RESTRICTION FRAGMENT LENGTH POLYMORPHISM（RFLP） ANALYSIS OF GENOMIC DNA OF 5 STRAINS OF TRICHINELLA SPIRALIS IN CHINA

Five restriction endonucleases were used to digest genomic DNA from 5 isolates of Trichinella spiralis obtained from Changchun, Tianjin, Xian, Henan and Yunnan. All the isolates were secured from pigs except the Changchun strain which came from dog. The DNA fragments digested by endonuclease were separated by agarose gel electrophoresis. The Changchun isolate had a EcoRI band at 1. 12kb and a DraI band at 1. 97kb which were unique to this isolate. A cloned specific repetitive DNA sequence (1. 12kb) from the Changchun strain was selected to prepare a probe for the Southern blotting of EcoRI restriction DNA fragments for the 5 isolates. The 1.12kb hybridizing band did not appear except in the Changchun isolate.These results seem to indicate that there are differences between the isolates obtained from hosts in different geographical regions.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Antibiotic-Resistant Bacterial Group in Field Soil Evaluated by a Newly Developed Method Based on Restriction Fragment Length Polymorphism Analysis

Spreading of antibiotic resistant bacteria into environment is becoming a major public health problem, implicating affair of the indirect transmission of antibiotic resistant bacteria to human through drinking water, or vegetables, or daily products. Until now, the risk of nosocomial infection of antibiotic resistant bacteria has mainly been evaluated using clinical isolates by phenotypic method. To evaluate a risk of community-acquired infection of antibiotic resistant bacteria, a new method has been developed based on PCR-RFLP without isolation. By comparing restriction fragment lengths of the 16S rDNA gene from bacterial mixture grown under antibiotic treatment to those simulated from the DNA sequence, bacterial taxonomies were elucidated using the method of Okuda and Watanabe [1] [2]. In this study, taxonomies of polymyxin B resistant bacteria group in field soils, paddy field with organic manure and upland field without organic manure were estimated without isolation. In the both field soils, the major bacteria grown under the antibiotic were B. cereus group, which had natural resistance to this antibiotic. In field applied with organic manure, Prevotella spp., and the other Cytophagales, which were suggested to be of feces origin and to acquire resistance to the antibiotic, were detected. When numbers of each bacterial group were roughly estimated by the most probable number method, B. cereus group was enumerated to be 3.30 × 106 MPN/g dry soil in paddy field soil and 1.32 × 106 MPN/g dry soil in upland filed. Prevotella spp. and the other Cytophagales in paddy field were enumerated to be 1.31 × 106 MPN, and 1.07 × 106 MPN·g-1 dry soil.

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS ASSOCIATED WITH FACTOR Ⅷ CARRIER DETECTION USING DNA RFLPs IN CHINESE

RFLPs for XbaI, BelI and BglI sites of human FⅧwere informative for 48%, 41% and 15% of females studied, respectively. BglI RFLP is different from that reported by Chan et al, a fact suggests Yangtze River region population of China would be at variance with the Southern Chinese population in certain RFLP distribution. TaqI allelic system Ⅰin the DXS52 region also shows the same variance among them, but heterozygous rate 0f 71% for system Ⅰ(alleles 1 to 8) and 49% for system Ⅱ(αand βalleles) were very similar. Using the Bell/XbaI RFLPs, accurate information could be obtained from this study for 56% of women who were at risk for hemophilia A (HA) carriership. The carrier of the remaining 44% could be determined by utilizing the TaqI RFLP. In addition, we report a new intergenie polymorphism (9%) at DXS115 as a marker for detection of heterozygotes in families at risk for HA. The advantage of using the XbaI/KpnI RFLP is that both the intragemie RFLP and the new intergenie RFLP can be evaluated on the same blot at the same time.

