用反转录—聚合酶链反应检测B组轮状病毒

轮状病毒（Ｒｏｔａｖｉｒｕｓ）是属于呼肠病毒科（Ｒｅｏｖｉｒｉｄａｅ）的双链ＲＮＡ（ｄｓＲＮＡ）病毒。至今已将轮状病毒分为七个组（Ａ～Ｇ）。已经发现的Ｂ组轮状病毒分别来自人、大鼠、牛、猪、羊。近十年来,通过轮状病毒的研究,轮状病毒Ｂ组已被公认为引起人...     

沙市婴幼儿腹泻中检出3株C群轮状病毒

1987～1988年在沙市165份婴幼儿急性腹泻标本中,用PAGE法检出轮状病毒37株(22.4％),其中3株为少见的轮状病毒,此种病毒经电镜观察,具有典型轮状病毒的形态结构,ELISA证实该病毒不具有A群和B群轮状病毒的群特异性抗原。RNA电泳分析表明,其基因组由11个双链RNA片段组成,电泳图型特殊,呈4:3:2:2的排列模式。上述试验表明,该病毒为世界上罕见的C群轮状病毒。免疫电镜证实,该病毒能被病人恢复期血清所凝集,提示该病毒是腹泻病儿的致病因子。     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

两种PCR方法对检测A组轮状病毒膜芯片杂交结果的影响

A组轮状病毒(rotavirus,RV)是导致拿世界婴幼儿腹泻的最主要病原,危害巨大。拟用RT-巢式PCR技术对A组RV的保守序列进行高度扩增,通过固本室内制的膜芯片杂交,实现对该病毒的检测。分别采用对称PCR和不对称PCR扩增,均可得到扩增的目的片段．对称式扩增产物杂交结果不理想。而不对称式扩增得到了大量待检单链产物,同膜芯片杂交获得了理想的杂交结果。显著地提高了对A组RV杂交检测的灵敏度。表明不对称式PCR扩增是一种制备用于芯片杂交大量单链产物的理想方法,尤其是针对富含AT的核酸检测区域。     

The Sequence of the Hartmannella vermiformis Small Subunit rRNA Coding Region

ABSTRACT The Hartmannella vermiformis small-subunit rRNA coding region was amplified, and the amplified DNA was cloned and sequenced. The coding region is 1,840 nucleotides long, and is typical of eukaryotic rRNA genes in both size and composition. Different clones contained different nucleotides at three positions.     

应用PCR技术检测病人血液标本中斑点热群立克次体DNA

本文以聚合酶链反应（PCR）,采用二次扩增法直接检测蜱传斑点热病人血液标本中斑点热群立克次体DNA,结果从7份标本中检出阳性1份,进一步的限制性片段多态性分析证明和斑点热群黑龙江立克次体基因型相同。实验证明：黑龙江立克次体对人具有致病性,同时也证明PCR技术快速、简单、敏感,用于斑点热群立克次体的早期诊断是可行的,并可作出分型鉴定。     

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed (for 2 h) to 2 × 10^5-7 cell culture infective dose (CCID₅₀)/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was ≤6.62 ± 1.57 copies/5 μL; 90% of the contaminated embryos had ≤4.64 ± 1.57 viral copies/5 μL of embryo-associated virus, using tolerance intervals (P < 0.05). The SEM was 0.33 and the mean of averages was 1.12/5 μL. Of the 87 in vitro-produced embryos, 42% were positive for virus. The range in amount of virus associated with 99% of the contaminated embryos was ≤3.44 ± 0.89 copies/5 μL; 90% of the contaminated embryos had ≤2.40 ± 0.89 viral copies/5 μL of embryo-associated virus using tolerance intervals (P < 0.05; S.E.M. was 0.14 and the mean of averages was 0.55/5 μL). Therefore, although many embryos were positive for virus, there were limited numbers of copies, thereby posing doubt regarding their potential for contamination following embryo transfer.     

