析NT－4基因，结果，我们获取的人NT－4基因与献报道有五个碱基不同，我们认为：直接采用PCR来获取目的基因，出现碱基错配的可能性较大；测序仪器和实验条件对结果有一定影响关系。     

PCR动力学数学模型在PCR产物直接核酸序列分析中的应用

使用ＰＣＲ动力学数学模型提供的ＰＣＲ产物数量计算方法计算经ＰＣＲ扩增获得的产物数量，纯化后用於ＰＣＲ产物的核酸序列分析。由於ＰＣＲ动力学数学模型提供了比较准确的产物数量计算，使测序模扳数量总能保持在比较适当的范围，因此测序结果的可读性、自显影条带的清晰度保持了较好的水平。提示了ＰＣＲ动力学数学模型是基本正确的。说明准确的定量计算ＰＣＲ产物数量对ＰＣＲ产物直接核酸序列分析是有益的。     

我国不同疫区杜氏利什曼原虫ITS片段的克隆及序列分析

为测定我国荒漠、山丘、平原疫区杜氏利什曼原虫分离株ITS序列并分析其序列差异,首先提取杜氏利什曼原虫基因组DNA进行PCR扩增,然后将扩增出ITS片段克隆入PMD18-Tvector上,再用双脱氧链末端终止法测序。结果用PCR成功扩增出约1000bp的ITS片段,测序结果表明:本文报道的荒漠、山丘、平原疫区的3株杜氏利什曼原虫(L.dXJ771,L.dSC10,L.dSD2)的ITS序列大小分别为:1086、1027、1028bp。序列分析表明:我国杜氏利什曼原虫荒漠分离株(L.dXJ771)的ITS序列与平原分离株(L.dSD2)及山丘分离株(L.dSC10)的ITS序列有明显差异,平原分离株(L.dSD2)与山丘分离株(L.dSC10)的ITS序列也存在较明显差异,荒漠分离株(L.dXJ771)与山丘分离株(L.dSC10)的ITS序列差异略小于与平原分离株(L.dSD2)的ITS序列差异。     

人单核细胞趋化蛋白－1（MCP－1）基因的PCR扩增、克隆及序列分析

本文报道用植物血凝素（ＰＨＡ）诱导健康人外周血单个核细胞（ＰＢＭＣ，包括单核细胞和淋巴细胞），提取细胞总ＲＮＡ，反转录合成ｃＤＮＡ第一链，再以其为模板进行ＰＣＲ，得到了编码成熟单核细胞趋化蛋白－１（ＭＣＰ－１）的ｃＤＮＡ。该ｃＤＮＡ用ＥｃｏＲＩ、ＢａｍＨＩ双酶切后，回收含ＭＣＰ－１基因的长约２８０ｂｐ的ＤＮＡ片段，插入到经ＥｃｏＲＩ、ＢａｍＨＩ双酶切的ｐＵＣ１９载体中，进行序列分析。结果表明，ＭＣＰ－１中第１２个氨基酸的编码序列与国外报道的不同。由ＴＧＴ变成了ＴＧＣ，但编码相同的氨基酸即半胱氨酸，其余的编码序列则完全相同，说明ＭＣＰ－１的基因型可能存在着多态性。     

菌落PCR和质粒PCR对转化菌的筛选

为建立简便,可靠转化菌的筛选方法。方法应用菌落ＰＣＲ和重组质粒ＰＣＲ方法对转化菌进行筛选和ＤＮＡ序列分析。克隆载体和表达载体筛选及鉴定采用与载体多克隆插入位眯序列互补的通用引物或免疫球蛋白家族特异性引物。结果菌落ＰＣＲ和质粒ＰＣＲ技术可作为基因克隆筛选和鉴定的方法。结论菌落ＰＤＲ和质粒ＰＣＲ方向简便,快速,可靠,其产可作为模板直接用于序列分析。     

