Association of VDR gene polymorphisms with risk of relapsing-remitting multiple sclerosis in an Iranian Kurdish population.

PubMed

Abdollahzadeh, Rasoul; Moradi Pordanjani, Parisa; Rahmani, Farideh; Mashayekhi, Fatemeh; Azarnezhad, Asaad; Mansoori, Yaser

2018-06-01

The purpose of this study was to evaluate the association of VDR Apa-I, Bsm-I, Fok-I, Taq-I single nucleotide polymorphisms (SNPs) with multiple sclerosis (MS) risk in an Iranian Kurdish population. A population including of 118 patients and 124 healthy matched controls were recruited to the study. Genotyping of the SNPs was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The frequency of allele T of Fok-I (P = 0.003) and allele C of Taq-I (P = 0.0003) was significantly different between case and control subjects and showed significant association with risk of MS (OR = 1.84, 95% CI = 1.23-2.76; OR = 1.98, 95% CI = 1.36-2.87, respectively). CT genotype (OR = 1.7, 95% CI = 1.05-2.99) of Fok-I and CC genotype (OR = 2.18, 95% CI = 1.05-4.52) of Taq-I showed a predisposing effect. Combined TT+TC vs. CC for Fok-I (OR = 2.15, 95% = CI 1.29-3.60) and combined CC+TC vs. TT for Taq-I (OR = 2.58, 95% CI 1.51-4.40) were susceptibility genotypes for MS. Apa-I and Bsm-I were not significantly associated with risk of MS (OR < 1, P > 0.05) and any genotypes in any genetic models were not significantly different between cases and controls (P > 0.05). As a result, Fok-I and Taq-I showed significant association with risk of MS, while Apa-I and Bsm-I were not observed to be related to the risk of the disease in this population.

Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide.

PubMed

Rachman, C N; Kabadjova, P; Prévost, H; Dousset, X

2003-01-01

The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.

Vitamin D receptor gene methylation is associated with ethnicity, tuberculosis and TaqI polymorphism

PubMed Central

Andraos, Charlene; Koorsen, Gerrit; Knight, Julian C; Bornman, Liza

2014-01-01

The Vitamin D Receptor (VDR) gene encodes a transcription factor which, on activation by vitamin D, modulates diverse biological processes including calcium homeostasis and immune function. Genetic variation involving VDR shows striking differences in allele frequency between populations and has been associated with disease susceptibility including tuberculosis and autoimmunity, although results have often been conflicting. We hypothesized that methylation of VDR may be population specific and that the combination of differential methylation and genetic variation may characterise TB predisposition. We use bisulphite conversion and/or pyrosequencing to analyse the methylation status of 17 CpGs of VDR and to genotype 7 SNPs in the 3′ CpG Island (CGI 1060), including the commonly studied SNPs ApaI (rs7975232) and TaqI (rs731236). We show that for lymphoblastoid cell lines from two ethnically diverse populations (Yoruba from HapMap, n=30 and Caucasians, n=30) together with TB cases (n=32) and controls (n=29) from the Venda population of South Africa there are methylation variable positions (MVPs) in the 3′ end that significantly distinguish ethnicity (9/17 CpGs) and TB status (3/17 CpGs). Moreover methylation status shows complex association with TaqI genotype highlighting the need to consider both genetic and epigenetic variants in genetic studies of VDR association with disease. PMID:21168462

Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment.

PubMed

Waleron, Małgorzata; Waleron, Krzysztof; Podhajska, Anna J; Lojkowska, Ewa

2002-02-01

Genotypic characterization, based on the analysis of restriction fragment length polymorphism of the recA gene fragment PCR product (recA PCR-RFLP), was performed on members of the former Erwinia genus. PCR primers deduced from published recA gene sequences of Erwinia carotovora allowed the amplification of an approximately 730 bp DNA fragment from each of the 19 Erwinia species tested. Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for most of the Erwinia species. Between one and three specific RFLP groups were identified among most of the species tested (Erwinia amylovora, Erwinia ananas, Erwinia cacticida, Erwinia cypripedii, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia persicina, Erwinia psidii, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera and Erwinia carotovora subsp. wasabiae). However, in two cases, Erwinia chrysanthemi and Erwinia carotovora subsp. carotovora, 15 and 18 specific RFLP groups were detected, respectively. The variability of genetic patterns within these bacteria could be explained in terms of their geographic origin and/or wide host-range. The results indicated that PCR-RFLP analysis of the recA gene fragment is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. carotovora subsp. carotovora and E. chrysanthemi.

Frequencies of VNTR and RFLP polymorphisms associated with factor VIII gene in Singapore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fong, I.; Lai, P.S.; Ouah, T.C.

1994-09-01

The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1more » = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.« less

An Examination of the Behavioral and Neuropsychological Correlates of Three ADHD Candidate Gene Polymorphisms (DRD4 7+, DBH TaqI A2, and DAT1 40bp VNTR) in Hyperactive and Normal Children Followed to Adulthood

PubMed Central

Barkley, Russell A.; Smith, Karen M.; Fischer, Mariellen; Navia, Bradford

2008-01-01

Several candidate gene polymorphisms have been implicated in attention deficit hyperactivity disorder (ADHD), including DAT1 40bp VNTR, DRD4 7+, and DBH TaqI A2 alleles. We used the Milwaukee longitudinal study of hyperactive (N=122) and normal (N=67) children to compare participants with and without these respective polymorphisms on ADHD-related behavioral ratings at childhood, 8 years later in adolescence, and 13+ years later into young adulthood. Neuropsychological tests were given at the adolescent and young adulthood follow-up. No differences were found between the DRD4-7+ and 7- repeat polymorphism. The DBH TaqI A2 allele, when homozygous, was associated with being more hyperactive in childhood, having more pervasive behavior problems at adolescence, and earning less money on a Card Playing Task in adulthood. At adolescence, poorer test scores were also found only in the hyperactive group with homozygous for this allele. The DAT1 40bp VNTR heterozygous 9/10 repeat, however, differed from the 10/10 repeat pair in many respects, having greater ADHD and externalizing symptoms at all three follow-ups, more cross-situational behavioral problems at both childhood and adolescence, poorer mother-teen relations at adolescence, and lower class rankings in high school. Participants with the 9/10 pair in the control group also had lower work performance, a lower grade point average in high school, greater teacher rated externalizing symptoms at adolescence, and greater omission errors on a continuous performance test in adulthood. The DAT1 40bp VNTR 9/10 polymorphism pairing appears to be reliably associated with greater symptoms of ADHD and externalizing behavior from childhood to adulthood, and with family, educational, and occupational impairments. We also present a contrary view on the appropriate endophenotypes for use in behavioral genetic research on ADHD. PMID:16741944

Vitamin D receptor gene polymorphisms and risk of polycystic ovary syndrome in South Indian women.

PubMed

Siddamalla, Swapna; Reddy, Tumu Venkat; Govatati, Suresh; Erram, Nagendram; Deenadayal, Mamata; Shivaji, Sisinthy; Bhanoori, Manjula

2018-02-01

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. Emerging evidence suggests that Vitamin D Receptor (VDR) might be a causal factor for characteristics associated with PCOS such as obesity and type 2 diabetes. Present study investigated association between VDR gene BsmI A/G (rs1544410), ApaI A/C (rs7975232) and TaqI T/C (rs731236) single nucleotide polymorphisms and PCOS risk in South Indian women. Genotyping of VDR gene SNPs was carried out in PCOS patients (n = 95) and controls (n = 130) by PCR-RFLP method and confirmed by sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pairwise linkage disequilibrium (LD) were assessed by Haploview software. Results showed significantly increased frequencies of BsmI G/G (p = .0197), ApaI C/C (p = .048), TaqI C/C (p = .044) genotypes and BsmI G (p = .0181), ApaI C (p = .0092), TaqI C (p = .0066) alleles in patients compared to controls. In addition, the frequency of the 'BsmI G, ApaI C, TaqI C' haplotype was also significantly elevated in patients (p = .0087). In conclusion, the VDR gene BsmI A/G ApaI A/C TaqI T/C and haplotype may constitute an inheritable risk factor for PCOS in South Indian women.

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis.

PubMed

Scholz, Holger Christian; Witte, Angela; Tomaso, Herbert; Al Dahouk, Sascha; Neubauer, Heinrich

2005-03-15

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.

Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait.

PubMed

Lin, Tzu Che; Yeh, Mau Shing; Cheng, Ya Ming; Lin, Li Chang; Sung, Jih Min

2012-03-15

Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)-generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR-RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR-RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR-RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. The present study demonstrates the usefulness of ITS2 PCR-RFLP coupled with pre-selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants.

Cleft lip with or without cleft palate: Associations with transforming growth factor alpha and retinoic acid receptor loci

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chenevix-Trench, G.; Jones, K.; Green, A.C.

1992-12-01

The first association study of cleft lip with or without cleft palate (CL/P), with candidate genes, found an association with the transforming growth-factor alpha (TGFA) locus. This finding has since been replicated, in whole or in part, in three independent studies. Here the authors extend their original analysis of the TGFA TaqI RFLP to two other TGFA RFLPs and seven other RFLPs at five candidate genes in 117 nonsyndromic cases of CL/P and 113 controls. The other candidate genes were the retinoic acid receptor (RARA), the bcl-2 oncogene, and the homeobox genes 2F, 2G, and EN2. Significant associations with themore » TGFA TaqI and BamHI RFLPs were confirmed, although associations of clefting with previously reported haplotypes did not reach significance. Of particular interest, in view of the known teratogenic role of retinoic acid, was a significant association with the RARA PstI RFLP (P = .016; not corrected for multiple testing). The effect on risk of the A2 allele appears to be additive, and although the A2A2 homozygote only has an odds ratio of about 2 and recurrence risk to first-degree relatives ([lambda][sub 1]) of 1.06, because it is so common it may account for as much as a third of the attributable risk of clefting. There is no evidence of interaction between the TGFA and RARA polymorphisms on risk, and jointly they appear to account for almost half the attributable risk of clefting. 43 refs., 1 fig., 4 tabs.« less

Detection and identification of Malassezia species in domestic animals and aquatic birds by PCR-RFLP

PubMed Central

Zia, M.; Mirhendi, H.; Toghyani, M.

2015-01-01

The present study aimed at detection and species-level identification of the Malassezia yeasts in domestic animals and aquatic birds by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Samples were collected using tape strips and swabs from 471 animals including 97 horses, 102 cattle, 105 sheep, 20 camels, 60 dogs, 30 cats, 1 hamster, 1 squirrel, 50 aquatic birds and 5 turkeys. Tape-strip samples were examined by direct microscopy. All samples were inoculated on modified Leeming and Notman agar medium. DNA extracted from the yeast colonies was amplified by PCR using primers specific for 26S rDNA. RFLP of the PCR products was performed using Hin6I enzyme, and PCR and RFLP products were visualized by agarose gel electrophoresis. Malassezia yeasts were detected at the following frequencies: 15.46% in horses, 12.74% in cattle, 12.38% in sheep, 28.33% in dogs, 26.66% in cats and 26% in aquatic birds. Eighty colonies of 6 species were isolated: Malassezia globosa 41.25%, Malassezia furfur 22.5%, Malassezia restricta 15%, Malassezia sympodialis 15%, Malassezia pachydermatis 5% and Malassezia slooffiae 1.25%. Therefore different lipophilic Malassezia species are found in a wide diversity of animals and aquatic birds. PCR-RFLP is a suitable technique for identification of different Malassezia species. PMID:27175148

Creatine kinase MM TaqI and methylenetetrahydrofolate reductase C677T and A1298C gene polymorphisms influence exercise-induced C-reactive protein levels.

PubMed

Miranda-Vilela, Ana Luisa; Akimoto, Arthur K; Lordelo, Graciana S; Pereira, Luiz C S; Grisolia, Cesar K; Klautau-Guimarães, Maria de Nazaré

2012-01-01

Physical training induces beneficial adaptations, but exhausting exercise increases reactive oxygen species, which can cause muscular injuries with consequent inflammatory processes, implying jeopardized performance and possibly overtraining. Acute strenuous exercise almost certainly exceeds the benefits of physical activity; it can compromise performance and may contribute to increased future risk of cardiovascular disease (CVD) in athletes. Polymorphisms in the muscle-type creatine kinase (CK-MM) gene may influence performance and adaptation to training, while many potentially significant genetic variants are reported as risk factors for CVD. Therefore, we investigated the influence of polymorphisms in CK-MM TaqI and NcoI, methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and C-reactive protein (CRP G1059C) genes on exercise-induced damage and inflammation markers. Blood samples were taken immediately after a race (of at least 4 km) that took place outdoors on flat tracks, and were submitted to genotyping and biochemical evaluation of aspartate aminotransferase (AST), CK, CRP and high-sensitivity CRP (hs-CRP). CK-MM TaqI polymorphism significantly influenced results of AST, CK and hs-CRP, and an association between MTHFR C677T and A1298C with CRP level was found, although these levels did not exceed reference values. Results indicate that these polymorphisms can indirectly influence performance, contribute to higher susceptibility to exercise-induced inflammation or protection against it, and perhaps affect future risks of CVD in athletes.

Creatine kinase MM TaqI and methylenetetrahydrofolate reductase C677T and A1298C gene polymorphisms influence exercise-induced C-reactive protein levels.

PubMed

Miranda-Vilela, Ana Luisa; Akimoto, Arthur K; Lordelo, Graciana S; Pereira, Luiz C S; Grisolia, Cesar K; Klautau-Guimarães, Maria de Nazaré

2012-03-01

Physical training induces beneficial adaptations, but exhausting exercise increases reactive oxygen species, which can cause muscular injuries with consequent inflammatory processes, implying jeopardized performance and possibly overtraining. Acute strenuous exercise almost certainly exceeds the benefits of physical activity; it can compromise performance and may contribute to increased future risk of cardiovascular disease (CVD) in athletes. Polymorphisms in the muscle-type creatine kinase (CK-MM) gene may influence performance and adaptation to training, while many potentially significant genetic variants are reported as risk factors for CVD. Therefore, we investigated the influence of polymorphisms in CK-MM TaqI and NcoI, methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and C-reactive protein (CRP G1059C) genes on exercise-induced damage and inflammation markers. Blood samples were taken immediately after a race (of at least 4 km) that took place outdoors on flat tracks, and were submitted to genotyping and biochemical evaluation of aspartate aminotransferase (AST), CK, CRP and high-sensitivity CRP (hs-CRP). CK-MM TaqI polymorphism significantly influenced results of AST, CK and hs-CRP, and an association between MTHFR C677T and A1298C with CRP level was found, although these levels did not exceed reference values. The results indicate that these polymorphisms can indirectly influence performance, contribute to higher susceptibility to exercise-induced inflammation or protection against it, and perhaps affect future risks of CVD in athletes.

Clinical application of RT-nested PCR integrated with RFLP in Hantavirus detection and genotyping: a prospective study in Shandong Province, PR China.

PubMed

Liu, Yun-Xi; Zhao, Zhong-Tang; Cao, Wu-Chun; Xu, Xiao-Qun; Suo, Ji-Jiang; Xing, Yu-Bin; Jia, Ning; Du, Ming-Mei; Liu, Bo-Wei; Yao, Yuan

2013-01-01

The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.

Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

PubMed

Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

2016-09-01

We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05). © 2016 The Author(s).

Risk factors associated with cluster size of Mycobacterium tuberculosis (Mtb) of different RFLP lineages in Brazil.

