Gene Restriction Fragment Length Polymorphism Research Articles

Peritoneal Pigmentation in Purebred and Hybrid River Herring

AbstractRiver herring (Alewives Alosa pseudoharengus and Blueback Herring A. aestivalis [bluebacks]) are congeneric, anadromous clupeid fishes that hybridize in areas of sympatry. Peritoneal pigmentation is the most diagnostic characteristic used to distinguish the species, but pigmentation has not been examined in hybrids. We developed a molecular‐based assay to distinguish purebred river herring species from F1 hybrids and compared the peritoneal pigmentation among captive‐raised purebred and hybrid individuals. Both wild‐caught Alewife and blueback females tank‐spawned with conspecific and congeneric males, but Alewives required exogenous hormone administration. Larvae of both species and hybrids were raised for ∼290 d posthatch, and peritoneal pigmentation was quantified. A nuclear gene (rag2) restriction fragment length polymorphism assay was used for species and hybrid identification. The peritoneal pigmentation was significantly darker in hatchery‐spawned bluebacks than in Alewives, and hybrids exhibited consistent, intermediate expression. Adult individuals collected from the wild, however, exhibited darker (Alewives) or more variable (bluebacks) peritoneal coloration than their respective, captive‐reared juveniles. These results indicated that peritoneal pigmentation alone is insufficient for diagnostic river herring species identification and should be coupled with genetic assays.

Study on genotype and virulence of Cryptococcus neoformans and Cryptococcus gattii clinical isolates in Guigang, Guangxi Zhuang Autonomous Region

To understand the species, genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii isolated from clinical samples in Guigang, Guangxi Zhuang Autonomous Region. A total of 20 Cryptococcus strains were isolated from clinical samples in Guigang from 2009 to 2012. The biological identification was conducted by polymerase chain reaction (PCR) to amplify internal transcribed spacer (ITS) sequences. The serotypes and mating types of C. neoformans and C. gattii were identified by PCR with serotype-specific and mating type-specific primers. The genotype was characterized by PCR fingerprinting and URA5 gene restriction fragment length polymorphism (URA5-RFLP). Phenotype study included growth test at 37 °C, melanin production test and urease test. Among the 20 strains, 19 (95%) were identified as C. neoformans varieties (var.) grubii (serotype A, mating type α, genotype VN I), and only 1 was identified as C. gattii (mating type α, genotype VG I). The results of virulence test showed that all the strains grew well at 37 °C and positive in both urease test and melanin production test. C. neoformans var. grubii (serotype A, genotype VN I and mating type α) was the predominant pathogen causing cryptococcosis in Guigang, and C. gattii strain was also detected.

Genetic Characteristics of Actinomycetes Isolates (GST, KP, KP11, Kp16, T24, T37 Code) by RFLP of NRPS Genes

Actinomycetes is the one of antibiotic producing microorganisms. Actinomycetes isolates with GST, KP, KP11, KP16, T24, T37 have been isolated from soil. This study aims to determine the diversity of actinomycetes isolates by RFLP (Restriction Fragment Length Polymorphism) of NRPS (Non Ribosomal Peptide Synthetase) genes from GST, KP, KP11, KP16, T24, and T37 isolates. This study is divided into several steps of the DNA isolation from isolate GST, KP16, T24, T37, KP, KP11, PCR of the 16S rRNA and NRPS genes and cutting the NRPS genes fragmens with HaeIII enzyme to determine the diversity of isolates. Results of DNA isolate, PCR of 16S rRNA genes and NRPS genes, and RFLP of NRPS genes performed by agarose gel electrophoresis. The diversity of the isolates were analyzed by multivariate analysis UPGMA with MVSP 2.0 applications. The results showed the diversity of 6 actinomycetes isolates are T37 and GST have 64 % similarity, KP11 and KP16 have 96 % similarity, KP has 76 % similarity with KP11 and KP16, while T24 has 60 % similarity with KP and 12 % similarity with T37 and GST. Based on RFLP analysis of the NRPS genes, among of 6 isolates can divided into 4 groups are T37 and GST isolates as group 1, KP11 and KP16 isolates as group 2, KP isolate as group 3, and T24 isolate as group 4. Keyword : Actinomycetes, diversity, 16S rRNA genes, NRPS genes, RFLP