Greater Amount of HCV-RNA in Tears Compared to Blood

Plasma, tear fluid and swabs from eye, nose and pharynx of 33 patients were examined for presence of hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR). All samples from plasma, tear fluid and eyeswabs were found to show a positive reaction in HCV-RNA PCR. Remarkably, we regularly found greater amounts of amplification products in tear fluid and eyeswabs compared to plasma using the same conditions for sample preparation.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

人头发DNA的提取及其基因扩增

头发是犯罪现场最容易得到的材料之一。本文报道从人的头发中提取DNA的方法以及利用PCR(聚合酶链反应)技术,从少量人发DNA扩增大量拷贝数的基因片段。对基因扩增得到的大量基因片段进行进一步的基因分析,将为法医物证学提供客观证据,具有重要价值。     

Simultaneous PCR amplification of two barley 5S rDNA sequence types in the same PCR reaction

Many researchers are currently using PCR technology to amplify individual members of multigene families, including 5S rDNA, for sequencing and related purposes. When members of the family differ in length, analyses would be facilitated if different units could be simultaneously and efficiently amplified. In the present paper we describe conditions that can be used to amplify simultaneously both the “long” and “short” 5S rDNA repeats found in barley (Hordeum vulgare L.).     

Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT‐PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42°C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT‐PCR is a valuable tool for optimization of the production of antimicrobial peptides.     

Development of A Rapid Method for the Diagnosis of Citrus Greening Disease using the Polymerase Chain Reaction

The fastidious bacterium causing citrus greening disease occurs in uneven and low concentrations in the sieve tubes of host plants. A rapid and sensitive assay based on the polymerase chain reaction (PCR) has been developed using the primers derived from the sequences of the cloned DNA fragment of greening fastidious bacterium (GFB) to detect GFB infection in citrus. One set of the primer pairs (named 226-primer pair), which generates a 226 bp GFB-specific fragment from total DNA templates purified from diseased citrus plants, was tested and chosen for PCR amplification. The PCR-based assay using this 226-primer pair effectively detected GFB infection in various citrus cultivars collected from different Asian countries. This detection technique, which can be completed within 6 h, offers a rapid and efficient method for accurate diagnosis of citrus greening disease.     

中国苗族人群中细胞色素P450 2C19基因多态性的研究

为研究细胞色素P45 0 2C19在中国苗族人群中的基因型 ,采用聚合酶链式反应与限制性内切核酸酶片段长度多态性技术分析了 98例无血缘关系苗族人群的基因型。结果显示 5 0人为CYP2C19野生型纯合子 (wt/wt) ;39人为CYP2C19m1杂合子 (wt/m1) ;9人为CYP2C19m1突变型纯合子 (m1/m1)。与国内外相关报道比较 ,本实验得出的CYP2C19m1突变频率与文献报道相符     

人B组轮状病毒vp7基因的克隆和序列分析

利用逆转录—聚合酶链反应(RT-PCR)技术,扩增了人B组轮状病毒WH—2vp7基因,连接到克隆载体pUCm—T,并对其基因序列进行了分析。WH—2与ADRV的同源性达98％,与印度加尔各达分离株CAL—1达92％,而与动物B组轮状病毒同源性差别较大,如与IDIR(鼠)同源性仅为58％,与WD653(牛)B组轮状病毒同源性为63％,与ATI(牛)同源性为61％。对vp7基因的二级结构分析发现其mRNA折叠形成多达18个发卡环状结构。VP7蛋白是249个氨基酸组成,分子：量为28．4kDa,含有3个潜在的N连接的糖基化位点和多个磷酰化位点,从氨基酸序列的同源性来看,WH—2与ADRV的同源性达99％,与CAL—1达95％,而IDIR仅为51％,说明了WH—2和ADRV的起源相同。此研究对了解B组轮状病毒基因的进化和变异规律具有重要意义,也将为B组轮状病毒预防性疫苗的研制提供科学的依据。     

Detection of Intra-Clonal Genetic Variability in Vegetatively Propagated Tea Using RAPD Markers

The role of random amplified polymorphic DNA (RAPD) markers in detecting intra-clonal genetic variability in vegetatively propagated UPASI-9 clone of tea (Camellia sinensis) was studied. Twenty five decamer primers were used, of which three did not amplify, three gave single bands and the rest of nineteen primers generated upto twelve bands (an average of 6.3 bands per primer). Twenty one primers exhibiting amplified products gave monomorphic banding patterns. Only one primer (OPE-17) gave a unique extra band of similar size in four plants.     

人α1/158Ⅴ型(α1c型)干扰素基因在大肠杆菌中的表达及其产物的初步纯化

采用DNA聚合酶链反应(PCR)技术,将本实验室从中国人胎肝细胞染色体DNA中发现和分离的IFN-α1/158V基因的原始克隆,改造成适于进行非融合蛋白原核表达的结构形式,并在大肠杆菌中获得高效表达。测得重组IFN-α1/158V的抗病毒活性为1.9×10~7单位/升菌液。随后又采用以单克隆抗体亲和层析为主的纯化流程对表达产物进行初步纯化,获得了在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带的纯化产物。