PubMed

Peres, Renata Lyrio; Vinhas, Solange Alves; Ribeiro, Fabíola Karla Correa; Palaci, Moisés; do Prado, Thiago Nascimento; Reis-Santos, Bárbara; Zandonade, Eliana; Suffys, Philip Noel; Golub, Jonathan E; Riley, Lee W; Maciel, Ethel Leonor

2018-02-08

Tuberculosis (TB) transmission is influenced by patient-related risk, environment and bacteriological factors. We determined the risk factors associated with cluster size of IS6110 RFLP based genotypes of Mycobacterium tuberculosis (Mtb) isolates from Vitoria, Espirito Santo, Brazil. Cross-sectional study of new TB cases identified in the metropolitan area of Vitoria, Brazil between 2000 and 2010. Mtb isolates were genotyped by the IS6110 RFLP, spoligotyping and RD Rio . The isolates were classified according to genotype cluster sizes by three genotyping methods and associated patient epidemiologic characteristics. Regression Model was performed to identify factors associated with cluster size. Among 959 Mtb isolates, 461 (48%) cases had an isolate that belonged to an RFLP cluster, and six clusters with ten or more isolates were identified. Of the isolates spoligotyped, 448 (52%) were classified as LAM and 412 (48%) as non-LAM. Our regression model found that 6-9 isolates/RFLP cluster were more likely belong to the LAM family, having the RD Rio genotype and to be smear-positive (adjusted OR = 1.17, 95% CI 1.08-1.26; adjusted OR = 1.25, 95% CI 1.14-1.37; crude OR = 2.68, 95% IC 1.13-6.34; respectively) and living in a Serra city neighborhood decrease the risk of being in the 6-9 isolates/RFLP cluster (adjusted OR = 0.29, 95% CI, 0.10-0.84), than in the others groups. Individuals aged 21 to 30, 31 to 40 and > 50 years were less likely of belonging the 2-5 isolates/RFLP cluster than unique patterns compared to individuals < 20 years of age (adjusted OR = 0.49, 95% CI 0.28-0.85, OR = 0.43 95% CI 0.24-0.77and OR = 0. 49, 95% CI 0.26-0.91), respectively. The extrapulmonary disease was less likely to occur in those infected with strains in the 2-5 isolates/cluster group (adjustment OR = 0.45, 95% CI 0.24-0.85) than unique patterns. We found that a large proportion of new TB infections in Vitoria is caused by prevalent Mtb genotypes

Association between the DRD2 A1 allele and response to methadone and buprenorphine maintenance treatments.

PubMed

Barratt, Daniel T; Coller, Janet K; Somogyi, Andrew A

2006-06-05

The TaqI A polymorphism (A(1)) of the dopamine D(2) receptor gene (DRD2), although not a specific predictor of opioid dependence, has been strongly associated with high levels of prior heroin use and poor treatment outcomes among methadone maintenance patients. The aims of this study were to confirm these findings via a retrospective analysis of A(1) allele frequency in methadone (n = 46) and buprenorphine (n = 25) patients, and non-opioid-dependent controls (n = 95). Subjects were genotyped at the DRD2 TaqI A locus using PCR amplification followed by TaqI restriction enzyme digestion and gel electrophoresis. For methadone and buprenorphine subjects, heroin use (prior to treatment), treatment outcomes, and withdrawal occurrence were determined from comprehensive case notes. No significant differences in A(1) allele frequency (%) were observed between: methadone (19.6%), buprenorphine (18.0%), and control (17.9%) groups (P > 0.7); successful and poor treatment outcome groups, methadone: 20.0% and 19.2%, respectively (P = 1.0); buprenorphine: 18.4% and 20.0%, respectively (P = 1.0). Also, there were no significant relationships between TaqI A genotype and prior heroin use (P = 0.47). However, among the successful methadone subjects, significantly fewer A(1) allele carriers experienced withdrawal than non-A(1) carriers (P = 0.04). In conclusion, the DRD2 genotype effects did not affect opioid maintenance treatment outcomes. This suggests the need for a further prospective investigation into the role of the DRD2 A(1) allele in heroin use and response to maintenance pharmacotherapies for opioid dependence.

Molecular Characterization of Hypoderma SPP. in Domestic Ruminants from Turkey and Pakistan.

PubMed

Ahmed, Haroon; Simsek, Sami; Saki, Cem Ecmel; Kesik, Harun Kaya; Kilinc, Seyma Gunyakti

2017-08-01

The aim of this study was to determine the morphological and molecular characterization of Hypoderma spp. in cattle and yak from provinces in Turkey and Pakistan. In total, 78 Hypoderma larvae were collected from slaughtered animals in Turkey and Pakistan from October 2015 to January 2016. Thirty-eight of these 78 Hypoderma larvae were morphologically classified as third instar larvae (L3s) of Hypoderma bovis, 37 were classified as Hypoderma lineatum, and 3 were classified as suspected or unidentified. The restriction enzyme TaqI was used to differentiate the Hypoderma spp. by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). According to the sequences and the PCR-RFLP results, all larval samples from cattle from Turkey were classified as H. bovis, except for 1 sample classified as H. lineatum. All Hypoderma larvae from Pakistan were classified as H. lineatum from cattle and as Hypoderma sinense from yak. This study provides the first molecular characterization of H. lineatum (cattle) and H. sinense (yak) in Pakistan based on PCR-RFLP and sequencing results.

Sensitive identification of mycobacterial species using PCR-RFLP on bronchial washings.

PubMed

Hidaka, E; Honda, T; Ueno, I; Yamasaki, Y; Kubo, K; Katsuyama, T

2000-03-01

In 98 patients (24 with active pulmonary tuberculosis [TB] lesions, 28 with cured TB lesions, and 46 with nontuberculous opacities [control group] in chest CT scans), we examined whether washing the bronchus after brushing the lesion, then applying polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to the bronchial washings might be useful for diagnosing TB and nontuberculous mycobacteriosis (NTMosis). After biopsy and brushing with a bronchoscope, the bronchus connecting to the lesion was washed with 20 ml saline. The saline used for washing the brushes (5 ml; brushing sample), and 3 to 10 ml saline aspirated through the forceps channel (washing sample) were examined by PCR-RFLP, which proved able to identify Mycobacterium tuberculosis and seven species of nontuberculous mycobacteria (NTM). The values obtained for the sensitivity of the PCR-RFLP with respect to the brushing sample, the washing sample, and both samples mixed together were 70, 76, and 91%, respectively, when only patients who were culture-positive or radiologically improved after antituberculous therapy were considered as showing true infection. A mixture of brushing and washing samples provides useful material for PCR and culture, and the PCR-RFLP used here is a good method for the simultaneous identification of several species of mycobacterium (including M. tuberculosis).

Standardization of PCR-RFLP analysis of nsSNP rs1468384 of NPC1L1 gene

PubMed Central

Balgir, Praveen P.; Khanna, Divya; Kaur, Gurlovleen

2008-01-01

Niemann-Pick C1-like 1 (NPC1L1) protein, a newly identified sterol influx transporter, located at the apical membrane of the enterocyte, which may actively facilitate the uptake of cholesterol by promoting the passage of sterols across the brush border membrane of the enterocyte. It effects intestinal cholesterol absorption and intracellular transport and as such is an integral part of complex process of cholesterol homeostasis. The study of population data for the distribution of these single nucleotide polymorphisms (SNP) of NPC1L1 has lead to the identification of six non-synonymous single nucleotide polymorphisms (nsSNP). The in vitro analysis using the software MuPro and StructureSNP shows that nsSNP M510I (rs1468384), which involves A→G base pair change leads to decrease in the stability of the protein. A reproducible and a cost-effective PCR-RFLP based assay was developed to screen for the SNP among population data. This SNP has been studied in Caucasian, Asian, and African American populations. Till date, no data is available on Indian population. The distribution of M510I NPC1L1 genotype was estimated in the North Western Indian Population as a test case. The allele distribution in Indian Population differs significantly from that of other populations. The methodology thus proved to be robust enough to bring out these differences. PMID:20300301

Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene.

PubMed

Kumar, Deepak; Singh, S P; Karabasanavar, Nagappa S; Singh, Rashmi; Umapathi, V

2014-11-01

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

Inheritance of RFLP loci in a loblolly pine three-generation pedigree

Treesearch

M.D. Devey; K.D. Jermstad; C.G. Tauer; D.B. Neale

1991-01-01

A high-density restriction fragment length polymorphism (RFLP) linkage map is being constructed for loblolly pine (Pinus taeda L.). Loblolly pine cDNA and genomic DNA clones were used as probes in hybridizations to genomic DNAs prepared from grandparents, parents, and progeny of a three-generation outbred pedigree. Approximately 200 probes were...

Identification of five sea cucumber species through PCR-RFLP analysis

NASA Astrophysics Data System (ADS)

Lv, Yingchun; Zheng, Rong; Zuo, Tao; Wang, Yuming; Li, Zhaojie; Xue, Yong; Xue, Changhu; Tang, Qingjuan

2014-10-01

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene ( COI) was used to identifing five sea cucumber species ( Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping.

PubMed

Chang, Hsueh-Wei; Cheng, Yu-Huei; Chuang, Li-Yeh; Yang, Cheng-Hong

2010-04-08

PCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2. The primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system. The user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.

Evaluation of a PCR-RFLP- ITS2 assay for discrimination of Anopheles species in northern and western Colombia

PubMed Central

Cienfuegos, Astrid V.; Rosero, Doris A.; Naranjo, Nelson; Luckhart, Shirley; Conn, Jan E.; Correa, Margarita M.

2011-01-01

Anopheles mosquitoes are routinely identified using morphological characters of the female that often lead to misidentification due to interspecies similarity and intraspecies variability. The aim of this work was to evaluate the applicability of a previously developed PCR-RFLP-ITS2 assay for accurate discrimination of anophelines in twelve localities spanning three Colombian malaria epidemiological regions: Atlantic Coast, Pacific Coast, and Uraba-Bajo Cauca-Alto Sinu Region. The evaluation of the stability of the PCR-RFLP patterns is required since variability of the ITS2 has been documented and may produce discrepancies in the patterns previously reported. The assay was used to evaluate species assignation of 939 mosquitoes identified by morphology. Strong agreement between the morphological and molecular identification was found for species An. albimanus, An. aquasalis, An. darlingi and An. triannulatus s.l. (p ≥ 0.05, kappa=1). However, disagreement was found for species An. nuneztovari s.l., An. neomaculipalpus, An. apicimacula and An. punctimacula (p ≤ 0.05; kappa ranging from 0.33–0.80). The ITS2-PCR-RFLP assay proved valuable for discriminating anopheline species of northern and western Colombia, especially those with overlapping morphology in the Oswaldoi Group. PMID:21345325

Linkage analysis of Norrie disease with an X-chromosomal ornithine aminotransferase locus.

PubMed

Bateman, J B; Kojis, T L; Cantor, R M; Heinzmann, C; Ngo, J T; Spence, M A; Inana, G; Kivlin, J D; Curtis, D; Sparkes, R S

1993-01-01

Norrie disease is a rare disease of newborn males caused by prenatal or perinatal retinal detachment, which may be associated with mental retardation, psychosis, and/or hearing loss. DXS7 (L1.28) and MAO A and B loci have been linked to the ND locus on the short arm of the X chromosome. Sequences homologous to OAT also have been mapped to the short arm of the X chromosome. We performed linkage analyses between the ND locus and one of the OAT-like clusters of sequences on the X chromosome (OATL1), using a ScaI RFLP in a ND family, and increased the previously calculated lod score (z) to over 3 (3.38; theta = 0.05). Similarly, we calculated a lod score of 4.06 (theta = 0.01) between the OATL1 and DXS7 loci. Alone, the OATL1 ScaI RFLP system is expected to be informative in 48% of females. If this system were used in combination with the DXS7 TaqI polymorphism, 71% of females would be informative for at least one of the markers and 21% would be informative for both. Because the OATL1 ScaI RFLP is a relatively common polymorphism, this system should be useful for the identification of ND carriers and affected male fetuses and newborns.

Linkage analysis of Norrie disease with an X-chromosomal ornithine aminotransferase locus.

PubMed Central

Bateman, J B; Kojis, T L; Cantor, R M; Heinzmann, C; Ngo, J T; Spence, M A; Inana, G; Kivlin, J D; Curtis, D; Sparkes, R S

1993-01-01

Norrie disease is a rare disease of newborn males caused by prenatal or perinatal retinal detachment, which may be associated with mental retardation, psychosis, and/or hearing loss. DXS7 (L1.28) and MAO A and B loci have been linked to the ND locus on the short arm of the X chromosome. Sequences homologous to OAT also have been mapped to the short arm of the X chromosome. We performed linkage analyses between the ND locus and one of the OAT-like clusters of sequences on the X chromosome (OATL1), using a ScaI RFLP in a ND family, and increased the previously calculated lod score (z) to over 3 (3.38; theta = 0.05). Similarly, we calculated a lod score of 4.06 (theta = 0.01) between the OATL1 and DXS7 loci. Alone, the OATL1 ScaI RFLP system is expected to be informative in 48% of females. If this system were used in combination with the DXS7 TaqI polymorphism, 71% of females would be informative for at least one of the markers and 21% would be informative for both. Because the OATL1 ScaI RFLP is a relatively common polymorphism, this system should be useful for the identification of ND carriers and affected male fetuses and newborns. PMID:7908152

Isolation and RFLP genotyping of Toxoplasma gondii from the gray wolf (Canis lupus).

PubMed

Dubey, J P; Choudhary, S; Ferreira, L R; Kwok, O C H; Butler, E; Carstensen, M; Yu, L; Su, C

2013-11-08

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study feral gray wolves (Canis lupus) from Minnesota were examined for T. gondii infection. Antibodies to T. gondii were detected in 130 (52.4%) of 248 wolves tested by the modified agglutination test (cut-off titer of 25). Tissues (hearts, brains or both) of 109 wolves were bioassayed in mice for protozoal isolation. Viable T. gondii was isolated from 25 and the isolates were further propagated in cell culture. T. gondii DNA from these isolates was characterized using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Four genotypes were detected. Twenty-one isolates were Type 12 (ToxoDB PCR-RFLP genotype #5), 2 were Type II clonal (ToxoDB #1), 1 was Type II variant (ToxoDB #3), and 1 was a new genotype designated as ToxoDB genotype #219. Published by Elsevier B.V.

Polymorphisms at the Ligand Binding Site of the Vitamin D Receptor Gene and Osteomalacia

PubMed Central

Ak, Duygu Gezen; Kahraman, Hakkí; Dursun, Erdinç; Duman, Belgin Süsleyici; Erensoy, Nevin; Alagöl, Faruk; Tanakol, Refik; Yılmazer, Selma

2005-01-01

Vitamin D receptor (VDR) gene polymorphisms have been suggested as possible determinants of bone mineral density (BMD) and calcium metabolism. In this study, our aim was to determine whether there is an association between VDR gene polymorphism and osteomalacia or not. We determined ApaI and TaqI polymorphisms in the vitamin D receptor gene in 24 patients with osteomalacia and 25 age-matched healthy controls. Serum calcium, phosphorus, ALP, PTH, 25OHD levels were also examined. We used PCR and RFLP methods to test for an association between osteomalacia and polymorphisms within, intron 8 and exon 9 of the VDR gene. When the control and patients were compared for their ApaI and TaqI genotypes there was no relationship between VDR gene allelic polymorphisms and osteomalacia. Whereas a nearly significant difference for A allele was found in the allellic distribution of the patients (p = 0.08). Also no association between biochemical data and VDR gene polymorphisms was observed. PMID:16403954

Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Part 1: Fungal field study

Treesearch

Grant T. Kirker; M. Lynn Prewitt; Tor P. Schultz; Susan V. Dieh

2012-01-01

The effects of chlorothalonil (CTN), butylated hydroxytoluene (BHT), and ammoniacal copper quat (ACQ-C) on the fungal community on southern yellow pine (SYP) were assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis over 15 months. Field stakes, treated with 0.25 and 0.37 % ACQ-C, 0.1 and 0.25 % CTN, 2 % BHT alone, 0.1 and 0.25 % CTN...

A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries.

PubMed

Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Amani, Hizem; Mezhoud, Habib; Babba, Hamouda

2016-10-01

Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1-G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6-G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.

A Rapid PCR-RFLP Method for Monitoring Genetic Variation among Commercial Mushroom Species

ERIC Educational Resources Information Center

Martin, Presley; Muruke, Masoud; Hosea, Kenneth; Kivaisi, Amelia; Zerwas, Nick; Bauerle, Cynthia

2004-01-01

We report the development of a simplified procedure for restriction fragment length polymorphism (RFLP) analysis of mushrooms. We have adapted standard molecular techniques to be amenable to an undergraduate laboratory setting in order to allow students to explore basic questions about fungal diversity and relatedness among mushroom species. The…

A new source of cytoplasmic male sterility in pearl millet: RFLP analysis of mitochondrial DNA.

