Restriction fragment length polymorphism for the Yc subunit gene of rat liver glutathione S-transferase

An altered expression of the Yc subunit gene of rat glutathione S-transferase (GST) in the liver of the LEC rat, which is a mutant strain with spontaneous hereditary hepatitis associated with severe jaundice, has been reported. To provide further information concerning the structure of the Yc subunit gene, we carried out the Southern blot hybridization analysis of DNA samples from rats of eight different inbred strains including LEC with cDNA complementary to mRNA specific for the Yc subunit of rat liver GST as a probe. The hybridization patterns of the DNA samples from rats belonging to the different inbred strains showed interstrain variation in the length of restriction fragments with four restriction endonucleases. Since the DNA samples prepared from several rats of one inbred strain gave an identical hybridization pattern, the restriction fragment patterns for the Yc gene could be used as markers for genetic monitoring of inbred rat strains. Although the altered expression of Yc-Yc activity of GST has been observed in the liver of the LEC rat, the characteristic changes in the gene structure of the Yc subunit of LEC rat were not detected in the present hybridization analysis.     

Restriction fragment length polymorphism and spacer oligonucleotide typing: A comparative analysis of fingerprinting strategies for Mycobacterium bovis

The combination of conventional investigation and DNA fingerprinting is yielding important insights into the epidemiology of Mycobacterium bovis infections. Various genetic markers used in restriction fragment length polymorphism (RFLP) have recently been exploited for fingerprinting of M. bovis isolates. The newly developed spacer oligonucleotide typing aimed to investigate the polymorphism of M. tuberculosis in the DR locus, has also been applied to the molecular typing of M. bovis isolates. This work compared the performance of the insertion sequence (IS) IS6110, IS1081 and the genetic elements polymorphic G+C-rich repeat (PGRS) and direct repeat (DR) used in RFLP analysis with spoligotyping using a group of 128 Spanish M. bovis isolates. In this study, the most sensitive technique for identifying polymorphism in M. bovis was PGRS–RFLP, closely followed by IS6110–RFLP. We propose several schemes for fingerprinting of these isolates, however, the clear geographical variations found by different authors makes the study of each local situation indispensable. An international consensus in the methods used would be desirable for efficient interlaboratory comparison of strains.     

Restriction fragment length patterns of DNA from parasitic nematodes of sheep

High molecular weight DNA obtained from sheep parasitic nematodes Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis, was digested with various restriction endonucleases. Digestion with Eco R1 produced the most informative pattern of repeat sequence bands. H contortus adult or larval DNA produced bands of 2.7, 3.0 and 1.4 kb. O circumcincta adult or larval DNA had common 2.7 and 1.4 kb bands with adult specific bands of 2.2 and 0.9 kb and a larval specific 2.08 kb band. T colubriformis adults or larval DNA produced 2.7, 1.4 and 0.79 kb bands. These preliminary results show that restriction patterns of repeat sequence DNA may be useful for the identification of various trichostrongylid species parasitic for sheep.     

猪链球菌荧光DNA扩增片段长度多态性检测方法的建立

利用荧光标记技术,采用2对引物初步建立检测猪链球菌荧光DNA扩增片段长度多态性方法,结果表明12株猪链球菌扩增的多态性位点数从51～98条不等,该方法能够检测猪链球菌的多态性,区分不同血清型以及同一血清型不同特性的菌株,可用于菌株鉴定及流行病学研究中细菌源的追踪。     

Testing of amplified fragment length polymorphism (AFLP) technique as a tool for molecular epidemiology of Trichinella nativa

A total of nine Trichinella nativa isolates were compared by amplified fragment length polymorphism (AFLP). Four hundred nanograms of genomic DNA from a pool of 10--20 larvae were digested using HindIII and MseI restriction endonucleases. Of the 16 primer combinations initially tested, Hind-C and Mse-C primers showed rich polymorphism with approximately 40--90 bands in the range of 30--270 bp. Genetic similarities were estimated visually. AFLP provided discriminatory banding patterns and may therefore be used as a method for detecting variation in T. nativa populations. However, the heterogeneous patterns obtained from pooled samples emphasize the need for further development of the sampling and numerical analysis of the patterns for epidemiological and taxonomical interpretation.     

吉戎兔微卫星DNA的研究

选用Sat2、Sat3、Sat4、Sat5、Sat7、Sat8、Sat12、Sat13、Sat16、Sol08、Sol28、Sol30、Sol03等13个微卫星位点PCR扩增60只吉戎兔的基因组DNA,然后在8%聚丙烯酰胺凝胶上电泳分型.检测结果,平均等位基因数为3.46个,平均杂合度(H)为0.578,平均多态信息含量(PIC)为0.531.结果表明,吉戎兔经多年选育,其品种的遗传结构稳定,品质均一,同时群体保持了较高的遗传多样性.     