Phylogenetic study of Japanese Dickeya spp. and development of new rapid identification methods using PCR–RFLP

Forty-one representative Japanese Dickeya spp. (Erwinia chrysanthemi) strains isolated from 24 plants in Japan were investigated using multilocus sequence analysis of recA, dnaX, rpoD, gyrB and 16S rDNA; PCR–RFLP (restriction fragment length polymorphism) of recA, rpoD and gyrB genes; PCR genomic fingerprinting; and biochemical tests. Based on the recA, dnaX, rpoD, gyrB and 16S rDNA sequences and PCR genomic fingerprinting, the strains were essentially divided into six groups (I–VI). Group I corresponded to D. chrysanthemi, group II corresponded to D. dadantii, group III to D. dianthicola and group IV to D. zeae. Meanwhile, group V and group VI could not be assigned to any existing Dickeya species, and they were deduced to be two putative new species. The PCR–RFLP analysis of gyrB, rpoD and recA clearly differentiated the six groups of Dickeya strains. From the results of the biochemical tests, the strains were assigned to biovars 1, 3, 5, 8 and 9; only one strain (SUPP 2525) was not assignable to the existing biovars. We also showed that the PCR–RFLP analysis of rpoD, gyrB and recA can be used as a rapid technique to identify Japanese Dickeya strains.

Vitamin D receptor polymorphisms and cancer.

It was suggested that vitamin D levels influence cancer development. The vitamin D receptor (VDR) is a crucial mediator for the cellular effects of vitamin D. In fact It has been hypothesized that polymorphisms in the VDR gene affect cancer risk and the relevance of VDR gene restriction fragment length polymorphisms for various types of cancer has been investigated by a great number of studies. However, results from previous studies on the association of VDR polymorphisms with different cancer types are somewhat contradictory, and the role of VDR in the etiology of cancer is still equivocal. We have performed a systematic review of the literature to analyze the relevance of more VDR polymorphisms (Fok1, Bsm1, Taq1, Apa1, and Cdx2) for individual malignancies, including cancer of the skin (melanoma and nonmelanoma skin cancer), ovarian cancer, renal cell carcinoma, bladder cancer, non-Hodgkin lymphoma, leukemia, thyroid carcinoma, esophageal adenocarcinoma, hepatocellular carcinoma, sarcoma, head and neck and oral squamous cell carcinoma. Up to June 2012, we identified 79 independent studies for a total of 52427 cases and 62225 controls. Significant associations with VDR polymorphisms have been reported for prostate (Fok1, Bsm1, Taq1), breast (Fok1, Bsm1, Apa1), colon-rectum (Fok1, Bsm1, Taq1) and skin cancer (Fok1, Bsm1, Taq1). Very few studies reported risk estimates for the other cancer sites. Conflicting data have been reported for most malignancies and at present it is still not possible to make any definitive statements about the importance of the VDR genotype for cancer risk. It seems probable that interactions with other factors such as calcium and vitamin D intake, 25(OH)D plasma levels and UV radiation exposure play a decisive role in cancer risk. To conclude, there is some indication that VDR polymorphisms may modulate the risk of some cancer sites and in future studies VDR genetic variation should be integrated also with prediagnostic indicator of vitamin D status.