PubMed

Sujata, V; Sivaramakrishnan, S; Rai, K N; Seetha, K

1994-06-01

A new source of cytoplasmic male sterility (cms) in pearl millet (Pennisetum glaucum (L.) R.Br.) derived from a half-sib progeny of the Early Gene Pool (EGP 261) and used in a male-sterile line, ICMA 90111, was compared with other known cms sources for RFLP of mitochondrial (mt) DNA. Southern blot hybridization of mtDNA from ICMA 90111 digested with several restriction enzymes and probed with homologous mtDNA clones from pearl millet and heterologous gene clones from maize and wheat revealed the RFLP patterns of ICMA 90111 distinct from others studied so far. The dendrogram of male-sterile lines constructed from the Southern blot hybridization patterns indicated that ICMA 90111 represents a separate group. Our results suggest that this source of cms is unique in several respects.

A Detailed RFLP Map of Cotton, Gossypium Hirsutum X Gossypium Barbadense: Chromosome Organization and Evolution in a Disomic Polyploid Genome

PubMed Central

Reinisch, A. J.; Dong, J. M.; Brubaker, C. L.; Stelly, D. M.; Wendel, J. F.; Paterson, A. H.

1994-01-01

We employ a detailed restriction fragment length polymorphism (RFLP) map to investigate chromosome organization and evolution in cotton, a disomic polyploid. About 46.2% of nuclear DNA probes detect RFLPs distinguishing Gossypium hirsutum and Gossypium barbadense; and 705 RFLP loci are assembled into 41 linkage groups and 4675 cM. The subgenomic origin (A vs. D) of most, and chromosomal identity of 14 (of 26), linkage groups is shown. The A and D subgenomes show similar recombinational length, suggesting that repetitive DNA in the physically larger A subgenome is recombinationally inert. RFLPs are somewhat more abundant in the D subgenome. Linkage among duplicated RFLPs reveals 11 pairs of homoeologous chromosomal regions-two appear homosequential, most differ by inversions, and at least one differs by a translocation. Most homoeologies involve chromosomes from different subgenomes, putatively reflecting the n = 13 to n = 26 polyploidization event of 1.1-1.9 million years ago. Several observations suggest that another, earlier, polyploidization event spawned n = 13 cottons, at least 25 million years ago. The cotton genome contains about 400-kb DNA per cM, hence map-based gene cloning is feasible. The cotton map affords new opportunities to study chromosome evolution, and to exploit Gossypium genetic resources for improvement of the world's leading natural fiber. PMID:7851778

PCR-RFLP genotypes associated with quinolone resistance in isolates of Flavobacterium psychrophilum.

PubMed

Izumi, S; Ouchi, S; Kuge, T; Arai, H; Mito, T; Fujii, H; Aranishi, F; Shimizu, A

2007-03-01

A novel genotyping method for epizootiological studies of bacterial cold-water disease caused by Flavobacterium psychrophilum and associated with quinolone resistance was developed. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was performed on 244 F. psychrophilum isolates from various fish species. PCR was performed with primer pair GYRA-FP1F and GYRA-FP1R amplifying the A subunit of the DNA gyrase (GyrA) gene, which contained the quinolone resistance determining region. Digestion of PCR products with the restriction enzyme Mph1103I showed two genotypes, QR and QS. The difference between these genotypes was amino acid substitutions at position 83 of GyrA (Escherichia coli numbering). The genotype QR indicated an alanine residue at this position associated with quinolone resistance in F. psychrophilum isolates. Of the 244 isolates tested in this study, the number of QR genotype isolates was 153 (62.7%). In isolates from ayu (n=177), 146 (82.5%) were genotype QR. With combination of this technique and previously reported PCR-RFLP genotyping, eight genotypes were observed in F. psychrophilum isolates. Using this genotyping system, the relationships between genotype and host fish species, or locality of isolation, were analysed and are discussed.

SNP detection in Na/K ATP-ase gene α1 subunit of bisexual and parthenogenetic Artemia strains by RFLP screening.

PubMed

Manaffar, R; Zare, S; Agh, N; Abdolahzadeh, N; Soltanian, S; Sorgeloos, P; Bossier, P; Van Stappen, G

2011-01-01

In order to find a marker for differentiating between a bisexual and a parthenogenetic Artemia strain, Exon-7 of the Na/K ATPase α(1) subunit gene was screened by RFLP technique. The results revealed a constant synonymous SNP (single nucleotide polymorphism) in digestion by the Tru1I enzyme that was consistent with these two types of Artemia. This SNP was identified as an accurate molecular marker for discrimination between bisexual and parthenogenetic Artemia. According to the Nei's genetic distance (1973), the lowest genetic distance was found between individuals from Artemia urmiana Günther 1890 and parthenogenetic populations, making the described marker the first marker to easily distinguish between these two cooccurring species. © 2010 Blackwell Publishing Ltd.

An Authentic RFLP Lab for High School or College Biology Students.

ERIC Educational Resources Information Center

Guilfoile, Patrick G.; Plum, Stephen

1998-01-01

Explains how students can perform an alternative authentic DNA fingerprinting analysis. Presents restriction fragment length polymorphism (RFLP) analysis and can serve as a simulated molecular epidemiology laboratory or as a simulated forensic laboratory exercise. (DDR)

Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species.

PubMed

Şakalar, Çağrı; Kuk, Salih; Erensoy, Ahmet; Dağli, Adile Ferda; Özercan, İbrahim Hanifi; Çetınkaya, Ülfet; Yazar, Süleyman

2014-01-01

To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

First Molecular Characterization of Hypoderma actaeon in Cattle and Red Deer (Cervus elaphus) in Portugal.

PubMed

Ahmed, Haroon; Sousa, Sérgio Ramalho; Simsek, Sami; Anastácio, Sofia; Kilinc, Seyma Gunyakti

2017-12-01

Hypoderma spp. larvae cause subcutaneous myiasis in several animal species. The objective of the present investigation was to identify and characterize morphologically and molecularly the larvae of Hypoderma spp. collected from cattle (Bos taurus taurus) and red deer (Cervus elaphus) in the district of Castelo Branco, Portugal. For this purpose, a total of 8 larvae were collected from cattle (n=2) and red deer (n=6). After morphological identification of Hypoderma spp. larvae, molecular characterization was based on PCR-RFLP and mitochondrial CO1 gene sequence analysis. All larvae were morphologically characterized as the third instar larvae (L3) of H. actaeon. Two restriction enzymes were used for molecular identification of the larvae. TaqI restriction enzyme was not able to cut H. actaeon. However, MboII restriction enzyme differentiated Hypoderma species showing 210 and 450 bp bands in H. actaeon. Furthermore, according to the alignment of the mt-CO1 gene sequences of Hypoderma species and to PCR-RFLP findings, all the identified Hypoderma larvae were confirmed as H. actaeon. This is the first report of identification of Hypoderma spp. (Diptera; Oestridae) from cattle and red deer in Portugal, based on morphological and molecular analyses.

Peculiarities of RFLP of highly repetitive DNA in crow genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chelomina, G.N.; Kryukov, A.P.; Ivanov, S.V.

1995-02-01

We present a study of the structural organization of highly repetitive DNA in genomes of hooded crow Corvus cornix L., carrion crow C. corone L., and jungle crow C. macrorhynchos Wagl. RFLP and blot-hybridization with {sup 32}P-labeled Msp I fragment from hooded crow nDNA suggest the interspecific structural conservatism of the most repetitive DNA. The family of repeats we studied had tandem organization and the same (210 bp) period of reiteration for a set of restriction enzymes. However, in parallel to the general similarity of restriction patterns, there are species-specific peculiarities. The repetitive family revealed (Alu I, BsuR I, andmore » Msp I fragments) has quantitative RFLP of nDNA and interspecific differences in the extent of the multimer {open_quotes}ladder{close_quotes} pattern of Msp I fragments. The latter is more pronounced in nDNA of carrion crow than in that of phylogenetically distant jungle crow and closely related hooded crow. This suggests a recent amplification event for highly organized homological repeats in crow genomes. 10 refs., 2 figs.« less

Juvenile idiopathic arthritis patients and their skeletal status: possible role of vitamin D receptor gene polymorphism.

PubMed

Kostik, M M; Smirnov, A M; Demin, G S; Scheplyagina, L A; Larionova, V I

2014-01-01

We evaluated bone mineralization and metabolism changes related to vitamin D receptor (VDR) polymorphic genotypes in children with juvenile idiopathic arthritis. One hundred and ninety eight children (82 boys and 116 girls) were included in our study. Bone mineral density (BMD) was measured by lumbar spine DXA. Osteocalcin, CTX, parathyroid hormone, total and ionized calcium, inorganic phosphate, total alkaline phosphatase activity was utilized for assessment of bone metabolism. Molecular testing: TaqI (rs731236) and Cdx2 (rs11568820) polymorphisms of VDR were detected by RFLP. No differences in TaqI and Cdx2 haplotypes, genotypes and alleles distribution related with normal and low BMD (Zscore <-2SD) were found. Children with low linear growth (<10th percentile) had more allele T-contained genotypes of TagI VDR (p = 0.037), compare with medium or high linear growth children. Children with high linear growth (>90th percentile) had the highest frequency of allele A-contained genotypes (GA+AA) of Cdx2 VDR (p = 0.009). Girls with TT TaqI VDR, who never been treated by glucocorticoides had lower BMD-Zscore than C allele carriers (TT = -0.94SD [IQR: -2.1;-0.5], TC+CC = -0.62SD [IQR: -1.26;0.39], p = 0.03). Girls with Tanner I with TT had higher total and ionized Ca level than carriers of C allele (Ca: TT = 2.43 ± 0.15 mmol/l, TC+CC = 2.28 ± 0.2 mmol/l, p = 0.024; Ca(2+): TT = 1.15 ± 0.08 mmol/l, TC+CC = 1.06 ± 0.13 mmol/l, p = 0.026). Presence of TT genotype negatively correlated with BMD-Zscore (r = -0.28, p = 0.04), and positively with frequency of LBMD (r = 0.3, p = 0.037). Boy with GG Cdx2 genotype had lower total Ca (GG = 2.3 ± 0.17 mmol/l, GA+AA = 2.43 ± 0.17 mmol/l, p = 0.004) compare with carriers of A allele. Pubertal boys (Tanner IV-V) with GG had higher CTX (GG = 1.75 ± 0.11 ng/ml, GA+AA = 1.06 ± 0.07 ng/ml, p = 0.04. TT genotype of TaqI and GG genotype of Cdx2 VDR is a negative factor impact bone mineralization metabolism and linear growth.

Comparison of Nested PCR and RFLP for Identification and Classification of Malassezia Yeasts from Healthy Human Skin

PubMed Central

Oh, Byung Ho; Song, Young Chan; Choe, Yong Beom; Ahn, Kyu Joong

2009-01-01

Background Malassezia yeasts are normal flora of the skin found in 75~98% of healthy subjects. The accurate identification of the Malassezia species is important for determining the pathogenesis of the Malassezia yeasts with regard to various skin diseases such as Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis. Objective This research was conducted to determine a more accurate and rapid molecular test for the identification and classification of Malassezia yeasts. Methods We compared the accuracy and efficacy of restriction fragment length polymorphism (RFLP) and the nested polymerase chain reaction (PCR) for the identification of Malassezia yeasts. Results Although both methods demonstrated rapid and reliable results with regard to identification, the nested PCR method was faster. However, 7 different Malassezia species (1.2%) were identified by the nested PCR compared to the RFLP method. Conclusion Our results show that RFLP method was relatively more accurate and reliable for the detection of various Malassezia species compared to the nested PCR. But, in the aspect of simplicity and time saving, the latter method has its own advantages. In addition, the 26S rDNA, which was targeted in this study, contains highly conserved base sequences and enough sequence variation for inter-species identification of Malassezia yeasts. PMID:20523823

RFLP and sequence analysis of the cytochrome b gene of selected animals and man: methodology and forensic application.

PubMed

Zehner, R; Zimmermann, S; Mebs, D

1998-01-01

To identify common animal species by analysis of the cytochrome b gene a method has been developed to obtain PCR products of a large domain of the cytochrome b gene (981 bp out of 1140 bp) in humans, selected mammals and birds using the same specifically designed primers. Species-specific RFLP patterns are generated by co-restriction with the restriction endonucleases ALU I and NCO I. The RFLP patterns obtained are conclusive even in mixtures of two or more species. The results were confirmed by sequence analysis which in addition explained intraspecies variations in the RFLP patterns. The method has been applied to forensic casework studies where the origin of roasted meat, stomach contents and a bone sample has been successfully identified.

PCR and RFLP analyses based on the ribosomal protein operon

USDA-ARS?s Scientific Manuscript database

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16Sr RNA gene. RFLP analysis of 16Sr RNA gene sequences has identified 31 16Sr RNA (16Sr) groups and more than 100 16Sr subgroups. Classification of phytoplasma strains can however, become more refin...

Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis.

PubMed

Dec, Marta; Urban-Chmiel, Renata; Gnat, Sebastian; Puchalski, Andrzej; Wernicki, Andrzej

2014-04-01

The objective of our study was to identify Lactobacillus sp. strains of goose origin using MALDI-TOF mass spectrometry, ITS-PCR and ITS-PCR/RFLP. All three techniques proved to be valuable tools for identification of avian lactobacilli and produced comparable classification results. Lactobacillus strains were isolated from 100% of geese aged 3 weeks to 4 years, but from only 25% of chicks aged 1-10 days. Among the 104 strains isolated, we distinguished 14 Lactobacillus species. The dominant species was Lactobacillus salivarius (35.6%), followed by Lactobacillus johnsonii (18.3%), Lactobacillus ingluviei (11.5%) and Lactobacillus agilis (7.7%). The intact-cell MALDI-TOF mass spectrometry enabled rapid species identification of the lactobacilli with minimal pretreatment. However, it produced more than one identification result for 11.5% examined strains (mainly of the species L. johnsonii). ITS-PCR distinguished 12 genotypes among the isolates, but was not able to differentiate closely related strains, i.e. between Lactobacillus amylovorus and Lactobacillus kitasatonis and between Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus zeae. These species were differentiated by ITS-PCR/RFLP using the restriction enzymes TaqI and MseI. The results obtained indicate that ITS-PCR and ITS-PCR/RFLP assays could be used not only for interspecific, but also for intraspecific, typing. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Genetic polymorphism in Leishmania infantum isolates from human and animals determined by nagt PCR-RFLP.

PubMed

El Hamouchi, Adil; El Kacem, Sofia; Ejghal, Rajaa; Lemrani, Meryem

2018-06-14

Leishmania infantum is the causative agent of human visceral leishmaniasis (VL) and sporadic human cutaneous leishmaniasis (CL) in the Mediterranean region. The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations. The main objective of this study was to explore the genetic polymorphism in L. infantum isolates from human and animal hosts in different regions of Morocco. The intraspecific genetic variability of 40 Moroccan L. infantum MON-1 strains isolated from patients with VL (n = 31) and CL (n = 2) and from dogs (n = 7) was evaluated by PCR-RFLP of nagt, a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase. For a more complete analysis of L. infantum polymorphism, we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database. Moroccan L. infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified. Three of the six genotypes were exclusively identified in the Moroccan population of L. infantum: variant M1 (15%), variant M2 (7.5%), and variant M3 (2.5%). The most common genotype (65%), variant 2 (2.5%), and variant 4 (7.5%), were previously described in several countries with endemic leishmaniasis. Phylogenetic analysis segregated our L. infantum population into two distinct clusters, whereas variant M2 was clearly distinguished from both cluster I and cluster II. This distribution highlights the degree of genetic variability among the Moroccan L. infantum population. The nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L. infantum strains isolated from human and canine reservoirs with 6 genotypes identified. Three of the six Moroccan nagt genotypes, have not been previously described and

Highly sensitive detection of the PIK3CA (H1047R) mutation in colorectal cancer using a novel PCR-RFLP method.

PubMed

Li, Wan-Ming; Hu, Ting-Ting; Zhou, Lin-Lin; Feng, Yi-Ming; Wang, Yun-Yi; Fang, Jin

2016-07-12

The PIK3CA (H1047R) mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CA (H1047R) mutation in high effectiveness. A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CA (H1047R) was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method. The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA (H1047R) mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CA (H1047R) mutation and the patients' age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods. We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CA (H1047R) mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy.

Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI.

PubMed

Diba, K; Mirhendi, H; Kordbacheh, P; Rezaie, S

2014-01-01

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species.

Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI

PubMed Central

Diba, K.; Mirhendi, H.; Kordbacheh, P.; Rezaie, S.

2014-01-01

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species. PMID:25242934

Genetic variation in Pythium myriotylum based on SNP typing and development of a PCR-RFLP detection of isolates recovered from Pythium soft rot ginger.

PubMed

Le, D P; Smith, M K; Aitken, E A B

2017-10-01

Pythium myriotylum is responsible for severe losses in both capsicum and ginger crops in Australia under different regimes. Intraspecific genomic variation within the pathogen might explain the differences in aggressiveness and pathogenicity on diverse hosts. In this study, whole genome data of four P. myriotylum isolates recovered from three hosts and one Pythium zingiberis isolate were derived and analysed for sequence diversity based on single nucleotide polymorphisms (SNPs). A higher number of true and unique SNPs occurred in P. myriotylum isolates obtained from ginger with symptoms of Pythium soft rot (PSR) in Australia compared to other P. myriotylum isolates. Overall, SNPs were discovered more in the mitochondrial genome than those in the nuclear genome. Among the SNPs, a single substitution from the cytosine (C) to the thymine (T) in the partially sequenced CoxII gene of 14 representatives of PSR P. myriotylum isolates was within a restriction site of HinP1I enzyme which was used in the PCR-RFLP for detection and identification of the isolates without sequencing. The PCR-RFLP was also sensitive to detect PSR P. myriotylum strains from artificially infected ginger without the need for isolation for pure cultures. This is the first study of intraspecific variants of Pythium myriotylum isolates recovered from different hosts and origins based on single nucleotide polymorphism (SNP) genotyping of multiple genes. The SNPs discovered provide valuable makers for detection and identification of P. myriotylum strains initially isolated from Pythium soft rot (PSR) ginger by using PCR-RFLP of the CoxII locus. The PCR-RFLP was also sensitive to detect P. myriotylum directly from PSR ginger sampled from pot trials without the need of isolation for pure cultures. © 2017 The Society for Applied Microbiology.

First isolation and RFLP genotyping of Toxoplasma gondii from crab-eating fox (Cerdocyon thous-Linnaeus, 1766).

PubMed

de Almeida, Jonatas Campos; de Melo, Renata Pimentel Bandeira; de Morais Pedrosa, Camila; da Silva Santos, Marcelo; de Barros, Luiz Daniel; Garcia, João Luis; Porto, Wagnner José Nascimento; Mota, Rinaldo Aparecido

2017-05-01

Wild animals may play an important role in the transmission and maintenance of Toxoplasma gondii in the environment. The purpose of the present study was to isolate and genotype T. gondii from a free-ranging crab-eating fox (Cerdocyon thous-Linnaeus, 1766). A crab-eating fox in critical health condition was attended in a veterinary hospital in Recife, Pernambuco State, Brazil. The animal died despite emergency treatment. The brain was collected aseptically and destined for mouse bioassay. One isolate of T. gondii was obtained, and Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) was used to assess genetic variability at 11 markers (SAG1, SAG2, altSAG2, SAG3, BTUB, GRA6, c228, c292, L358, PK1 and APICO). A murine model was used to assess the virulence of the isolate. Using the PCR-RFLP, genotype ToxoDB #13 was identified, which is considered an atypical strain. The isolate was classified as avirulent in the murine model. This is the first study to report T. gondii infection in the crab-eating fox. Copyright © 2017 Elsevier B.V. All rights reserved.

PCR-RFLP of ribosomal internal transcribed spacers highlights inter and intra-species variation among Leishmania strains native to La Paz, Bolivia.

PubMed

Buitrago, Rosio; Cupolillo, Elisa; Bastrenta, Brigitte; Le Pont, Francois; Martinez, Eddy; Barnabé, Christian; Brenière, Simone Frédérique

2011-04-01

Human leishmaniasis is highly endemic in Bolivia and shows a growing incidence. This report reveals the genetic variability of 35 isolates mainly belonging to Leishmania braziliensis and Leishmania amazonensis species. Among them, 31 were from human patients with different clinical presentations, 3 strains from Lutzomya nuneztovari anglesi (the proven vector of L. amazonensis) and 1 strain of a mammal (Conepatus chinga). The isolates were analyzed by isoenzyme electrophoresis (MLEE) and PCR-RFLP of ITS rRNA genes, a genetic marker highly polymorphic and better adapted to sub-structuring of populations. MLEE and RFLP-ITS were in agreement to discriminate the species, 12 belong to L. (V.) braziliensis, 21 to L. (L.) amazonensis, 1 to Leishmania (V.) lainsoni and 1 to Leishmania (L.) chagasi. Among L. (V.) braziliensis the RFLP-ITS only highlights variability. Ten isolates from either cutaneous or mucocutaneous clinical forms, were grouped together (bootstrap value of 99.8%) apart from two others, one from a mammal (C. chinga), the other from a patient with a cutaneous form. Among L. (L.) amazonensis both markers detect variability but no significant sub-division was identified including isolates from different clinical forms. Moreover, the high frequency of several isolates from cutaneous forms occurred during an outbreak, with putative hybrid character (multiloci heterozygous patterns depicted by MLEE) could be linked to better fitness of these parasites. However, in the absence of observation of hypothetical parents, their hybrid status remains a question. Copyright © 2010. Published by Elsevier B.V.

COMPARISON OF TAXONOMIC, COLONY MORPHOTYPE AND PCR-RFLP METHODS TO CHARACTERIZE MICROFUNGAL DIVERSITY

EPA Science Inventory

We compared three methods for estimating fungal species diversity in soil samples. A rapid screening method based on gross colony morphological features and color reference standards was compared with traditional fungal taxonomic methods and PCR-RFLP for estimation of ecological ...

A PCR-RFLP Assay targeting RPS8 gene for the discrimination between bovine Babesia and Theileria species in China.

PubMed

Tian, Zhancheng; Du, Junzheng; Yang, Jifei; Liu, Aihong; Liu, Xiaocui; Liu, Guangyuan; Yin, Hong

2015-09-17

Bovine babesiosis and theileriosis is an important hemoprotozoal disease in cattles and yaks in tropical and subtropical regions leading to significant economic losses. In the field, the risk of co-infection between the bovine Babesia and Theileria species is very high. Thus, it is necessary to develop a simple, accurate, rapid and cost-effective method for large-scale epidemic investigation, in particular for the detection of co-infection in field. In this study, DNA sequences of a ribosomal protein S8 (RPS8) gene from eight species of cattle piroplasms in China were used to develop a species-specific PCR-RFLP diagnostic tool. The eight Theileria and Babesia species could be differentiated by digesting the RPS8 PCR product with Mbo I. The sensitivity of the PCR assays was 0.1 pg DNA for Babesia species but 1 pg DNA for Theileria species. The clearly different size of the PCR-RFLP products allowed for a direct discrimination between eight bovine Theileria and Babesia species (T. annulata, T. sinensis, T. sergenti, B. ovata, B. bovis, B. bigemina, B. major and Babesia species Kashi isolate). Our results indicated that the established method based on the RPS8 gene was a reliable molecular diagnostic tool for the simultaneous detection and identification of bovine Babesia and Theileria species in China, which could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.

Analysis of ELA-DQB exon 2 polymorphism in Argentine Creole horses by PCR-RFLP and PCR-SSCP.

PubMed

Villegas-Castagnasso, E E; Díaz, S; Giovambattista, G; Dulout, F N; Peral-García, P

2003-08-01

The second exon of equine leucocyte antigen (ELA)-DQB genes was amplified from genomic DNA of 32 Argentine Creole horses by PCR. Amplified DNA was analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformation polymorphism (SSCP). The PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns, five MspI patterns and two HinfI patterns. EcoRI showed no variation in the analysed sample. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel ELA-DQB alleles. PCR-SSCP analysis exhibited seven different band patterns, and the number of bands per animal ranged from four to nine. Both methods indicated that at least two DQB genes are present. The presence of more than two alleles in each animal showed that the primers employed in this work are not specific for a unique DQB locus. The improvement of this PCR-RFLP method should provide a simple and rapid technique for an accurate definition of ELA-DQB typing in horses.

Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

PubMed

Montalvo, Ana M; Fraga, Jorge; Tirado, Dídier; Blandón, Gustavo; Alba, Annia; Van der Auwera, Gert; Vélez, Iván Darío; Muskus, Carlos

2017-07-01

Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.

Identification of Pit-1 Gen Using PCR-RFLP of Padjadjaran Sheep and Evaluate of Growth Rate

NASA Astrophysics Data System (ADS)

Prajoga, S. B. K.; Andriani, L.; Subhandiana, H.

2018-02-01

The objectives of this research were to evaluate variation of Pit-1 gen of Padjadjaran Sheep and evaluate of their growth rate. The data comprised of 15 lambs female and 15 lambs male records of 0 - 12 mouths old lambs. Variation of Padjadjaran Sheep PIT-1 gen was analyzed using PCR-RFLP and used primer by 5’-GAGGGATAATTACAAATGGTCC-3’ and 5’-TGTTAACAGCTGTGGGACACAC-3’, length fragment analyse using HinfI restriction enzyme. Presence or absence of deference bands in a PCR fragment of the result can be distinguished by differences in the electrophoretic migration of the fragment. The result showed that the variation Pit1 gene showed that there was in the position 345 bp, 137 bp and 115 bp. In this experiment the difference migration did occur in all samples (monomorphic), that was amplificated with reverse and forward primer. The average male birth weight (BW), weaning weight (WN) and Yearling Weight (YW) was 2.29 ±0.42kg 8.96 ±1,89 kg and 30.12 ±5,65 kg. The average female birth weight (BW), weaning weight (WN) and Yearling Weight (YW) was 2.33 ±0.49 kg; 8.23 ±1.99 kg and 26.11 ±5.50 kg. Correlation value between age and body weight was 0,99 for female and 0,97 for male. The highest body weight gain per month was 2.85 kg for female at the age of six months and 3.4 kg for male at the age of one year. The best equition of growth rate was Ŷ = 1,862 + 0,127X1 0,076X2

Software for optimization of SNP and PCR-RFLP genotyping to discriminate many genomes with the fewest assays

PubMed Central

Gardner, Shea N; Wagner, Mark C

2005-01-01

Background Microbial forensics is important in tracking the source of a pathogen, whether the disease is a naturally occurring outbreak or part of a criminal investigation. Results A method and SPR Opt (SNP and PCR-RFLP Optimization) software to perform a comprehensive, whole-genome analysis to forensically discriminate multiple sequences is presented. Tools for the optimization of forensic typing using Single Nucleotide Polymorphism (SNP) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses across multiple isolate sequences of a species are described. The PCR-RFLP analysis includes prediction and selection of optimal primers and restriction enzymes to enable maximum isolate discrimination based on sequence information. SPR Opt calculates all SNP or PCR-RFLP variations present in the sequences, groups them into haplotypes according to their co-segregation across those sequences, and performs combinatoric analyses to determine which sets of haplotypes provide maximal discrimination among all the input sequences. Those set combinations requiring that membership in the fewest haplotypes be queried (i.e. the fewest assays be performed) are found. These analyses highlight variable regions based on existing sequence data. These markers may be heterogeneous among unsequenced isolates as well, and thus may be useful for characterizing the relationships among unsequenced as well as sequenced isolates. The predictions are multi-locus. Analyses of mumps and SARS viruses are summarized. Phylogenetic trees created based on SNPs, PCR-RFLPs, and full genomes are compared for SARS virus, illustrating that purported phylogenies based only on SNP or PCR-RFLP variations do not match those based on multiple sequence alignment of the full genomes. Conclusion This is the first software to optimize the selection of forensic markers to maximize information gained from the fewest assays, accepting whole or partial genome sequence data as input. As more sequence data becomes

Genetic characterization of Toxoplasma gondii from pigs from different localities in China by PCR-RFLP.

PubMed

Jiang, Hai-Hai; Huang, Si-Yang; Zhou, Dong-Hui; Zhang, Xiao-Xuan; Su, Chunlei; Deng, Shun-Zhou; Zhu, Xing-Quan

2013-08-07

Toxoplasma gondii is a widely prevalent protozoan parasite that causes serious toxoplasmosis in humans and animals. The present study aimed to determine the genetic diversity of T. gondii isolates from pigs in Jiangxi, Sichuan, Guangdong Provinces and Chongqing Municipality in China using multilocous polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. A total of 38 DNA samples were extracted from hilar lymph nodes of pigs with suspected toxoplasmosis, and were detected for the presence of T. gondii by semi-nested PCR of B1 gene. The positive DNA samples were typed at 11 genetic markers, including 10 nuclear loci, namely, SAG1, 5'-SAG2 and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast locus Apico. Twenty-five of the 38 DNA samples were T. gondii B1 gene positive. Complete genotyping data for all loci could be obtained for 17 of the 25 samples. Two genotypes were revealed (ToxoDB PCR-RFLP genotypes #9 and #3). Sixteen samples belong to genotype #9 which is the major lineage in mainland China and one sample belongs to genotype #3 which is Type II variant. To our knowledge, this is the first report of genetic typing of T. gondii isolates from pigs in Jiangxi, Sichuan Province and Chongqing Municipality, and the first report of ToxoDB #3 T. gondii from pigs in China. These results have implications for the prevention and control of foodborne toxoplasmosis in humans.

Aspergillus tubingensis and Aspergillus niger as the dominant black Aspergillus, use of simple PCR-RFLP for preliminary differentiation.

PubMed

Mirhendi, H; Zarei, F; Motamedi, M; Nouripour-Sisakht, S

2016-03-01

This work aimed to identify the species distribution of common clinical and environmental isolates of black Aspergilli based on simple restriction fragment length polymorphism (RFLP) analysis of the β-tubulin gene. A total of 149 clinical and environmental strains of black Aspergilli were collected and subjected to preliminary morphological examination. Total genomic DNAs were extracted, and PCR was performed to amplify part of the β-tubulin gene. At first, 52 randomly selected samples were species-delineated by sequence analysis. In order to distinguish the most common species, PCR amplicons of 117 black Aspergillus strains were identified by simple PCR-RFLP analysis using the enzyme TasI. Among 52 sequenced isolates, 28 were Aspergillus tubingensis, 21 Aspergillus niger, and the three remaining isolates included Aspergillus uvarum, Aspergillus awamori, and Aspergillus acidus. All 100 environmental and 17 BAL samples subjected to TasI-RFLP analysis of the β-tubulin gene, fell into two groups, consisting of about 59% (n=69) A. tubingensis and 41% (n=48) A. niger. Therefore, the method successfully and rapidly distinguished A. tubingensis and A. niger as the most common species among the clinical and environmental isolates. Although tardy, the Ehrlich test was also able to differentiate A. tubingensis and A. niger according to the yellow color reaction specific to A. niger. A. tubingensis and A. niger are the most common black Aspergillus in both clinical and environmental isolates in Iran. PCR-RFLP using TasI digestion of β-tubulin DNA enables rapid screening for these common species. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis

Treesearch

Grant T. Kirker; M. Lynn Prewitt; Walter J. Diehl; Susan V. Diehl

2012-01-01

The effects of wood preservatives on the bacterial community in southern yellow pine were assessed by the molecular method ‘terminal restriction fragment length polymorphism’ (T-RFLP). Stakes, treated with 0.25 % and 0.37 % ammoniacal copper quat (ACQ-C), 0.1 % and 0.25 % chlorothalonil (CTN), 0.1 % and 0.25 % CTN with 2 % butylated hydroxytoluene (BHT), and 2 % BHT...

[Differentiation of species within the Mycobacterium tuberculosis complex by molecular techniques].