猪TLR2基因Bi-PASA遗传标记的建立及多态性研究

根据人和小鼠TLR2基因序列设计了特异性PCR引物,优化PCR条件后扩增到中国梅山猪、欧洲约克夏猪、PIC-2系及PIC-3系商品猪的TLR2基因690 bp的基因片段,并对其进行序列分析。序列分析结果显示,猪TLR2基因多态性程度低,发现在猪TLR2基因编码区第1 255位点上存在1个单碱基突变位点。利用双向特定等位基因PCR扩增法(B i-PASA)建立了检测猪TLR2基因变异的遗传标记。利用猪TLR2基因的B i-PASA标记,分析了TLR2基因在大河乌猪、撒坝猪和黔邵花猪中的基因频率和多态性。研究建立的B i-PASA遗传标记和基因变异信息,将为进一步分析猪TLR2基因变异与疾病抵抗力及经济性状的相关分析提供基础资料。     

Genotypic change of porcine circovirus type 2 on Japanese pig farms as revealed by restriction fragment length polymorphism analysis

Porcine circovirus type 2 (PCV2) has been recognized as the causal agent of postweaning multisystemic wasting syndrome and can be divided into two major genotypic groups. We developed a method of restriction fragment length polymorphism (RFLP) analysis of PCV2 open reading frame 2 for easy discrimination between the two major groups. Genotyping of PCV2 isolates from 10 Japanese commercial pig farms was performed, and the analysis revealed that both PCV2 groups and at least five RFLP types of PCV2 are prevalent in Japan. On two farms, the genotypes of the PCV2 isolates in the spring of 2007 were different from those in the autumn of 2006. One genotype may have become dominant within only six months on these farms.     

Effect of ruminal protozoa on fecal microbiota in cattle using terminal restriction fragment length polymorphism

The effect of the presence of ruminal protozoa on the composition of fecal microbiota in cattle was investigated. Six castrated Holstein cattle (mean bodyweight 137 kg) were divided into two groups: three faunated and three unfaunated. The fecal bacterial composition of the faunated and unfaunated cattle was compared using a culture method and by terminal restriction fragment length polymorphism (T‐RFLP) analysis. Approximately 0.4 to 2.3% of the bacterial cells detected by microscopy formed colonies on medium 10. The major terminal restriction fragments were detected in the T‐RFLP profiles generated by Hha I and Msp I digestion in both the faunated and unfaunated cattle. In particular, the Bacteroides group, the Clostridium coccoides group and the Clostridium leptum subgroup might be the known bacterial groups that protozoa influence by Msp I digestion. From the dendrogram analysis by T‐RFLP patterns, the faunated and unfaunated cattle were divided into two clusters, I and II, respectively. These results suggest that absence of protozoa in the rumen changes the composition of fecal bacteria.     

Identification of the variant C of β-lactoglobulin in sheep using a polymerase chain reaction-restriction fragment length polymorphism method

β-Lactoglobulin (β-LG) is the major whey protein in the milk of ruminants and is able to bind and transport small hydrophobic molecules. However, its biological role is mainly unknown (G odovac -Z immermann 1988). Previously three genetic variants have been found in sheep: A, B and C. The genetic variants A and B differ at amino acid position 20, where variant A has a His and variant B has a Thr (K olde and B raunitzer 1983). The variant C is a subtype of variant A with a single amino acid exchange of Arg→Glu at position 148 (E rhardt et al. 1989). The genotype β-LG BB was found to be associated with higher milk yield, whereas genotypes AA and AB had a higher milk protein and casein content as well as yielding more curd (G arzon and M artinez 1992). No data is available concerning the relationship between the β-LG C allele and production traits or milk properties. Since DNA-based genotyping has already been performed for alleles A and B (S chlee et al. 1993), the aim of the present study was to develop a DNA-based method for identifying the β-LG C variant. However polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing has been established (P rinzenberg and E rhardt 1999) recently, and this study shows an alternative method to detect β-LG C.     

Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by sequencing and restriction fragment length polymorphism analysis of the pvpA gene

Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host-parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host-parasite relationship.     

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.     

An up-date on the differentiation of Brachyspira species from pigs with nox-PCR-based restriction fragment length polymorphism

Different Brachyspira (B.) species colonize the porcine intestinal tract, some of which are pathogens of significant clinical and economic importance. In 2002 we published a novel molecular method for differentiation of Brachyspira species from pigs based on the amplification of the nox-gene and the generation of species-specific restriction patterns (nox-RFLP) using the enzymes BfmI and DpnII (Rohde et al., 2002). We applied this method for identification in addition to biochemical testing in doubtful cases until 2008. Since 2009 we have used it as the first line method of identification. The current study documents the results of examining 2050 Brachyspira isolates collected from January 2009 to December 2011. In addition to identifying isolates with previously described patterns, four novel restriction fragment length patterns were observed, and isolates with these patterns could be assigned to the species B. intermedia and the B. innocens/murdochii complex on the basis of their phenotypic properties and by nox-sequence analysis. In 2007 a potentially new Brachyspira species, "B. suanatina", was described in Swedish pigs (R?sb?ck et al., 2007). From the published nox-gene sequence it could be expected that this Brachypira species should show a new restriction pattern making nox-RFLP a suitable technique for identification of "B. suanatina". In this study the new restriction fragment length pattern could be demonstrated in one of the strains described by R?sb?ck et al. (AN4859/03). Nevertheless, no isolates with this new pattern corresponding to "B. suanatina" were identified amongst the 2050 Brachyspira isolates examined from northern Germany.     

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.