Aeromonas hydrophila and Aeromonas veronii Predominate among Potentially Pathogenic Ciprofloxacin- and Tetracycline-Resistant Aeromonas Isolates from Lake Erie

Members of the genus Aeromonas are ubiquitous in nature and have increasingly been implicated in numerous diseases of humans and other animal taxa. Although some species of aeromonads are human pathogens, their presence, density, and relative abundance are rarely considered in assessing water quality. The objectives of this study were to identify Aeromonas species within Lake Erie, determine their antibiotic resistance patterns, and assess their potential pathogenicity. Aeromonas strains were isolated from Lake Erie water by use of Aeromonas selective agar with and without tetracycline and ciprofloxacin. All isolates were analyzed for hemolytic ability and cytotoxicity against human epithelial cells and were identified to the species level by using 16S rRNA gene restriction fragment length polymorphisms and phylogenetic analysis based on gyrB gene sequences. A molecular virulence profile was identified for each isolate, using multiplex PCR analysis of six virulence genes. We demonstrated that Aeromonas comprised 16% of all culturable bacteria from Lake Erie. Among 119 Aeromonas isolates, six species were identified, though only two species (Aeromonas hydrophila and A. veronii) predominated among tetracycline- and ciprofloxacin-resistant isolates. Additionally, both of these species demonstrated pathogenic phenotypes in vitro. Virulence gene profiles demonstrated a high prevalence of aerolysin and serine protease genes among A. hydrophila and A. veronii isolates, a genetic profile which corresponded with pathogenic phenotypes. Together, our findings demonstrate increased antibiotic resistance among potentially pathogenic strains of aeromonads, illustrating an emerging potential health concern.

Indoleamine 2,3 dioxygenase gene polymorphisms correlate with CD8+ Treg impairment in systemic sclerosis

Systemic sclerosis (SSc) is characterized by tissue fibrosis, vasculopathy and autoimmunity. Indoleamine 2,3 dioxygenase (IDO) plays a pivotal role in immunological tolerance modulating regulatory T cell (Treg) generation and function. Single nucleotide polymorphisms (SNPs) of IDO gene could impact on Treg function and predispose to autoimmunity. Here, the existence of an association between specific IDO SNPs and SSc was analyzed. Five specific SNPs in IDO gene were searched in 31 SSc patients and 37 healthy controls by gene sequencing or restriction fragment length polymorphism. The function of both CD4+CD25+ and CD8+ Treg from SSc patients was analyzed by proliferation suppression assay. SNP rs7820268 was statistically more frequent in SSc patients than in controls. Notably, SSc patients bearing the T allelic variant of the rs7820268 SNP showed impaired CD8+ Treg function. Our unprecedented data show that a specific IDO gene SNP is associated with an autoimmune disease such as SSc.

Sweet potato little leaf strain V4 phytoplasma associated with snake bean in the Northern Territory, Australia

Sweet potato little leaf strain V4 phytoplasma (SPLL-V4) was found to be associated with 12-week old snake bean plants (Vigna unguiculata ssp. sesquipedalis) from a block with 890 plants in Lambells Lagoon, Northern Territory. Symptoms included witches’ broom, big bud, phyllody and little leaf. There was a 19.9 % incidence of symptoms on 366 plants assessed. The same phytoplasma was also detected from a single plant of Phyllanthus amarus with witches’ broom symptoms growing near the snake bean plants. The SPLL-V4 phytoplasma was identified based upon the 16S ribosomal RNA gene and intergenic spacer region nucleotide sequence and restriction fragment length polymorphism.

Molecular epidemiology and clinical manifestations of human cryptosporidiosis in Sweden

This study describes the epidemiology and symptoms in 271 cryptosporidiosis patients in Stockholm County, Sweden. Species/genotypes were determined by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) of the Cryptosporidium oocyst wall protein (COWP) and 18S rRNA genes. Species were C. parvum (n=111), C. hominis (n=65), C. meleagridis (n=11), C. felis (n=2), Cryptosporidium chipmunk genotype 1 (n=2), and a recently described species, C. viatorum (n=2). Analysis of the Gp60 gene revealed five C. hominis allele families (Ia, Ib, Id, Ie, If), and four C. parvum allele families (IIa, IIc, IId, IIe). Most C. parvum cases (51%) were infected in Sweden, as opposed to C. hominis cases (26%). Clinical manifestations differed slightly by species. Diarrhoea lasted longer in C. parvum cases compared to C. hominis and C. meleagridis cases. At follow-up 25-36 months after disease onset, 15% of the patients still reported intermittent diarrhoea. In four outbreaks and 13 family clusters, a single subtype was identified, indicating a common infection source, which emphasizes the value of genotyping for epidemiological investigations.