PubMed

Herrera-León, Laura; Pozuelo-Díaz, Rodolfo; Molina Moreno, Tamara; Valverde Cobacho, Azucena; Saiz Vega, Pilar; Jiménez Pajares, María Soledad

2009-11-01

The Mycobacterium tuberculosis complex includes the following species: Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis-BCG, Mycobacterium microti, Mycobacterium caprae, Mycobacterium pinnipedii, and Mycobacterium canettii. These species cause tuberculosis in humans and animals. Identification of mycobacterial strains has classically been performed by phenotype study. Over the last years, laboratories have developed several molecular techniques to differentiate between these species. The aim of this study is to evaluate these methods and develop a simple, fast, identification scheme. We analyzed 251 strains randomly obtained from the strains studied in 2004, and 797 strains received by the Reference Laboratory between 2005 and 2007. Phenotype characterization of 4183 strains isolated during that period was done by studying the colony morphology, characteristics in culture, nitrate reduction, niacin accumulation, and growth in the presence of thiophen-2-carboxylic acid hydrazide 10 microg/mL and pyrazinamide 50 microg/mL. The molecular identification scheme designed was as follows: 1) gyrB PCR-RFLP with RsaI, TaqI or SacII and hsp65 RFLP/PCR with HhaI., and 2) multiplex-PCR to determine the presence/absence of the RD9 and RD1 regions. The results showed 100% agreement between phenotype study and the molecular scheme. This molecular identification scheme is a simple and fast method, with 100% sensitivity and specificity, that can be implemented in most clinical laboratories at a low cost.

Partial DNA sequencing of Douglas-fir cDNAs used in RFLP mapping

Treesearch

K.D. Jermstad; D.L. Bassoni; C.S. Kinlaw; D.B. Neale

1998-01-01

DNA sequences from 87 Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) cDNA RFLP probes were determined. Sequences were submitted to the GenBank dbEST database and searched for similarity against nucleotide and protein databases using the BLASTn and BLASTx programs. Twenty-one sequences (24%) were assigned putative functions; 18 of which...

Typing clinical and animal environment Aspergillus fumigatus gliotoxin producer strains isolated from Brazil by PCR-RFLP markers.

PubMed

Soleiro, C A; Pena, G A; Cavaglieri, L R; Coelho, I; Keller, L M; Dalcero, A M; Rosa, C A R

2013-12-01

Aspergillus fumigatus, a well-known human and animal pathogen causing aspergillosis, has been historically identified by morphological and microscopic features. However, recent studies have shown that species identification on the basis of morphology alone is problematic. The aim of this work was to confirm the taxonomic state at specie level of a set of clinical (human and animal) and animal environment A. fumigatus strains identified by morphological criteria applying a PCR-RFLP assay by an in silico and in situ analysis with three restriction enzymes. The A. fumigatus gliotoxin-producing ability was also determined. Previous to the in situ PCR-RFLP analysis, an in silico assay with BccI, MspI and Sau3AI restriction enzymes was carried out. After that, these enzymes were used for in situ assay. All A. fumigatus strains isolated from corn silage, human aspergillosis and bovine mastitis and high per cent of the strains isolated from cereals, animal feedstuff and sorghum silage were able to produce high gliotoxin levels. Also, all these strains identified by morphological criteria as A. fumigatus, regardless of its isolation source, had band patterns according to A. fumigatus sensu stricto by PCR-RFLP markers. Aspergillus fumigatus is a well-known human and animal pathogen causing aspergillosis. In this study, clinical (human and animal) and animal environment strains were able to produce high gliotoxin levels and had band profiles according to A. fumigatus sensu stricto by PCR-RFLP markers. The results obtained here suggest that strains involved in human and animal aspergillosis could come from the animal environment in which A. fumigatus is frequently found. Its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs. © 2013 The Society for Applied Microbiology.

Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting.

PubMed

Romi, Wahengbam; Keisam, Santosh; Ahmed, Giasuddin; Jeyaram, Kumaraswamy

2014-02-28

Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes. We targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping. We herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used

Genetic differentiation of Colletotrichum gloeosporioides and C. truncatum associated with Anthracnose disease of papaya (Carica papaya L.) and bell pepper (Capsium annuum L.) based on ITS PCR-RFLP fingerprinting.

PubMed

Maharaj, Ariana; Rampersad, Sephra N

2012-03-01

Members of the genus Colletotrichum include some of the most economically important fungal pathogens in the world. Accurate diagnosis is critical to devising disease management strategies. Two species, Colletotrichum gloeosporioides and C. truncatum, are responsible for anthracnose disease in papaya (Carica papaya L.) and bell pepper (Capsicum annuum L.) in Trinidad. The ITS1-5.8S-ITS2 region of 48 Colletotrichum isolates was sequenced, and the ITS PCR products were analyzed by PCR-RFLP analysis. Restriction site polymorphisms generated from 11 restriction enzymes enabled the identification of specific enzymes that were successful in distinguishing between C. gloeosporioides and C. truncatum isolates. Species-specific restriction fragment length polymorphisms generated by the enzymes AluI, HaeIII, PvuII, RsaI, and Sau3A were used to consistently resolve C. gloeosporioides and C. truncatum isolates from papaya. AluI, ApaI, PvuII, RsaI, and SmaI reliably separated isolates of C. gloeosporioides and C. truncatum from bell pepper. PvuII, RsaI, and Sau3A were also capable of distinguishing among the C. gloeosporioides isolates from papaya based on the different restriction patterns that were obtained as a result of intra-specific variation in restriction enzyme recognition sites in the ITS1-5.8S-ITS2 rDNA region. Of all the isolates tested, C. gloeosporioides from papaya also had the highest number of PCR-RFLP haplotypes. Cluster analysis of sequence and PCR-RFLP data demonstrated that all C. gloeosporioides and C. truncatum isolates clustered separately into species-specific clades regardless of host species. Phylograms also revealed consistent topologies which suggested that the genetic distances for PCR-RFLP-generated data were comparable to that of ITS sequence data. ITS PCR-RFLP fingerprinting is a rapid and reliable method to identify and differentiate between Colletotrichum species.

Characterization and quantification of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR

PubMed Central

Jin, Tao; Yan, Qingmei

2010-01-01

Using ammonia monooxygenase α-subunit (amoA) gene and 16S rRNA gene, the community structure and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in a nitrogen-removing reactor, which was operated for five phases, were characterized and quantified by cloning, terminal restriction fragment length polymorphism (T-RFLP), and quantitative polymerase chain reaction (qPCR). The results suggested that the dominant AOB in the reactor fell to the genus Nitrosomonas, while the dominant AOA belonged to Crenarchaeotal Group I.1a in phylum Crenarchaeota. Real-time PCR results demonstrated that the levels of AOB amoA varied from 2.9 × 103 to 2.3 × 105 copies per nanogram DNA, greatly (about 60 times) higher than those of AOA, which ranged from 1.7 × 102 to 3.8 × 103 copies per nanogram DNA. This indicated the possible leading role of AOB in the nitrification process in this study. T-RFLP results showed that the AOB community structure significantly shifted in different phases while AOA only showed one major peak for all the phases. The analyses also suggested that the AOB community was more sensitive than that of AOA to operational conditions, such as ammonia loading and dissolved oxygen. PMID:20405121

Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment

PubMed Central

Millard, Julie T.; Chuang, Edward; Lucas, James S.; Nagy, Erzsebet E.; Davis, Griffin T.

2013-01-01

A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to three weeks. This experiment, which has an engaging and versatile scenario, provides students with exposure to key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context. PMID:24363455

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

PubMed

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

Polymorphic haplotypes and recombination rates at the LDL receptor gene locus in subjects with and without familial hypercholesterolemia who are from different populations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miserez, A.R.; Chiodetti, N.; Keller, U.

1993-04-01

RFLPs at the low-density lipoprotein (LDL) receptor locus for TaqI, StuI, HincII, AvaII, ApaLI (5[prime] and 3[prime]), PvuII, and NcoI were studied in Swiss and German families with familial hypercholesterolemia (FH). A total of 1,104 LDL receptor alleles were analyzed using Southern blotting and new PCR-based techniques for detection of the TaqI, StuI, HincII, AvaII, NcoI RFLPs. Two hundred fifty-six independent haplotypes from 368 individuals of 61 unrelated Swiss families, as well as 114 independent haplotypes from 184 subjects of 25 unrelated German families, were constructed. In 76 families, clinical diagnosis of FH was confirmed by cosegregation analysis. Of themore » 43 unique haplotypes consisting of seven RFLPs detected in the Swiss and Germans, only 9 were common in both population samples. Analysis of linkage disequilibrium revealed nonrandom associations between several of the investigated RFLPs. ApaLI (5[prime]), NcoI, PvuII, TaqI, and AvaII or HincII were particularly informative. Relative frequencies, heterozygosity indexes, and PICs of the RFLPs from the Swiss and Germans were compared with values calculated from reported haplotype data for Italians, Icelanders, North American Caucasians, South African Caucasians, and Japanese. Pairwise comparisons of population samples by common RFLPs demonstrated unexpected differences even between geographically adjacent populations (e.g., the Swiss and Germans). Furthermore, genetic distances from the Germans to the other Caucasians were larger than to the Japanese. An unexpected lack of correlation between linkage disequilibria and physical distances was detected for the German and Japanese data, possibly because of nonuniform recombination with excessively high rates between exon 13 and intron 15. Hence, the present study revealed a striking variety of polymorphic haplotypes and heterogeneity of RFLP frequencies and recombination rates among the seven population samples. 60 refs., 2 figs., 8 tabs.« less

Bovine Papillomavirus in Brazil: Detection of Coinfection of Unusual Types by a PCR-RFLP Method

PubMed Central

Carvalho, R. F.; Sakata, S. T.; Giovanni, D. N. S.; Mori, E.; Brandão, P. E.; Richtzenhain, L. J.; Pozzi, C. R.; Arcaro, J. R. P.; Miranda, M. S.; Mazzuchelli-de-Souza, J.; Melo, T. C.; Comenale, G.; Assaf, S. L. M. R.; Beçak, W.; Stocco, R. C.

2013-01-01

Bovine papillomavirus (BPV) is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil. PMID:23865043

First genotyping of Cryptosporidium spp. in pre-weaned calves, broiler chickens and children in Syria by PCR-RFLP analysis.

PubMed

Kassouha, Morshed; Soukkarieh, Chadi; Alkhaled, Abdulkarim

2016-07-30

In this study, PCR-RFLP was used for the first time in Syria for genotyping Cryptosporidium species of man, calves and chickens. The total of 391 fecal samples included 213 from children with diarrhea (<5years), 67 from pre-weaned calves with diarrhea and 111 from broiler chicken farms. All samples were collected and examined with acid fast stain to detect the positive samples. Subsequently a nested-PCR test was performed on 35 positive samples (17 from calves, 11 from chicken, and 7 from children) targeting SSU rRNA gene, and was followed by RFLP analysis using three restriction enzymes SspI, VspI and MboII. Results showed that C. parvum was the only identified species in children and calves, on the other hand C. baileyi was identified in broilers in addition to another species with unknown RFLP profile in comparison to those which have been described in chicken. Further studies using more genes are needed to sequence and detect subtypes of this parasite. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a PCR-RFLP assay for the detection and differentiation of canine parvovirus and mink enteritis virus.

PubMed

Zhang, Chuanmei; Yu, Yongle; Yang, Haiyan; Li, Guimei; Yu, Zekun; Zhang, Hongliang; Shan, Hu

2014-12-15

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay has been developed to detect and differentiate between canine parvovirus (CPV) and mink enteritis virus (MEV). Eight CPV and three MEV epidemic strains isolated from 28 pathological samples from dogs and minks suspected of being infected with parvovirus were amplified by PCR using a pair of specific primers designed based on the CPV-N strain (M19296). PCR amplified a fragment of 1016bp from the genomic DNA of both MEV and CPV. The MEV-derived fragment could be digested with the restriction enzyme BSP1407I into three fragments of 102bp, 312bp and 602bp, while the fragment amplified from the CPV genomic DNA was digested into only two fragments of 414bp and 602bp. The lowest DNA concentration of CPV and MEV that could be detected using this assay was 0.004μg/ml and 0.03μg/ml, respectively. The PCR-RFLP assay developed in the present study can, therefore, be used to detect and differentiate MEV from CPV with high specificity and sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes psient1 and psient2 and the selu or seluv gene using PCR-RFLP.

PubMed

Collery, Mark M; Smyth, Cyril J

2007-02-01

The egc locus of Staphylococus aureus harbours two enterotoxin genes (seg and sei) and three enterotoxin-like genes (selm, seln and selo). Between the sei and seln genes are located two pseudogenes, psient1 and psient2, or the selu or seluv gene. While these two alternative sei-seln intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between egc loci bearing the pseudogenes and the selu or seluv gene. In silico restriction enzyme digestion of genomic regions encompassing the egc locus from the 3' end of the sei gene through the 5' first quarter of the seln gene allowed pseudogene- and selu- or seluv-bearing egc loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease HindIII cleaved PCR amplimers bearing pseudogenes but not those with a selu or seluv gene, while selu- or seluv-bearing amplimers were susceptible to cleavage by endonuclease HphI, but not by endonuclease HindIII. The restriction enzyme BccI cleaved selu- or seluv-harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these egc loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an egc locus type, and can also be used for the primary identification of the intergenic sei-seln egc locus type.

Relative diversity and community structure analysis of rumen protozoa according to T-RFLP and microscopic methods.

PubMed

Tymensen, Lisa; Barkley, Cindy; McAllister, Tim A

2012-01-01

Protozoa are common inhabitants of the rumen where they play roles in host nutrition and methanogenesis. Knowledge of how changes in the composition of protozoa communities affect these processes is limited in part due to a lack of efficient methods for protozoa community analysis. In this study, a terminal-restriction fragment length polymorphism (T-RFLP) assay targeting the 18S rRNA gene was developed for comparative analysis of rumen protozoa communities. Comparison of diversity and structure of protozoa communities from hay-fed versus silage/grain-fed cattle via T-RFLP analysis yielded similar overall results to microscopy analysis. According to both methods, Entodinium spp. were more abundant in the silage/grain-fed cattle and protozoa diversity (as calculated using the Shannon index) was higher for the hay-fed cattle due to greater species evenness. Type B protozoa were more prevalent in the hay-fed cattle, whereas Type A protozoa were more prevalent in the silage/grain-fed cattle. Analysis of similarity (ANOSIM) indicated that the protozoa communities from hay-fed and silage/grain-fed cattle were different, and multivariate analysis indicated that pen mates (i.e., cattle fed the same diet and housed together) tended to have similar protozoa communities types. In summary, we present a T-RFLP method for analyzing rumen protozoa communities which complements traditional microscopy approaches but has the advantage of being amenable to high-throughput. Copyright © 2011. Published by Elsevier B.V.

Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

PubMed

Daniel, Hubert D-J; David, Joel; Raghuraman, Sukanya; Gnanamony, Manu; Chandy, George M; Sridharan, Gopalan; Abraham, Priya

2017-05-01

Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P < 0.00) and 96% (κ = 0.794, P < 0.00) correlated with NS5b sequencing, respectively. Sixty percent and 75% of discordant samples by 5'NCR PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing. © 2016 Wiley Periodicals, Inc.

Genetic diversity pattern of microeukaryotic communities and its relationship with the environment based on PCR-DGGE and T-RFLP techniques in Dongshan Bay, southeast China

NASA Astrophysics Data System (ADS)

Zhang, Wenjing; Mo, Yuanyuan; Yang, Jun; Zhou, Jing; Lin, Yuanshao; Isabwe, Alain; Zhang, Jian; Gao, Xiu; Yu, Zheng

2018-07-01

Microeukaryotes play important roles in aquatic ecosystems, and could act as drivers of the biological nutrient cycling processes. However, compared with prokaryotic ones, little is known about the genetic diversity pattern of their community, and the environmental factors affecting their ecological pattern, especially in marine ecosystems. In this study, we used denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) to explore the genetic diversity structure of microeukaryotic communities in Dongshan Bay, southeast China. Our results revealed that microeukaryotic diversity ranged from 31 to 48 phylotypes (on average, 42) using the DGGE approach, while from 22 to 38 phylotypes (on average, 30) based on T-RFLP method, and the Shannon-Wiener diversity (DGGE-based) was relatively higher, suggesting that DGGE displayed a slightly higher resolution than T-RFLP. Surprisingly, the DGGE showed significant horizontal difference among microeukaryotic communities, but was similar with T-RFLP analysis that had no significant influence on microeukaryotic diversity at vertical scale. Further, redundancy analysis (RDA) indicated that the DGGE-based microeukaryotic communities distribution was significantly correlated with three environmental factors (Chl-a, TP and salinity), whereas microeukaryotic community revealed by T-RFLP was affected by four environmental factors namely salinity, temperature, depth and NOX-N. Compared with RDA, BIO-ENV analysis showed that heterotrophic bacteria and NOX-N were important environmental variable influencing microeukaryotic communities in both methods. These differences may be attributed to the noisy effects caused by the relatively large number of environmental variables. Generally speaking, despite differences in beta-diversity ordination for both DGGE and T-RFLP methods, there exists some consistency in the results of both techniques in terms of microeukaryotes responses to the

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines

PubMed Central

Asing; Ali, Md. Eaqub; Abd Hamid, Sharifah Bee; Hossain, M. A. Motalib; Mustafa, Shuhaimi; Kader, Md. Abdul; Zaidul, I. S. M.