Bacterial Groups Concerned with Maturing Process in Manure Production Analyzed by a Method Based on Restriction Fragment Length Polymorphism Analysis

Composting is a biological aerobic decomposition process consisted from different phases. Although the Japanese Standards for manure recommended that it took at least 6 months to complete the maturing phase, there was no reliable ground. In order to find out shortening method of the maturing phase, the microorganisms concerned with a progress of the maturing was determined by using the most probable number method (MPN) and PCR-RFLP of the 16S rDNA, which was found effective to provide numbers and taxonomy of polymyxin B resistant bacterial groups in the former paper [1]. Compared to the numbers after thermophilic phase, those of Actinobacteria, δ-proteobacteria, and the other gram negative bacteria increased to 50 times, 20 times, and 105 times, respectively, after maturing phase, while those of Bacillus spp., and α and β-proteobacteria decreased to 1/10, and 1/105 after maturing phase. Numbers of the other Fumicutes, and γ-proteobacteria remained in the same revel. Actinobacteria, δ-proteobacteria, and the other gram negative bacteria might be concerned with a progress of the maturing phase, because these bacterial groups were detected and enumerated due to their proliferation ability. Although number of Acitinobacteria might be underestimated because of a PCR bias, the method was found effective for the purpose to monitor bacteria actively proliferated in culture medium.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g-1) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.

Diversity of Microflora in Colonic Mucus from Severe Ulcerative Colitis Patients Analyzed by Terminal Restriction Fragment Length Polymorphism and Clone Libraries of Bacterial 16S rRNA Gene Sequences

Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.

Comparing the frequencies of restriction fragment length polymorphisms for dystrophin gene in Chinese with those from Japanese and Caucasian populations

The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuII/1a, Taq I/2b-3, Taq I/5b-7, and Xba I/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuII/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq I/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq I/2b-3 and Xba I/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq I/8 and A2 (10.7kb) in EcoR V/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq I/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1a, there was no significant difference between Chinese and Japanese.

The Use of Restriction Fragment Length Polymorphism and Fluorescence in Situ Hybridization to Investigate Microbiota of Piglets after Feeding Oregano

A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93.9 mg carvacrol/kg DM. After the experimental period of 5 weeks, 20 piglets of both extreme feeding groups were slaughtered: 10 animals of the control group and 10 animals of the group that received 8 g oregano/kg. Ingesta samples of jejunum, caecum and colon were collected and analyzed by FISH and PCR RFLP to compare the diversity of microbiota. The results showed no significant changes in microbiota in response to oregano. The patterns of the PCR-RFLP showed a similarity of 61.8% - 91.8% in both feeding groups. In conclusion, an effect of oregano on the in- testinal microbiota could not be shown under the methods used.

鸡金银羽位点基因型PCR-RFLP鉴定方法的建立及其应用研究

为了建立一个快速、简便、有效鉴定鸡金银羽位点基因型的方法,试验通过对金银羽等位基因位点核苷酸序列进行分析,设计合成一对引物,应用该对引物对海兰褐、伊莎褐和大午金凤等采用金银羽羽色自别雌雄的蛋鸡配套系商品代公鸡(金银羽等位基因杂合,Ss)和母鸡(金羽等位基因半合子,s-)进行了PCR-RFLP基因型鉴定分析。结果显示,PCR-RFLP方法的鉴定结果与实际基因型一致;采用该方法从960只白羽太行鸡母鸡中鉴定得到3只不携带显性白羽等位基因的银羽母鸡(银羽等位基因半合子,S-);以海兰褐父母代父本公鸡(金羽等位基因纯合,ss)作为父本与用本方法鉴定得到的3只银羽太行鸡母鸡进行杂交,来模拟金银羽自别雌雄的海兰褐蛋鸡商品代鸡的生产,获得的后代雏鸡绒羽颜色表型和性别符合海兰褐商品代鸡金银羽羽色自别雌雄的规律。上述结果表明,建立的PCR-RFLP方法能够快速、简便、有效且低成本地鉴定鸡金银羽位点基因型,为银羽纯合品系的建立提供了一个有效的个体金银羽位点基因型鉴定的方法。

RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIALDNA FROM HUMAN LUNG ADENOCARCINOMA CELLLINE SPC-A-1

Objective: To understand the role of mitochondrial DNA (mtDNA) in carcinogenesis. Methods: single-step method was used to isolate the mtDNA from human lung adenocarcinoma cell line SPC-A-1. The mtDNA was analyzed by restriction fragment length polymorphism (RFLP) with 11 kinds of restriction endonuclease, which were Pvu II, Xho I, Pst I, EcoR I, BstE II, Hind III, Hpa I, Bc1 I, EcoR V, Sca I and Xba I. Restriction map of mtDNA from SPC-A-1 cell was obtained by the single and double-digestion method. Results: It was found that no variation at 32 restriction-sites could be detected in the coding region of mtDNA from SPC-A-1 cell line. But a new site was found at nucleotide 16276 (EcoR V) within the noncoding region. Conclusion: These results indicate that the primary structure of gene coding region of mtDNA isolated from SPC-A-1 cell is highly stable. While the major variation of nucleotide is probably located in the noncoding region.