O3-S1.02 Deciphering the code of Treponema pallidum in the UK: implications for treatment and prevention

BackgroundThe efficacy and convenience of single-dose oral azithromycin meant it became the preferred treatment for syphilis in many countries and a suitable alternative to penicillin in many more. However, two...

벚나무 빗자루병균 Taphrina wiesneri의 유전적 특성

자낭균인 Taphrina wiesneri는 한국의 공원과 가로수에 주로 식재되는 왕벚나무에 빗자루병을 일으키는 병원균이다. 한국과 일본에서 분리한 13개의 병원균에 대해 18S rDNA 염기서열 분석을 통한 계통학적 분석과 rDNA-IGS 영역에 대한 RFLP 분석을 실시하였다. 18S rDNA 염기서열 분석을 통한 계통도 분석결과 병원균은 2그룹으로 분류되었다. Hha I 제한효소를 이용한 rDNA-IGS 영역에 대한 RFLP 분석결과 B, C, D, G 4개의 패턴으로 나타났으며, 그중 G 패턴은 새로운 패턴이었다. The ascomycetous fungus Taphrina wiesneri, the pathogen of cherry witches' broom, is highly pathogenic to Prunus yedoensis, the most widely planted cherry trees in Korea as park and roadside trees. A collection of 13 strains of the pathogen in Korea and Japan was characterized by 18S rDNA gene sequence and restriction fragment length polymorphism (RFLP) analysis. In cluster analysis based on 18S rDNA gene sequence the strains were divided into 2 clusters. In RFLP analysis of the rDNA-IGS region using HhaI, the strains were separated into four patterns, B, C, D and G, of which pattern G was new.

Association between GH encoding gene polymorphism and semen characteristics in Iranian Holstein bulls

The objective of this present study was to investigate relationships between the growth hormone gene restriction fragment length polymorphism (RFLP) and bull sperm characteristics. A total of 89 bulls from two semen evaluation stations were genotyped for the bovine growth hormone (bGH)-AluI polymorphism by polymerase chain reaction and followed by 4% metaphor agaros gel electrophoresis. The overall allele frequencies for two L and V alleles were 0.50, respectively. The relationship between the bGH-AluI polymorphism and semen characteristics was evaluated according 1500 ejaculated records. Five sperm characteristics were analyzed. Sperm characteristics showed significant variability in relation to bGH genotypes. LL bulls had a lower ejaculated volume and higher percentage of live sperm, and VV bulls had higher fresh sperm concentration and minimum effect after cryopreservation. This polymorphism could be further used for semen evaluation process in artificial insemination program in Iranian Holstein bulls. Key word : Semen characteristics, growth hormone (GH) polymorphism, Holstein bulls.

Association between GH encoding gene polymorphism and semen characteristics in Iranian Holstein bulls

The objective of this present study was to investigate relationships between the growth hormone gene restriction fragment length polymorphism (RFLP) and bull sperm characteristics. A total of 89 bulls from two semen evaluation stations were genotyped for the bovine growth hormone (bGH)-AluI polymorphism by polymerase chain reaction and followed by 4% metaphor agaros gel electrophoresis. The overall allele frequencies for two L and V alleles were 0.50, respectively. The relationship between the bGH-AluI polymorphism and semen characteristics was evaluated according 1500 ejaculated records. Five sperm characteristics were analyzed. Sperm characteristics showed significant variability in relation to bGH genotypes. LL bulls had a lower ejaculated volume and higher percentage of live sperm, and VV bulls had higher fresh sperm concentration and minimum effect after cryopreservation. This polymorphism could be further used for semen evaluation process in artificial insemination program in Iranian Holstein bulls. Key word : Semen characteristics, growth hormone (GH) polymorphism, Holstein bulls.