两种细蚤rDNA—ITS2序列的研究

本文首次测定和分析缓慢细蚤Leptopsylla segnis（Schonherr,1811）和距细蚤Leptopsylla lauta （Rothschild,1913）的rDNA—ITS2序列,为蚤的分类和系统发育研究提供有意义的基础资料。通过聚合酶链反应（Polymerase chain reaction,PCR）扩增缓慢细蚤L．segnis和距细蚤L．lauta各3个个体的rDNA．ITS2序列,并对扩增产物进行PCR直接测序和分析。结果显示,缓慢细蚤和距细蚤的rDNA．ITS2序列的长度均为309bp;缓慢细蚤序列的CG含量为50．9％～52．1％;距细蚤序列的CG含量为49．5％～50．6％。缓慢细蚤与距细蚤的rDNA—ITS2序列有16个碱基不同,其中7个是颠换,9个为转换,两种细蚤之间有一定差异。     

人脑源性神经营养因子基因扩增及序列测定

目的：从基因组DNA获取编码脑源性神经营养因子基因,并对目的基因进行序列测定。方法：本研究直接从脑组织中提取人的基因组DNA,根据人的脑源性神经营养因子（BDNF）的cDNA序列,设计一对寡核苷酸引物,采用聚合酶链反应（RCR）,体外扩增编码人脑源性神经营养因子基因;扩增产物用自动分析仪进行序列测定。结果：从基因组DNA体扩增出脑源性神经营养因子基因,序列与献报道一致。结论：利用PCR直接从基因组获取目的基因,表明人脑神经营养因子基因为单一外显子,目的基因的扩增成功为进一步研究打下了基础。     

小鼠BMP—McAb可变区基因的体外扩增,克隆与序列分析

从体外培养的ＢＭＰＭｃＡｂ杂交瘤中提取细胞总ＲＮＡ，反转录成ｃＤＮＡ。以ｃＤＮＡ为模板，利用两对根据免疫球蛋白重链与轻链可变区５′端序列和Ｊ区序列设计合成的引物，通过ＰＣＲ方法，扩增出抗体重链、轻链可变区基因片段（ＶＨ，ＶＬ）。将扩增产物分别克隆入ｐＵＣ１８，ｐＵＣ１９质粒，筛选出阳性克隆，利用双脱氧链终止法进行序列测定，经计算机分析，ＶＨ基因全长为３４５ｂｐ，编码１１５个氨基酸；ＶＬ基因全长为３１５ｂｐ，编码１０５个氨基酸。     

应用改进的SSP-抑制PCR技术扩增cDNA片段旁侧序列

结合抑制PCR和单链特异引物PCR技术，降低RACE过程中由公用引物引发的非特异扩增、增加目的cDNA在原始模板中的相对比例，从而提高RACE特异性，有效地扩增靶序列。     

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66 – 100% for isolates of different subgroups within an AG, and 55 – 96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance. Received: 11 February /16 May 1997     

Differentiation of Sheep Pox and Goat Poxviruses by Sequence Analysis and PCR-RFLP of P32 Gene

Sheep pox and Goat pox are highly contagious viral diseases of small ruminants. These diseases were earlier thought to be caused by a single species of virus, as they are serologically indistinguishable. P32, one of the major immunogenic genes of Capripoxvirus, was isolated and Sequenced from two Indian isolates of goat poxvirus (GPV) and a vaccine strain of sheep poxvirus (SPV). The sequences were compared with other P32 sequences of capripoxviruses available in the database. Sequence analysis revealed that sheep pox and goat poxviruses share 97.5 and 94.7% homology at nucleotide and amino acid level, respectively. A major difference between them is the presence of an additional aspartic acid at 55th position of P32 of sheep poxvirus that is absent in both goat poxvirus and lumpy skin disease virus. Further, six unique neutral nucleotide substitutions were observed at positions 77, 275, 403, 552, 867 and 964 in the sequence of goat poxvirus, which can be taken as GPV signature residues. Similar unique nucleotide signatures could be identified in SPV and LSDV sequences also. Phylogenetic analysis showed that members of the Capripoxvirus could be delineated into three distinct clusters of GPV, SPV and LSDV based on the P32 genomic sequence. Using this information, a PCR-RFLP method has been developed for unequivocal genomic differentiation of SPV and GPV.     