Analysis of Rb gene Xba Ⅰ polymorphism in Shaanxi aged atherosclerosis population

Objective:To investigate variable number tandem repeat (VNTR) polymorphism of the 17th intron of Rb gene in Shaanxi aged population and the relationship between the polymorphism of Rb gene and atherosclerosis(AS) genetic suscepti- bility. Methods: VNTR polymorphism of the 17th intron of Rb gene were examined in 100 Shaanxi aged AS patients and 100 Shaanxi aged control individuals by PCR-Rb-Xba Ⅰ-RFLP. Results::Two alleles were found both in AS group and control group, which were separately 945 bp(S1) and 630bp + 315bp(S2). S1S2 genotype was the most frequent one in the two populations. Significant difference in allele frequency was not found between AS group and control group, and allele frequency was no significant difference between Chinese and Caucasian. Conclusion: Xba Ⅰ enzyme site of Rb gene could have been certainly stable in AS population, and it was inferred that the polymorphism locus was not liable to cause mutation, which might not implicated in the formation of AS.

RFLP Analysis of Nov Gene in Tumor and Normal Cells

Using human nov (nephroblastoma overexpressed,nov) DNA as probe,hybridization to total cellular DNAs of tumor and normal cells digested by restriction enzymes BamHI or EcoRI respectively was carried out through Southern blot. It was observed that nov gene in these cells is not only highly conserved, but also certains RFLP characteristic . The correlation between RFLP characteristic of nov gene and its function was analyzed .

TLR4基因多态性与小儿反复呼吸道感染的关系

目的探究Toll样受体4(TLR4)基因多态性与小儿反复呼吸道感染(RRTI)的关系。方法选择107例RRTI病儿作为观察组,另选103例体检健康儿童作为对照组(近1年未出现RRTI),采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)技术分析两组TLR4基因rs2737190位点多态性,比较不同病情严重程度病儿TLR4基因多态性的差异,并分析不同TLR4基因多态性病儿临床特征的差异。结果观察组TLR4基因rs2737190位点AG基因型、G等位基因频率均明显高于对照组,AA基因型频率明显低于对照组(χ^(2)=4.129~8.564,P<0.05);中重度RRTI病儿GG基因型、G等位基因频率明显高于轻度病儿,AA基因型频率显著低于轻度病儿(χ^(2)=9.233~13.357,P<0.05);AA基因型病儿年发作次数、合并呼吸困难比例,以及C反应蛋白、白细胞介素-6、γ-干扰素和丙二醛等指标均显著低于AG和GG基因型病儿,超氧化物歧化酶则显著高于AG和GG基因型病儿(χ^(2)=7.573,F=4.668~24.317,P<0.05)。结论TLR4基因rs2737190位点AG基因型、G等位基因频率会增加小儿RRTI易感性,并促进其炎症反应升高和病情进展。

Analysis of polymorphisms in the promoter region of interleukin-1 β by restriction fragment length polymorphism-PCR

Objective: To investigate the frequencies of -1470, (-511) and -31 single nucleotide polymorphisms (SNPs) in the promoter of IL-1β and its haplotype constitution in Chongqing population. Methods: One hundred and twelve healthy Chongqing people were enrolled in this study. Polymorphisms at -1470 (G to C), (-511) (T to C) and -31 (C to T) of IL-1β were genotyped with the method of restriction fragment length polymorphism (RFLP). Haplotype frequencies were analyzed by Arlequine software.Results: Frequencies of IL-1β -1470, -511 and -31 SNPs were (41.67)%, 50% and (45.33)%, respectively. Genotype frequencies of -1470 locus were (39.81)%, (37.04)% and (23.15)% for G/G, G/C and C/C respectively. As for T-511C SNP, genotype frequencies of (T/T), T/C and C/C were (29.91)%, (40.18)% and (29.91)%, respectively. Genotyping results of C/C, C/T, and T/T of -31 locus were (35.51)%, (38.32)% and (26.71)% respectively. Haplotype analysis found that there were mainly three haplotypes constituted by three SNPs, ie., G-T-C, C-T-C and G-C-T. Conclusions: Polymorphisms exist in the promoter of IL-1β in Chongqing population. Three SNPs locate in the same haplotype block.