2016-01-01

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition. PMID:27716792

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

PubMed

Asing; Ali, Md Eaqub; Abd Hamid, Sharifah Bee; Hossain, M A Motalib; Mustafa, Shuhaimi; Kader, Md Abdul; Zaidul, I S M

2016-01-01

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

Genetic polymorphisms variants in interleukin-6 and interleukin-1beta patients with obstructive sleep apnea syndrome in East Northern Turkey.

PubMed

Gok, Ilhami; Huseyinoglu, Nergiz; Ilhan, Dogan

2015-08-01

To investigate the relationship of IL-1β and IL-6 cytokine gene polymorphisms with obstructive sleep apnea syndrome (OSAS) in 61 patients admitted to the neurology clinic in Kafkas University Hospital with insomnia problem who were diagnosed with OSAS in sleeping labs, and 80 healthy subjects not associated with the syndrome. METHODS :Blood samples were taken to isolate DNA from patients diagnosed with OSAS based on polysomnography results and healthy controls. DNA amplification of the genes was performed with PCR. Amplification products were cut with the restriction enzymes in order to determine IL-1 gene (TaqI) and IL-6 gene (Lwel) polymorphisms. The cut DNA fragments were carried out in agarose gel electrophoresis, and RFLP analysis was performed by utilizing the images with gel imaging system. PCR products were sequenced with an Applied Biosystems Automated Sequencer. Polymorphic changes were observed for IL-1β gene in 26 of 62 patients (41.9%), and 16 of the 80 (25.8%) in the control group. The incidence of polymorphic changes in IL-6 gene was in seen in seven (of the 62 patients) (11.3%), and in the 16 (20%) controls. The findings on the genomic level in OSAS may provide an important contribution to diagnosis of obstructive sleep apnea syndrome in clinical practice, as well as it helps to obtain the results easily about environmental and genetic interaction of OSAS patients. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

Association of Vitamin D receptor gene polymorphisms with metabolic syndrome and its components among adult Arabs from the United Arab Emirates.

PubMed

Hasan, Hayder A; AbuOdeh, Ra'ed O; Muda, Wan Abdul Manan Bin Wan; Mohamed, Hamid Jan Bin Jan; Samsudin, Ab Rani

2017-12-01

The aim was to investigate relationships of Vitamin D receptor gene (VDR) polymorphisms to the components of MetS among Arabs adult residing in the United Arab Emirates. A cross-sectional study of 198 Arabs adult (50 males and 148 females). Serum levels of glucose, vitamin D, HDL-C, and TG, and blood pressure were measured. FokI, BsmI & TaqI genotyping of VDR were investigated using PCR-RFLP technique. Age of the participants was 21(9) years with a BMI of 26.8(7.8) kg/m 2 . About 15% had MetS with serum vitamin D levels of 25.5(18.2) nmol/L. VDR genotyping yielded: FokI: 57.1% FF and 38.9% Ff, BsmI: 29.8% bb and 51.5% Bb, while TaqI showed 39.4% TT and 43.4% Tt. The ff carriers had higher total cholesterol [174(12.4) mg/dl] than FF and Ff genotypes. Bb carriers showed higher BMI and LDL-C than BB and bb genotypes. In females, FokI VDR polymorphism showed significant association with systolic blood pressure (SBP) and F allele carriers were at higher risk of developing high SBP [x 2 =4.4, df1, OR=0.29 (95%CI: 0.087-0.98), p=0.035]. VDR gene polymorphisms were not associated with MetS, yet it may affect the severity of some of components of MetS, namely the association of BsmI with obesity, FokI and BsmI with dyslipidemia and FokI with SBP. Copyright © 2017 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Association between SLC11A1 (NRAMP1) polymorphisms and susceptibility to tuberculosis in Chinese Holstein cattle.

PubMed

Liu, Kaihua; Zhang, Bin; Teng, Zhaochun; Wang, Youtao; Dong, Guodong; Xu, Cong; Qin, Bo; Song, Chunlian; Chai, Jun; Li, Yang; Shi, Xianwei; Shu, Xianghua; Zhang, Yifang

2017-03-01

We investigated the associations between SLC11A1 polymorphisms and susceptibility to tuberculosis (TB) in Chinese Holstein cattle, using a case-control study of 136 animals that had positive reactions to TB tests and showed symptoms and 96 animals that had negative reactions to tests and showed no symptoms. Polymerase chain reaction (PCR) sequencing and the restriction fragment length polymorphism (RFLP) technique were used to detect and determine SLC11A1 polymorphisms. Association analysis identified significant correlations between SLC11A1 polymorphisms and susceptibility/resistance to TB, and two genetic markers for SLC11A1 were established using PCR-RFLP. Sequence alignment of SLC11A1 revealed seven single-nucleotide polymorphisms (SNPs). This is the first report of MaeII PCR-RFLP markers for the SLC11A1-SNP3 site and PstI PCR-RFLP markers for the SLC11A1-SNP5 and SLC11A1-SNP6 sites in Chinese Holstein cattle. Logistic regression analysis indicated that SLC11A1-SNP1, SLC11A1-SNP3, and SLC11A1-SNP5 were significantly associated with susceptibility/resistance to TB. Two genotypes of SLC11A1-SNP3 were susceptible to TB, whereas one genotype of SLC11A1-SNP1 and two genotypes of SLC11A1-SNP5 were resistant. Haplotype analysis showed that nine haplotypes were potentially resistant to TB. After Bonferroni correction, three of the haplotypes remained significantly associated with TB resistance. SLC11A1 is a useful candidate gene related to TB in Chinese Holstein cattle. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel Phl-producing genotypes of finger millet rhizosphere associated pseudomonads and assessment of their functional and genetic diversity.

PubMed

Sekar, Jegan; Prabavathy, Vaiyapuri Ramalingam

2014-07-01

Genetic diversity of phlD gene, an essential gene in the biosynthesis of 2,4-diacetylphloroglucinol, was studied by restriction fragment length polymorphism (RFLP) in 20 Phl-producing pseudomonads isolated from finger millet rhizosphere. RFLP analysis of phlD gene displayed three patterns with HaeIII and TaqI enzymes. phlD gene sequence closely correlated with RFLP results and revealed the existence of three new genotypes G, H and I. Further, the phylogenetic and concatenated sequence analysis of the 16S rRNA, rpoB, gyrB, rpoD genes supported the hypothesis that these genotypes G, H and I were different from reported genotypes A-F. In all phylogenetic studies, the genotype G formed a distant clade from the groups of Pseudomonas putida and P. aeruginosa (sensu strictu), but the groups H and I were closely related to P. aeruginosa/P. stutzeri group. The Phl-producing pseudomonads exhibited antagonistic activity against Pyricularia grisea (TN508), Gaeumannomyces graminis (DSM1463), Fusarium oxysporum (DSM62297), Xanthomonas campestris (DSM3586) and Erwinia persicina (HMGU155). In addition, these strains exhibited various plant growth-promoting traits. In conclusion, this study displays the existence of novel Phl-producing pseudomonads genotypes G, H and I from finger millet rhizosphere, which formed taxonomically outward phylogenetic lineage from the groups of P. putida and P. aeruginosa (sensu strictu). © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Bcıı--RFLP profiles for serum amiloid A1 and mutated MEFV gene prevalence in chronic renal failure patients requiring long-term hemodialysis.

PubMed

Ozdemir, Ozturk; Kayatas, Mansur; Cetinkaya, Selma; Yildirim, Malik Ejder; Silan, Fatma; Kurtulgan, Hande Kucuk; Koksal, Binnur; Urfali, Mine; Candan, Ferhan

2015-03-01

There is an increased mortality risk in long-term hemodialysis patients of renal failure due to the chronic inflammation. The relationship between the chronic renal failure (CRF) and the role of familial genetic markers remains incompletely understood. In the current study, it was aimed to find out the prevalence of common MEFV gene mutations and BcII polymorphism in serum amyloid A1 (SAA1) gene in chronic renal patients (CRF) who require long-term hemodialysis. Current cohort includes 242 CRF patients and 245 healthy individuals from the same population. Total genomic DNA was isolated from peripheral blood-EDTA samples and genotyping of target MEFV gene was carried out by reverse hybridization Strip Assay and real-time techniques. The SAA1 gene was genotyped by the BclI-RFLP method. Increased mutated MEFV genotypes were found in current CRF patients when compared with the control group from the same ethnicity and the difference was statistically significant (Table 2) (OR: 4.9401, 95% CI: 3.0694-7.9509), p<0.0001. The most frequent point mutations were M694V and E148Q. The mutated T allel frequency in the SAA1 gene was also different when compared with the healthy controls and the difference was found to be statistically significant (χ2: 13.18; p=0.000). The current results indicate the germ-line mutations in both genetic biomarkers (MEFV and SAA1 genes) that are related to inflammation and amyloidosis processes may play a crucial role in CRF pathogenesis due to the long-term chronic inflammation.

Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

PubMed

Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

2017-08-01

Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

Delimitation of Umbelopsis (Mucorales, Umbelopsidaceae fam. nov.) based on ITS sequence and RFLP data.

PubMed

Meyer, Wieland; Gams, Walter

2003-03-01

In a continuation of studies started by de Ruiter et al. (1993), all known species of the Mortierella isabellina-group (Micromucor/Umbelopsis clade of O'Donnell et al. 2001) and a few other Mucorales and species of Mortierella were investigated by RFLP (including ITS1, 5.8S, ITS2 and the 5' end of the large subunit rDNA gene) and ITS1 sequence analyses. This monophyletic group is unrelated to Mortierella and is only distantly related to the core group of the Mucoraceae. M. longicollis falls outside the Umbelopsis clade. Molecular data resolved two subclades within the M. isabellina-group; however, they are not correlated with any differences in sporangial wall and shape, spore pigmentation and shape, or sporangiophore branching. Therefore we subsume all taxa in one genus, Umbelopsis. The new family Umbelopsidaceae and the new combinations U. isabellina, U. ramanniana, and U. autotrophica are proposed.

RFLP Analysis and Allelic Discrimination with Real-Time PCR Using the Human Lactase Persistence Trait: A Pair of Molecular Genetic Investigations

ERIC Educational Resources Information Center

Weinlander, Kenneth M.; Hall, David J.; De Stasio, Elizabeth A.

2010-01-01

We describe here two open-ended laboratory investigations for an undergraduate laboratory course that uses students' DNA as templates for quantitative real-time PCR and for traditional PCR followed by RFLP analysis. Students are captivated by the immediacy of the application and the relevance of the genotypes and traits, lactase persistence or…

Norrie disease: linkage analysis using a 4.2-kb RFLP detected by a human ornithine aminotransferase cDNA probe.

PubMed

Ngo, J T; Bateman, J B; Cortessis, V; Sparkes, R S; Mohandas, T; Inana, G; Spence, M A

1989-05-01

Previous study has shown that the usual DNA marker for Norrie disease, the L1.28 probe which identifies the DXS7 locus, can recombine with the disease locus. In this study, we used a human ornithine aminotransferase (OAT) cDNA which detects OAT-related DNA sequences mapped to the same region on the X chromosome as that of the L1.28 probe to investigate the family with Norrie disease who exhibited the recombinational event. When genomic DNA from this family was digested with the PvuII restriction endonuclease, we found a restriction fragment length polymorphism (RFLP) of 4.2 kb in size. This fragment was absent in the affected males and cosegregated with the disease locus; we calculated a lod score of 0.602, at theta = 0.00. No deletion could be detected by chromosomal analysis or on Southern blots with other enzymes. These results suggest that one of the OAT-related sequences on the X chromosome may be in close proximity to the Norrie disease locus and represent the first report which indicates that the OAT cDNA may be useful for the identification of carrier status and/or prenatal diagnosis.

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

PubMed Central

Wenzl, Peter; Li, Haobing; Carling, Jason; Zhou, Meixue; Raman, Harsh; Paul, Edie; Hearnden, Phillippa; Maier, Christina; Xia, Ling; Caig, Vanessa; Ovesná, Jaroslava; Cakir, Mehmet; Poulsen, David; Wang, Junping; Raman, Rosy; Smith, Kevin P; Muehlbauer, Gary J; Chalmers, Ken J; Kleinhofs, Andris; Huttner, Eric; Kilian, Andrzej

2006-01-01

Background Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding

Associations between vitamin D receptor polymorphisms and susceptibility to ulcerative colitis and Crohn's disease: a meta-analysis.

PubMed

Xue, Le-Ning; Xu, Ke-Qun; Zhang, Wei; Wang, Qiang; Wu, Jia; Wang, Xiao-Yong

2013-01-01

Several polymorphisms have been identified in the vitamin D receptor (VDR) gene, while their roles in the incidence of ulcerative colitis (UC) and Crohn's disease (CD) are conflicting. This meta-analysis was designed to clarify the impact of these polymorphisms on UC and CD risk. The PubMed, Embase, and Cochrane electronic databases were searched from February 1995 to August 2011 for studies on the four VDR polymorphisms: TaqI, BsmI, FokI, and ApaI. Data were extracted and pooled odd ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Nine studies were included. In Asians, the ff genotype of FokI was associated with increased UC risk (OR = 1.65; 95% CI, 1.11- 2.45). The "a" allele carrier status of ApaI appeared to be a protective factor for CD (OR = 0.81; 95% CI, 0.67-0.97). The tt genotype increased the risk of CD in Europeans (OR = 1.23; 95% CI, 1.02-1.49). Moreover, the tt genotype of TaqI in males had a moderate elevated risk of UC (OR = 1.56; 95% CI, 1.02-2.39) and CD (OR = 1.84; 95% CI, 1.19-2.83). The meta-analysis reveals a significant increase in CD risk for Europeans carrying TaqI tt genotype and a significant decrease in CD risk for all carriers of the Apal "a" allele. For Asians, the VDR FokI polymorphism appears to confer susceptibility to UC. For males, the TaqI tt genotype is associated with susceptibilities to both UC and CD. Our study explored the genetic risk prediction in UC and CD, and may provide valuable insights into IBD therapy.

Molecular Characterization and Chronobiology of Hypodermosis in Cattle Slaughtered in the Diyarbakir Province of Turkey.

PubMed

Sayın İpek, Duygu Neval

2016-06-01

The aim of present study was to investigate the chronobiology and identification of Hypoderma species in cattle slaughtered in the Diyarbakir Province of Turkey. In total, 736 hides and subcutaneous tissue of slaughtered cattle were examined for the presence of second- and third-instar larvae in the slaughterhouse between November 2012 and May 2013. Third-instar larvae were collected from the slaughterhouses, and gDNA isolates were examined by PCR-RFLP analysis of the cytochrome c oxidase I (COI) gene of mt-DNA using TaqI enzyme. In total, 62 out of 736 cattle (8.42%) were found to be positive for Hypoderma larvae. A total of 328 (90 second- and 238 third-instar) Hypoderma larvae were detected in the hide and subcutaneous tissue of the back of infested cattle. All the 238 third-instar larvae (100%) were identified as H. bovis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The mean number of Hypoderma larvae in each cow was 5.29% (62/736). In the examined cattle, second-instar larvae were observed starting from the second week of January and subcutaneous nodules were found until the last week of April. H. bovis was the dominant species detected in the Diyarbakir Province.

PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus.