Sequence Analysis of the NSP4 Gene from Human Rotavirus Strains Isolated in the United States

Two major and one minor genotype of the rotavirus NSP4 gene have been described. The sequences of 29 NSP4 genes from rotavirus isolates obtained in the United States during the 1996–1997 rotavirus season (types P[8]G1, P[8]G9, P[4]G2 and P[6]G9) and 10 strains isolated during previous rotavirus seasons (types P[8]G1 and P[4]G2) were determined. All NSP4 genes from strains with short E types (6 P[4]G2, 4 P[6]G9) belonged to genotype NSP4A, whereas all 19 strains with long E types (16 P[8]G1, 3 P[8]G9) had NSP4 genes of genotype NSP4B. Genetic variation within genotypes was low (2.3% for both NSP4A and NSP4B), confirming that the NSP4 genes are highly conserved. Nonetheless, at least two distinct sub-lineages could be detected within each genotype: strains isolated in the same year, regardless of geographic location, were more closely related or even identical at the deduced amino acid level; strains isolated in different years were more distinct. Thus, geographic distance did not affect genetic distance. Northern hybridization analysis with NSP4A and NSP4B total gene probes failed to detect any unusual combinations of the VP6 and NSP4 genes in 31 additional isolates from the 1996–1997 rotavirus season.     

Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR

This report describes the development of a real-time LightCycler assay for the detection and identification of Candida and Aspergillus spp., using the MagNa Pure LC Instrument for automated extraction of fungal DNA. The assay takes 5-6 h to perform. The oligonucleotide primers and probes used for species identification were derived from the DNA sequences of the 18S rRNA genes of various fungal pathogens. All samples were screened for Aspergillus and Candida to the genus level in the real-time PCR assay. If a sample was Candida-positive, typing to species level was performed using five species-specific probes. The assay detected and identified most of the clinically relevant Aspergillus and Candida spp. with a sensitivity of 2 CFU/mL blood. Amplification was 100% specific for all Aspergillus and Candida spp. tested. To assess clinical applicability, 1,650 consecutive samples (1,330 blood samples, 295 samples from other body fluids and 25 biopsy samples) from patients with suspected invasive fungal infections were analysed. In total, 114 (6.9%) samples were PCR-positive, 5.3% for Candida and 1.7% for Aspergillus spp. In patients with positive PCR results for Candida and Aspergillus, verification with conventional methods was possible in 83% and 50% of cases, respectively. In conclusion, the real-time PCR assay allows sensitive and specific detection and identification of fungal pathogens in vitro and in vivo.     

Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp.

A simple real-time fluorescence resonance energy transfer (FRET) PCR, targeting the gyrA gene outside the quinolone resistance-determining region, was developed to identify Campylobacter jejuni and Campylobacter coli. These species were distinguished easily, as the corresponding melting points showed a difference of 15 degrees C. A second assay using the same biprobe and PCR conditions, but different PCR primers, was also developed to identify the less frequently encountered Campylobacter fetus. These assays were applied to 807 Campylobacter isolates from clinical specimens. Compared to phenotypic identification tests, the FRET assay yielded the same results for all except three of the isolates. Analysis by standard PCR and 16S rDNA sequencing demonstrated that two of these isolates were hippurate-negative C. jejuni strains, resulting in an erroneous phenotypic identification, while the third was an isolate of C. coli that contained a gyrA gene typical of C. jejuni, resulting in misidentification by the FRET assay. The FRET assay identified more isolates than standard PCR, which failed to yield amplification products with c. 10% of isolates. It was concluded that the FRET assays were rapid, reliable, reproducible and relatively cost-efficient, as they require only one biprobe and can be performed directly on boiled isolates.     

Identification at strain level ofRhizoctonia solani AG4 isolates by direct sequence of asymmetric PCR products of the ITS regions

The relatedness of nine isolates ofRhizoctonia solani, belonging to anastomosis group (AG) 4, and one isolate of AG1 was determined by comparative sequence analysis based on direct sequencing of PCR-amplified ribosomal DNA [the internal transcribed spacer (ITS) region and the 5.8 s ribosomal DNA]. The 5.8s rDNA is completely conserved, but both ITS regions show variation among strains. AG1 was an outgroup based on anastomosis ability and RFLP analyses. Phylogenetic analyses based on the ITS sequences suggest that the analyzed AG4 strains can be divided into three groups that correlate with habitat and virulence.