利用RFLP和SSR标记划分玉米自交系杂种优势群的研究

利用 RFL P和 SSR标记对 2 9个玉米自交系进行杂种优势群划分 ,筛选出 56个多态性 RFL P探针酶组合 ,6 6对多态性 SSR引物 ,分别在供试材料中检测到 187个和 2 32个等位基因变异。两种方法比较表明 ,SSR标记的平均多态性信息量 (PIC,0 .54)高于 RFLP(0 .4 2 ) ;但对供试材料的遗传多样性评价基本一致 ,平均遗传相似系数 (GS)分别为 0 .6 4和 0 .6 2。综合 RFL P和 SSR分析结果进行聚类分析 ,将供试材料划分为四平头 ,旅大红骨 ,L SC,BSSS和 PA五个类群 ,划分结果与系谱分析基本一致 ,并把系谱来源不清的种质划分到相应的杂种优势群。其中 PN群的确认 ,进一步完善了我国玉米种质杂种优势群的基本框架 ,为育种实践提供了有价值的信息。

PCR-RFLP鉴定隐孢子虫种类研究

为快速、准确鉴别人畜隐孢子虫种类,建立了巢式PCR扩增隐孢子虫18SrRNA基因的特殊区域,扩增片段测序结果表明:安徽牛源分离株(Cryptosporidiummuris)781bp;北京鸡源分离株(C.baileyi)776bp;北京牛源分离株(C.muris)781bp;河南牛源分离株(C.muris)725bp;长春牛源分离株(C.muris)776bp;宁夏鸡源分离株(C.baileyi)725bp,该片段位于18SrRNA全序列271～1103bp之间。使用Ssp 限制性内切酶消化发现C.muris产生418～420bp和305～363bp两个片段,C.baileyi产生544～545bp和185～231bp两个片段。所检测的6个分离株可以显著区分为C.muris和C.baileyi两个种,所建立的PCR-RFLP可以有效鉴别隐孢子虫种类。

水稻籼粳特异性RFLP标记及广亲和品种亲缘关系分析

应用１６０个ＲＦＬＰ标记分析了２１个水稻广亲和品种和６个籼粳测验种．发现其中６８个标记对以区分籼粳测验种。２１个广亲和品种根据与籼粳测验种共有片段比率的大小可以分为籼、粳和籼粳中间型三类。６８个标记在１５个籼粳品种中进一步筛选，得到２４个籼粳特异性ＲＦＬＰ标记，它们在亚种内杂文带型相同而亚种间则不一样。其中ＲＧ３５８，Ｇ３１８为籼稻专一性探针，在粳稻中发现为零等位。以此２４个探针为基础构建了广亲和品种亲缘关系的树状图。讨论了籼粳特异性ＲＦＬＰ标记在水稻遗传育种实践中的应用。

T-RFLP技术分析油藏微生物多样性

应用T-RFLP(末端限制性片段长度多态性)技术分析和比较了胜利油田单12区块的一口注水井(S12-zhu)和三口采油井(S12-4、S12-5和S12-19)的油藏微生物多样性。基于T-RFLP图谱的多样性指数表明注入水样品具有更丰富的细菌和古菌多样性。相似性指数表明,样品间细菌群落结构的相似性介于22.4%～30.8%之间,古菌群落结构的相似性介于20.8%～34.5%之间。查询RDP数据库推测这4个油藏样品所共有的优势微生物可能为Pseudomonas属,Marinobacter属和产甲烷微生物。T-RFLP技术能方便快捷的分析微生物多样性,其在油藏微生物多样性研究上的应用可以为MEOR提供有用的信息。