PubMed

Nasri, Tuba; Hedayati, Mohammad Taghi; Abastabar, Mahdi; Pasqualotto, Alessandro C; Armaki, Mojtaba Taghizadeh; Hoseinnejad, Akbar; Nabili, Mojtaba

2015-10-01

Aspergillus species are important agents of life-threatening infections in immunosuppressed patients. Proper speciation in the Aspergilli has been justified based on varied fungal virulence, clinical presentations, and antifungal resistance. Accurate identification of Aspergillus species usually relies on fungal DNA sequencing but this requires expensive equipment that is not available in most clinical laboratories. We developed and validated a discriminative low-cost PCR-based test to discriminate Aspergillus isolates at the species level. The Beta tubulin gene of various reference strains of Aspergillus species was amplified using the universal fungal primers Bt2a and Bt2b. The PCR products were subjected to digestion with a single restriction enzyme AlwI. All Aspergillus isolates were subjected to DNA sequencing for final species characterization. The PCR-RFLP test generated unique patterns for six clinically important Aspergillus species, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus and Aspergillus nidulans. The one-enzyme PCR-RFLP on Beta tubulin gene designed in this study is a low-cost tool for the reliable and rapid differentiation of the clinically important Aspergillus species. Copyright © 2015 Elsevier B.V. All rights reserved.

Haplotype diversity and linkage disequilibrium at DRD2 locus--a study on four population groups of Andhra Pradesh, India.

PubMed

Saraswathy, Kallur Nava; Mukhopadhyay, Rupak; Shukla, Deepti; Kaur, Harpreet; Sachdeva, Mohinder Pal; Rao, A P; Saksena, Deepti; Kalla, Aloke Kumar

2009-02-01

Dopamine receptor D2 (DRD2) is expressed in the central nervous system and has a high affinity for many antipsychotic drugs. Besides several epidemiological investigations on association of DRD2 locus polymorphism(s) with neuropsychiatric problems and addictive behavior, a few polymorphisms in this locus have also been used to understand genomic diversity and population migratory histories globally. The present study attempts to understand the genomic diversity/affinity among four endogamous groups of Andhra Pradesh (India) against the backdrop of diversity studies from other parts of India and the rest of the world, with special reference to DRD2 locus. The four population groups from Adilabad District of Andhra Pradesh, namely, Brahmin (n=50), Nayakpod (n=49), Thoti (n=52), and Kolam (n=53), were included in the study. The DRD2 markers typed for the present study are three biallelic restriction fragments, that is, TaqI A (rs1800497), TaqI B (rs1079597), and TaqI D (rs1800498). Scoring of DRD2 haplotypes with respect to the three TaqI sites shows that five out of eight possible haplotypes are shared by the four populations. Ancestral haplotype B2D2A1 is most frequent among Thotis (0.359). The results of the present study indicate a differential gene flow into South India followed by certain important demographic events resulting in diversified peopling of India.

Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study.

PubMed

Dooley, John J; Sage, Helen D; Clarke, Marie-Anne L; Brown, Helen M; Garrett, Stephen D

2005-05-04

Identification of 10 white fish species associated with U.K. food products was achieved using PCR-RFLP of the mitochondrial cytochrome b gene. Use of lab-on-a-chip capillary electrophoresis for end-point analysis enabled accurate sizing of DNA fragments and identification of fish species at a level of 5% (w/w) in a fish admixture. One restriction enzyme, DdeI, allowed discrimination of eight species. When combined with NlaIII and HaeIII, specific profiles for all 10 species were generated. The method was applied to a range of products and subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine admixture samples was achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials.

Using T-RFLP data on denitrifier community composition to inform understanding of denitrification in stream sediments (Invited)

NASA Astrophysics Data System (ADS)

Wang, S.; Somers, K.; Sudduth, E.; Hassett, B.; Bernhardt, E. S.; Urban, D. L.

2010-12-01

We used terminal restriction fragment length polymorphism (T-RFLP), a molecular fingerprinting method, to characterize denitrifier communities in sediments taken from 48 study streams in North Carolina, USA. In addition to characterizing denitrifier communities, we also used denitrification enzyme activity (DEA) assays to measure potential denitrification rates. Due to differences in watershed land-use, study streams covered a gradient of nitrogen and carbon concentrations, as well as a gradient of contaminant loading from stormwater and sanitary sewers. Nitrogen and carbon (i.e., substrate) concentrations are commonly used to make predictions about denitrification rates in streams. Such models do not take into account denitrifier community composition, which may be an important, independent control of denitrification rates, particularly under stressful conditions (e.g., high contaminant loading) that prevent communities from capitalizing on high substrate availability. Our results indicate that substrate availability by itself was a weak predictor of denitrification rates; the same was also true for denitrifier community composition. However, when both factors were incorporated in a multiple regression model, the percent variation explained increased substantially. These findings suggest that T-RFLP, a relatively cost-effective method, can be used to improve our understanding of controls on denitrification rates in streams with varying watershed land-uses.

Hemi-nested PCR and RFLP methodologies for identifying blood meals of the Chagas disease vector, Triatoma infestans.

PubMed

Roellig, Dawn M; Gomez-Puerta, Luis A; Mead, Daniel G; Pinto, Jesus; Ancca-Juarez, Jenny; Calderon, Maritza; Bern, Caryn; Gilman, Robert H; Cama, Vitaliano A

2013-01-01

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by hematophagous reduviid bugs within the subfamily Triatominae. These vectors take blood meals from a wide range of hosts, and their feeding behaviors have been used to investigate the ecology and epidemiology of T. cruzi. In this study we describe two PCR-based methodologies that amplify a fragment of the 16S mitochondrial rDNA, aimed to improve the identification of blood meal sources for Triatoma infestans: a.--Sequence analyses of two heminested PCRs that allow the identification of mammalian and avian species, and b.--restriction fragment length polymorphism (RFLP) analysis from the mammalian PCR to identify and differentiate multi-host blood meals. Findings from both methodologies indicate that host DNA could be detected and the host species identified in samples from laboratory reared and field collected triatomines. The implications of this study are two-fold. First, these methods can be used in areas where the fauna diversity and feeding behavior of the triatomines are unknown. Secondly, the RFLP method led to the identification of multi-host DNA from T. infestans gut contents, enhancing the information provided by this assay. These tools are important contributions for ecological and epidemiological studies of vector-borne diseases.

Screening of cytoplasmic DNA diversity between and within Lupinus mutabilis Sweet and Lupinus albus sensu lato by restriction fragment length polymorphism (RFLP).

PubMed

Olczak, T; Rurek, M; Jańska, H; Augustyniak, H; Sawicka-Sienkiewicz, E J

2001-01-01

Seven populations and five mutant lines of the Andean lupin and four species from the section Albus were screened for their mitochondrial and chloroplast polymorphisms. For this purpose the RFLP method with EcoRI as a restriction enzyme was used. Lupinus luteus, Lupinus albus and Phaseolus vulgaris organellar clones as well as amplified fragments were used as probes. We found that mitochondrial probes were more suitable than chloroplast probes for identification of inter- and intra-specific variations within the examined material. Most mitochondrial probes differentiate the two species investigated. A high level of mitochondrial polymorphism was observed among the populations of L. mutabilis in contrast to monomorphism among the species in the section Albus. A limited polymorphism was detected between the mutant lines of L. mutabilis. We conclude from this study that the mitochondrial RFLP analysis is a valuable tool for identification of variability among Andean lupin populations.

Development of a PCR-RFLP method based on the transcription elongation factor 1-α gene to differentiate Fusarium graminearum from other species within the Fusarium graminearum species complex.

PubMed

Garmendia, Gabriela; Umpierrez-Failache, Mariana; Ward, Todd J; Vero, Silvana

2018-04-01

Fusarium head blight (FHB) is a destructive disease of cereals crops worldwide and a major food safety concern due to grain contamination with trichothecenes and other mycotoxins. Fusarium graminearum, a member of the Fusarium graminearum species complex (FGSC) is the dominant FHB pathogen in many parts of the world. However, a number of other Fusarium species, including other members of the FGSC, may also be present for example in Argentina, New Zealand, Ethiopia, Nepal, Unites States in cereals such as wheat and barley. Proper species identification is critical to research aimed at improving disease and mycotoxin control programs. Identification of Fusarium species is are often unreliable by traditional, as many species are morphologically cryptic. DNA sequence-based methods offer a reliable means of species identification, but can be expensive when applied to the analyses of population samples. To facilitate identification of the major causative agent of FHB, this work describes an easy and inexpensive method to differentiate F. graminearum from the remaining species within the FGSC and from the other common Fusarium species causing FHB in cereals. The developed method is based on a PCR-RFLP of the transcription elongation factor (TEF 1-α) gene using the restriction enzyme BsaHI. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and RFLP Genotyping of Toxoplasma gondii in Free-Range Chickens (Gallus domesticus) in Grenada, West Indies, Revealed Widespread and Dominance of Clonal Type III Parasites.

PubMed

Chikweto, Alfred; Sharma, Ravindra N; Tiwari, Keshaw P; Verma, Shiv K; Calero-Bernal, Rafael; Jiang, Tiantian; Su, Chunlei; Kwok, Oliver C; Dubey, Jitender P

2017-02-01

The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondii in free-range chickens from Grenada, West Indies. Using the modified agglutination test, antibodies to T. gondii were found in 39 (26.9%) of 145 free-range chickens with titers of 25 in 7 chickens, 50 in 6 chickens, 100 in 2 chickens, and 200 or higher in 24 chickens. The hearts of the 39 seropositive chickens were bioassayed in mice; viable T. gondii was isolated from 20 and further propagated in cell culture. Genotyping of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed 4 genotypes, including ToxoDB PCR-RFLP no. 2 (Type III), no. 7, no. 13, and no. 259 (new). These results indicated that T. gondii population genetics in free-range chickens seems to be moderately diverse with ToxoDB no. 2 (Type III) as the most frequent (15/20 = 75%) compared to other genotypes in Grenada.

PCR/RFLP-based analysis of genetically distinct Plasmodium vivax population of Pvmsp-3α and Pvmsp-3β genes in Pakistan.

PubMed

Khan, Shahid Niaz; Khan, Asif; Khan, Sanaullah; Ayaz, Sultan; Attaullah, Sobia; Khan, Jabbar; Khan, Muhammad Asim; Ali, Ijaz; Shah, Abdul Haleem

2014-09-09

Plasmodium vivax is one of the widespread human malarial parasites accounting for 75% of malaria epidemics. However, there is no baseline information about the status and nature of genetic variation of Plasmodium species circulating in various parts of Pakistan. The present study was aimed at observing the molecular epidemiology and genetic variation of Plasmodium vivax by analysing its merozoite surface protein-3α (msp-3α) and merozoite surface protein-3β (msp-3β) genes, by using suballele, species-specific, combined nested PCR/RFLP detection techniques. A total of 230 blood samples from suspected subjects tested slide positive for vivax malaria were collected from Punjab, Sindh, Khyber Pakhtunkhwa, and Balochistan during the period May 2012 to December 2013. Combined nested PCR/RFLP technique was conducted using Pvmsp-3α and Pvmsp-3β genetic markers to detect extent of genetic variation in clinical isolates of P. vivax in the studied areas of Pakistan. By PCR, P. vivax, 202/230 (87.82%), was found to be widely distributed in the studied areas. PCR/RFLP analysis showed a high range of allelic variations for both msp-3α and msp-3β genetic markers of P. vivax, i.e., 21 alleles for msp-3α and 19 for msp-3β. Statistically a significant difference (p ≤ 0.05) was observed in the genetic diversity of the suballelic variants of msp-3α and msp-3β genes of P. vivax. It is concluded that P. vivax populations are highly polymorphic and diverse allelic variants of Pvmsp-3α and Pvmsp-3β are present in Pakistan.

Association Between Polymorphisms of VDR, COL1A1, and LCT genes and bone mineral density in Belarusian women with severe postmenopausal osteoporosis.

PubMed

Marozik, Pavel; Mosse, Irma; Alekna, Vidmantas; Rudenko, Ema; Tamulaitienė, Marija; Ramanau, Heorhi; Strazdienė, Vaidilė; Samokhovec, Volha; Ameliyanovich, Maxim; Byshnev, Nikita; Gonchar, Alexander; Kundas, Liubov; Zhur, Krystsina

2013-01-01

BACKGROUND AND OBJECTIVE. Variation of osteoporosis in the population is the result of an interaction between the genotype and the environment, and the genetic causes of osteoporosis are being widely investigated. The aim of this study was to analyze the association between the polymorphisms of the vitamin D receptor (VDR), type I collagen (COL1A1), and lactase (LCT) genes and severe postmenopausal osteoporosis as well as bone mineral density (BMD). MATERIAL AND METHODS. A total of 54 women with severe postmenopausal osteoporosis and 77 controls (mean age, 58.3 years [SD, 6.2] and 56.7 years [SD, 7.42], respectively) were included into the study. The subjects were recruited at the City Center for Osteoporosis Prevention (Minsk, Belarus). Dual-energy x-ray absorptiometry was used to measure bone mineral density at the lumbar spine and the femoral neck. Severe osteoporosis was diagnosed in the women with the clinical diagnosis of postmenopausal osteoporosis and at least 1 fragility fracture. The control group included women without osteoporosis. Polymorphic sites in osteoporosis predisposition genes (ApaI, BsmI, TaqI, and Cdx2 of the VDR gene, G2046T of the COL1A1 gene, and T-13910C of the LCT gene) were determined using the polymerase chain reaction on the deoxyribonucleic acid isolated from dried bloodspots. RESULTS. The data showed that the ApaI and BsmI polymorphisms of the VDR gene and T- 13910C of the LCT gene were associated with severe postmenopausal osteoporosis in the analyzed Belarusian women (P<0.01). A statistically significant positive correlation between the VDR risk genotypes ApaI and TaqI and bone mineral density was found (P<0.05). CONCLUSIONS. The findings of this study suggest that at least the ApaI and BsmI polymorphisms of the VDR gene and T-13910C of the LCT gene are associated with the risk of postmenopausal osteoporosis in our sample of the Belarusian women.

Positive association of vitamin D receptor gene variations with multiple sclerosis in South East Iranian population.

PubMed

Narooie-Nejad, Mehrnaz; Moossavi, Maryam; Torkamanzehi, Adam; Moghtaderi, Ali

2015-01-01

Among the factors postulated to play a role in MS susceptibility, the role of vitamin D is outstanding. Since the function of vitamin D receptor (VDR) represents the effect of vitamin D on the body and genetic variations in VDR gene may affect its function, we aim to highlight the association of two VDR gene polymorphisms with MS susceptibility. In current study, we recruited 113 MS patients and 122 healthy controls. TaqI (rs731236) and ApaI (rs7975232) genetic variations in these two groups were evaluated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. All genotype and allele frequencies in both variations showed association with the disease status. However, to find the definite connection between genetic variations in VDR gene and MS disease in a population of South East of Iran, more researches on gene structure and its function with regard to patients' conditions are required.

Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis.

PubMed

Chim Chan, On; Casper, Peter; Sha, Li Qing; Feng, Zhi Li; Fu, Yun; Yang, Xiao Dong; Ulrich, Andreas; Zou, Xiao Ming

2008-06-01

Bacterial community structure is influenced by vegetation, climate and soil chemical properties. To evaluate these influences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from high-plateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced influences than climate and soil chemical properties.

Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP.

PubMed

Montalvo Alvarez, Ana Margarita; Nodarse, Jorge Fraga; Goodridge, Ivón Montano; Fidalgo, Lianet Monzote; Marin, Marcel; Van Der Auwera, Gert; Dujardin, Jean-Claude; Bernal, Iván Darío Velez; Muskus, Carlos

2010-05-01

Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist. Copyright 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Effects of dopamine D2 receptor (DRD2) and transporter (SLC6A3) polymorphisms on smoking cue-induced cigarette craving among African-American smokers.