Identification of causative agents and species in shrimp implicated in a food poisoning case in Taiwan.

The possible causative agent and shrimp species involved in a bait shrimp poisoning case that occurred in northern Taiwan was determined. Because the patient's symptoms were similar to those caused by boric acid and slightly similar to those caused by sulfite, the concentrations of boric acid and sulfite (as sulfur dioxide) in the patient's vomitus and in shrimp collected from bait stores and markets were analyzed. The concentration of boric acid was 36.8 to 37.1 mg/g in the patient's vomitus, 1.4 to 3.8 mg/g in shrimp meats obtained from bait stores, and not detectable (less than 0.001 mg/g) in shrimp meat obtained from commercial markets. No significant differences in sulfur dioxide concentrations (0.067 to 0.088 mg/g) were found in patient's vomitus and the shrimp meat from both bait stores and commercial markets. A fragment of the cytochrome b gene (∼406 bp) was amplified by PCR using a pair of primers (UCYTB151F and UCYTB270R) from shrimp meat of two species and the vomitus. The vomited shrimp was identified as Parapenaeus fissuroides on the basis of gene sequencing and restriction fragment length polymorphism patterns after treatment with endonuclease Alu I. Based on the patient's symptoms and analytical data, we concluded that boric acid at toxic levels had been illegally added to the bait shrimp P. fissuroides.

Study of fecal bacterial diversity in Yunnan snub-nosed monkey (Rhinopithecus bieti) using phylogenetic analysis of cloned 16S rRNA gene sequences

The bacterial diversity in fecal samples from Yunnan snub-nosed monkey ( Rhinopithecus bieti ) was investigated by constructed 16S rRNA gene clone library and restriction fragment length polymorphism analysis. As a result, a total of 156 representative clones for each profile, comprising nearly full length sequences (with a mean length of 1.5 kb) were sequenced and submitted to an on-line similarity search and neighbor-joining phylogenetic analysis. Using the criterion of 97%, these 16S rRNA gene sequences were binned in 129 OTUs. 11 sequences whose similarity is 97% were affiliated to the cultured bacteria and accounted for 7.05% of the total clones. For 23 sequences (14.74%), the similarity with the database was in the range of 89 - 97%. The remaining 122 sequences (78.21%) were uncultured and unidentified bacteria. Based on the phylogenetic analysis, the fecal bacteria of R. bieti distributed mainly in 6 bacteriophyta of Firmicutes, Proteobacteria, Bacteroidetes, Fibrobacteres, Spirochaetes and Actinobacteria , and belonged to 17 genera. Besides, there were a large number of uncultured and unidentified bacteria. These results illustrate the fecal bacteria diversity of R. bieti. Key Words : Rhinopithecus bieti, fecal bacterial diversity, 16S rRNA gene, phylogenetic analysis.

Characterization of macrolide resistance in Mycoplasma pneumoniae isolated from children in Shanghai, China

One hundred Mycoplasma pneumoniae strains were isolated from pediatric patients from March 2008 to July 2009. Of 100 isolates, 90 (90%) were resistant to erythromycin (MICs >128 μg/mL for 88 strains and 64 μg/mL for 2 strains), azithromycin, and clarithromycin. Fluoroquinolones and tetracyclines maintain good activities against clinical M. pneumoniae isolates. Of 90 macrolide-resistant M. pneumoniae strains, 88 (98%) harbored an A-to-G transition mutation at position 2063 in 23S rRNA genes, and the remaining 2 showed either A2064G or A2063T mutation; the latter point mutation is newly discovered and reported. Ninety-three (93%) clinical isolates were classified into the P1 gene restriction fragment length polymorphism (RFLP) type I, and 7 (7%) were type II.

Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes, mating types, molecular types, virulence factors, and antifungal susceptibility

This study investigated the microbiological characteristics of 100 clinical isolates of Cryptococcus neoformans species complex, including serotypes, mating types, molecular types, antifungal susceptibility and virulence. The isolates were collected at National Taiwan University Hospital from 1999 to 2004. Eight isolates of C. neoformans from pigeon droppings were also evaluated. Among these isolates, 99 were C. neoformans var. grubii serotype A and one was C. neoformans var. gattii serotype B. All of these isolates were α mating types. PCR fingerprinting, generated by primers M13 and (GACA)4, and URA5 gene restriction fragment length polymorphism analysis revealed that C. neoformans var. grubii isolates belonged to the VNI (98 isolates) and the VNII (one isolate) types, and the single C. neoformans var. gattii was VGI type. The similar profiles of clinical and environmental isolates suggest that patients might acquire these yeasts from the environment. The MIC90 for fluconazole, itraconazole, 5-flucytosine, voriconazole and amphotericin B against all C. neoformans isolates were 8, 0.5, 4, 0.125 and 0.5 mg/L, respectively. All clinical isolates produced urease, phospholipase, capsule and melanin, but these activities varied with individual isolates. Analysis of six clinical and two environmental isolates with various levels of phospholipase activity indicated a correlation between phospholipase activity and the ability to adhere to the lung epithelial cell line, A549. The extent of cell damage, as indicated by lactate dehydrogenase release, also paralleled the phospholipase activity of these isolates. In addition, production of melanin contributed significant protection against amphotericin B killing of the isolates tested.

Antimicrobial Susceptibility of Mycoplasma pneumoniae Isolates and Molecular Analysis of Macrolide-Resistant Strains from Shanghai, China

Fifty-three Mycoplasma pneumoniae strains were isolated from pediatric patients in Shanghai, China, from October 2005 to February 2008. Of 53 clinical isolates, 44 (83%) were resistant to erythromycin (MICs of >128 microg/ml for all 44 strains), azithromycin, and clarithromycin. All macrolide-resistant M. pneumoniae strains harbored an A-to-G transition mutation at position 2063 in 23S rRNA genes. Forty-five (85%) clinical isolates were classified into the P1 gene restriction fragment length polymorphism type I, and six (11%) were type II.

Molecular typing of Staphylococcus aureus isolated from bovine mastitis based on polymorphism of the coagulase gene in the north west of Iran

The objective was to investigate the genotypic characteristics and distribution of Staphylococcus aureus in 9 dairy herds of Tabriz and Urmia regions which are located in east and west Azerbaijan provinces, respectively, Iran. In this study 58 S. aureus isolates were recovered from 370 milk samples of cows with clinical and subclinical mastitis. S. aureus isolates were identified on the basis of the cultural and biochemical properties as well as by amplification of the aroA gene specific to S. aureus and then were analyzed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the coagulase gene ( coa). Amplification of the variable region of the coa gene from these isolates produced five different PCR products ranging in size from approximately 490 bp to 850 bp. To obtain RFLP patterns of the PCR products they were subjected to digestion with restriction endonuclease HaeIII and the fragments separated by gel electrophoresis. Nine coa gene RFLP patterns, numbered I–IX, were observed, with 23 isolates (39.66%) assigned to RFLP pattern I and 14 isolates (24.14%) assigned to RFLP pattern III. Five out of nine patterns were found in both regions and four of nine patterns were only found in one region. The results demonstrated that several variants of the coa gene are present in the studied regions, but only a few of them were predominant, suggesting contagious transmission, a common source, or host adaptation of subset of the population of S. aureus strains. This study also indicated that genetic heterogeneity among S. aureus isolates recovered from bovine mastitis may be exists within and among herds in different regions.

Novel Diagnostic Algorithm Using tuf Gene Amplification and Restriction Fragment Length Polymorphism is Promising Tool for Identification of Fragment Length Polymorphism is Promising Tool for Identification of Fragment Length Polymorphism is Promising Tool for Identification of Nontuberculous Mycobacteria

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84- 58) and M. chelonae (477-84-80-58), and M. kansasii (141- 136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level.