PubMed

Erblich, J; Lerman, C; Self, D W; Diaz, G A; Bovbjerg, D H

2005-04-01

Cue-induced craving for addictive substances has long been known to contribute to the problem of persistent addiction in humans. Research in animals over the past decade has solidly established the central role of dopamine in cue-induced craving for addictive substances, including nicotine. Analogous studies in humans, however, are lacking, especially among African-American smokers, who have lower quit rates than Caucasian smokers. Based on the animal literature, the study's objective was to test the hypothesis that smokers carrying specific variants in dopamine-related genes previously associated with risk for addictive behaviors would exhibit heightened levels of cigarette craving following laboratory exposure to cues. To this end, cigarette craving was induced in healthy African-American smokers (n=88) through laboratory exposure to smoking cues. Smokers carrying either the DRD2 (D2 dopamine receptor gene) TaqI A1 RFLP or the SLC6A3 (dopamine transporter gene) 9-repeat VNTR polymorphisms had stronger cue-induced cravings than noncarriers (Ps <0.05 and 0.01, respectively). Consistent with the separate biological pathways involved (receptor, transporter), carriers of both polymorphisms had markedly higher craving responses compared to those with neither (P<0.0006), reflecting additive effects. Findings provide support for the role of dopamine in cue-induced craving in humans, and suggest a possible genetic risk factor for persistent smoking behavior in African-American smokers.

Renin Gene Polymorphisms in Bangladeshi Hypertensive Population

PubMed Central

Afruza, Rownock; Islam, Laila N; Banerjee, Sajal; Hassan, Md. Mahbub; Suzuki, Fumiaki; Nabi, AHM Nurun

2014-01-01

Objective: Linkages of renin gene polymorphisms with hypertension have been implicated in several populations with contrasting results. Present study aims to assess the pattern of renin gene polymorphisms in Bangladeshi hypertensive individuals. Methodology: Introns 1, 9 of renin gene and 4063 bases upstream of promoter sequence of renin gene were amplified from the genomic DNA of the total 124 (hypertensive and normotensive) subjects using respective primers. Polymerase chain reaction-based restriction fragment length polymorphisms were performed using BglI, MboI and TaqI restriction enzymes. Results: Homozygosity was common in renin gene regarding BglI (bb=48.4%, Bb=37.9%, BB=13.7%, χ2 =1.91, P>0.05), TaqI (TT=81.5%, Tt=14.5%, tt=4.0%, χ2 =7.50, P<0.01) and MboI (mm=63.7%, Mm=32.3%, MM=4.0%, χ2=0.00, P>0.05) polymorphisms among total study population. For BglI and TaqI genotype distribution, hypertensive subjects (BglI: χ2 =6.66, P<0.05; TaqI: χ2 = 10.28, P<0.005) significantly deviate from Hardy-Weinberg Equilibrium law compared to normotensive subjects (BglI: χ2=0.51, P>0.05; TaqI: χ2=0.20, P>0.05). On the other hand, with respect to MboI polymorphisms of renin gene, only normotensive subjects deviate from the law (patients: χ2=1.28, P>0.05; vs controls: χ2=6.81, P<0.01). In the context of allelic frequency, common T allele was clearly prevalent (T frequency=0.86, t frequency = 0.14) for TaqI, but rare alleles b and m were more frequent for both BglI (b frequency=0.69, B frequency=0.31) and MboI (m frequency=0.80 M frequency=0.20) polymorphisms, respectively. Conclusion: Thus, we report that Bangladeshi hypertensive subjects did not show any distinct pattern of renin gene polymorphisms compared to their healthy control subjects with regard to their genotypic and allelic frequencies. PMID:25057323

Using PCR-RFLP technology to teach single nucleotide polymorphism for undergraduates.

PubMed

Zhang, Bo; Wang, Yan; Xu, Xiaofeng; Guan, Xingying; Bai, Yun

2013-01-01

Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a serial experiment in molecular biology laboratory course to teach single nucleotide polymorphism (SNP) to undergraduate students majoring in molecular biology or genetics. The flanking sequence of rs4382766 was located in Prdx6 gene, which contained a restriction site of SspI, and was used as a target in this lab course. The students could mimic real research by integrating different techniques, such as database retrieving, genomic DNA isolation, PCR, and restriction enzyme assay. This serial experiment of PCR-RFLP helps students set up intact idea of molecular biology and understand the relation among individual experiments. Students were found to be more enthusiastic during the laboratory classes than those in the former curriculum. Copyright © 2013 Wiley Periodicals, Inc.

Multilocus PCR-RFLP profiling in Trypanosoma cruzi I highlights an intraspecific genetic variation pattern.

PubMed

Ramírez, Juan David; Duque, María Clara; Montilla, Marleny; Cucunubá, Zulma M; Guhl, Felipe

2012-12-01

Chagas disease represents a serious problem in public health. This zoonotic pathology is caused by the kinetoplastid Trypanosoma cruzi which displays a high genetic diversity falling into six Discrete Typing Units (TcI-TcVI). In Colombia, the prevalent DTU is TcI with findings of TcII, TcIII and TcIV in low proportions. The aim of this work was to observe the genetic variability within TcI using a multilocus PCR-RFLP strategy. We analyzed 70 single-celled clones from triatomines, reservoirs and humans that were amplified and restricted via ten PCR-RFLPs targets across TcI genome, the restriction fragments were used to construct phylograms according to calculated genetic distances. We obtained five polymorphic targets (1f8, HSP60, HSP70, SAPA and H1) and the consensus tree constructed according to these regions allowed us to observe two well-defined groups with close association to the transmission cycles (domestic/peridomestic and sylvatic) of Chagas disease in Colombia. Our findings allowed us to corroborate the previous reported genotypes based on the intergenic region of mini-exon gene. More studies examining the genetic diversity among T. cruzi I populations must be conducted in order to obtain a better understanding in regions where this DTU is endemic. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of Ancylostoma ceylanicum in children from a tribal community in Tamil Nadu, India using a semi-nested PCR-RFLP tool.

PubMed

George, Santosh; Kaliappan, Saravanakumar Puthupalayam; Kattula, Deepthi; Roy, Sheela; Geldhof, Peter; Kang, Gagandeep; Vercruysse, Jozef; Levecke, Bruno

2015-04-01

It is generally assumed that hookworm infections in humans are caused by Necator americanus and Ancylostoma duodenale. However, previous studies have also reported the presence of the animal hookworm A. ceylanicum in human stools. We determined hookworm infections in children in a tribal community in Tamil Nadu, India, using a semi-nested PCR-RFLP approach. The results indicate that human species account for a majority of the hookworm infections (N. americanus 39/41 [95%]; A. duodenale 6/41 [15%]), whereas the animal hookworm A. ceylanicum only accounts for a minority of the infections (5%; 2/41). The results emphasize the need to consider zoonotic ancylostomiasis while developing strategies to control hookworm infections. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Association of alcohol-metabolizing genes with alcoholism in a Mexican Indian (Otomi) population.

PubMed

Montano Loza, Aldo J; Ramirez Iglesias, Maria Teresa; Perez Diaz, Ivan; Cruz Castellanos, Socorro; Garcia Andrade, Consuelo; Medina Mora, Maria Elena; Robles Díaz, Guillermo; Kershenobich, David; Gutierrez Reyes, Gabriela

2006-06-01

Association studies provide a powerful approach to link DNA variants and genetic predisposition to complex diseases. In this study, we determined the genotype and allelic frequencies of genes encoding enzymes involved in alcohol metabolism in alcoholic and nonalcoholic subjects of related ethnicity. A total of 118 individuals of Otomi Mexican Indian ancestry were included. Fifty-nine were chronic alcoholics according to WHO criteria and alcohol dependents according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM IV) criteria. They were compared to 59 teetotalers or alcohol consumers of <10 g per day. The restriction fragment length polymorphisms analyzed were ADH1B/MaeIII, ALDH2/MboII, CYP2E1/DraI, CYP2E1/RsaI, and CYP2E1/TaqI. Of the studied polymorphisms, a significant difference between alcoholic and nonalcoholic Otomies was observed only in the CYP2E1/TaqI. The common genotype in alcoholics was A1/A2 (54%), and in nonalcoholics the homozygous A2/A2 (63%) (odds ratio [OR]: 0.28; 95% confidence interval [CI]: 0.13-0.60; P=.002). The frequency of the mutant allele A1 was significantly higher in alcoholics than in nonalcoholics (41 vs. 21%; OR: 2.4; 95% CI: 1.3-4.3; P=.003). This documents the presence of a polymorphism of CYP2E1 that is overexpressed in alcoholic Otomies, in which the variant allele (A1 of CYP2E1/TaqI) is associated with increased susceptibility to alcoholism. The appreciation that this finding may be an additional factor contributing to the high frequency of liver cirrhosis in Otomies requires further investigation.

Isolation and RFLP genotyping of Toxoplasma gondii from the mongoose (Herpestes auropunctatus) in Grenada, West Indies.

PubMed

Choudhary, Shanti; Zieger, Ulrike; Sharma, Ravindra N; Chikweto, Alfred; Tiwari, Keshaw P; Ferreira, Leandra R; Oliveira, Solange; Barkley, Lovell J; Verma, Shiv Kumar; Kwok, Oliver C H; Su, Chunlei; Dubey, J P

2013-12-01

Little is known of the genetic diversity and epidemiology of Toxoplasma gondii infection in wildlife in Caribbean Islands. The prevalence and genetic diversity of T. gondii in mongooses (Herpestes auropunctatus) was investigated. During 2011 and 2012, 91 mongooses were trapped in different parts of Grenada, bled, euthanized, and examined at necropsy. Antibodies to T. gondii were found in 27 mongooses tested by the modified agglutination test (cut-off titer 25). Muscles (heart, tongue, neck) of 25 of the seropositive mongooses were bioassayed for T. gondii infection in mice. Viable T. gondii was isolated by bioassay in mice from four mongooses with MAT titers of 1:50 in two, 1:200 for one, and 1:400 for one mongoose. The four T. gondii isolates were further propagated in cell culture. Strain typing of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed one isolate belongs to the Type III (ToxoDB #2) lineage, two to ToxoDB#7 lineage, and one to the ToxoDB #216 lineage. This is the first report of T. gondii isolation and genotyping in H. auropunctatus worldwide.

Regulatory role of Cdx-2 and Taq I polymorphism of vitamin D receptor gene on chemokine expression in pulmonary tuberculosis.

PubMed

Harishankar, M; Selvaraj, P

2016-06-01

Vitamin D receptor (VDR) gene variants have been shown to be regulating the immune response in tuberculosis. We studied the regulatory role of VDR promoter Cdx-2 and 3'UTR TaqI gene variants on chemokine levels from culture filtrate antigen (CFA) stimulated with or without 1,25(OH)2D3 treated peripheral blood mononuclear cells of 50 pulmonary tuberculosis patients (PTB) and 51 normal healthy controls (HCs). In CFA with 1,25(OH)2D3 treated cultures, the MIP-1α, MIP-1β, RANTES levels were significantly decreased in Cdx-2 AA genotype compared to GG genotype, while a significantly increased MIG level was observed in Cdx-2 AA genotype (p<0.05). In TaqI polymorphism, tt genotype significantly decreased MIP-1β and RANTES levels compared to TT genotype. Moreover, a significantly increased level of IP-10 and MIG was observed in TaqI tt genotype compared with TT genotype (p<0.05). The results suggests that the 1,25(OH)2D3 may alter the chemokine response through the VDR polymorphic variants during infection. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Vitamin D receptor variants and uncontrolled asthma.

PubMed

Hutchinson, K; Kerley, C P; Faul, J; Greally, P; Coghlan, D; Louw, M; Elnazir, B; Rochev, Y

2017-11-28

Background. Asthma is a common childhood respiratory disease, affecting around 20% of Irish children. In other populations, vitamin D receptor (VDR) polymorphisms have been associated with asthma risk. We aimed to investigate the association between 2 VDR polymorphisms and uncontrolled paediatric asthma. Methods. 44 asthmatic children and 57 healthy volunteers were studied. The VDR TaqI gene variant in exon 9 (T/C) (rs731236) and ApaI (rs7975232) in intron 8 (C/T) were determined, using TaqMan® Assays. The lung function, serum 25-hydroxyvitamin D (25OHD) levels and other biomarkers of allergy, immunity, airway and systemic inflammation were as-sessed. Results. The distribution of T and C alleles and genotype frequencies differed significantly between asthmatics and controls for both polymorphisms (p < 0.05). A significant association was found between both TaqI [OR = 2.37, 95% CI (1.27 - 4.45), p = 0.007] and ApaI polymorphisms, and asthma risk [OR = 2.93, 95% CI (1.62 - 5.3), p = 0.0004]. No association was observed between genotypes and 25OHD levels, lung function and other biomarkers, with the exception of Interleukin-10 (IL-10) and white blood cells count (WBC). IL-10 levels were lower in asthmatics with TC genotype for TaqI polymorphism (p < 0.01) and were higher in patients with TT genotype for ApaI (p < 0.01). WBC were higher in patients with TC and CC genotypes for TaqI (p < 0.05) and lower in TT genotype for ApaI (p < 0.05). Conclusion. TaqI and ApaI polymorphisms are associated with asthma in Irish children. Further studies are warranted to investigate the importance of decreased IL-10 levels in paediatric asthmatics with specific genotypes.

Strong association between a splice mutation (IVS12+5G{r_arrow}A) and haplotype 6 in hereditary tyrosinemia type I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanguay, R.M.; St-Louis, M.; Gibson, K.

1994-09-01

Hereditary tyrosinemia type I (HT I; McKusick 276700) is a severe inborn error of tyrosine catabolism pathway caused by a deficiency of fumarylacetoacetate hydrolase (FAH). The highest frequency reported is the one in Saguenay-Lac St-Jean (Quebec, Canada) where 1:1,846 births are affected. The FAH gene has been cloned and several mutations have been described. Allele specific oligonucleotide (ASO) hybridization was used to examine the frequency of a splice (IVS12-5G{r_arrow}A) mutation recently reported and RFLP analysis was done to identify haplotypes related to HT I. The splice mutation was found on 45/50 alleles (90%) in patients from SLSJ and 12/66 (18%)more » alleles from patients world-wide. All 25 patients from the SLSJ region were positive with 20 being homozygous, indicating that this mutation is the major cause of HT I in French Canada. Of these 25 patients, 96% were positive for one haplotype called no 6 which is these 25 patients, 96% were positive for one haplotype called no 6 which is identified by TaqI, RsaI, BglII, MspI and KpnI digestions. These data show a really strong association between the mutation (IVS12+5G{r_arrow}A) and haplotype 6. Among our patients from around the world, {approximately}52% were positive for haplotype 6 indicating its strong relation with HT I. These results provide the rationale for DNA-based carrier testing for HT I in the F-C population at risk as well as in HT I patients in general.« less

Mapping of the bcl-2 oncogene on mouse chromosome 1.

PubMed

Mock, B A; Givol, D; D'Hoostelaere, L A; Huppi, K; Seldin, M F; Gurfinkel, N; Unger, T; Potter, M; Mushinski, J F

1988-01-01

Two bcl-2 alleles have been identified in inbred strains of mice by restriction fragment length polymorphism (RFLP). Analysis of a bcl-2 RFLP in a series of bilineal congenic strains (C.D2), developed as a tool for chromosomal mapping studies, revealed linkage of bcl-2 to the Idh-1/Pep-3 region of murine chromosome 1. The co-segregation of bcl-2 alleles with allelic forms of two other chromosome 1 loci, Ren-1,2 and Spna-1, in a set of back-cross progeny, positions bcl-2 7.8 cM centromeric from Ren-1,2.

Possibilities in identification of genomic species of Burkholderia cepacia complex by PCR and RFLP.

PubMed

Navrátilová, Lucie; Chromá, Magdalena; Hanulík, Vojtech; Raclavský, Vladislav

2013-01-01

The strains belonging to Burkholderia cepacia complex are important opportunistic pathogens in immunocompromised patients and cause serious diseases. It is possible to obtain isolates from soil, water, plants and human samples. Taxonomy of this group is difficult. Burkholderia cepacia complex consists of seventeen genomic species and the genetic scheme is based on recA gene. Commonly, first five genomovars occurre in humans, mostly genomovars II and III, subdivision IIIA. Within this study we tested identification of first five genomovars by PCR with following melting analysis and RFLP. The experiments were targeted on eubacterial 16S rDNA and specific gene recA, which allowed identification of all five genomovars. RecA gene appeared as more suitable than 16S rDNA, which enabled direct identification of only genomovars II and V; genomovars I, III and IV were similar within 16S rDNA sequence.

C. trachomatis in female reproductive tract infections and RFLP-based genotyping: a 16-year study from a tertiary care hospital.

PubMed

Gita, Satpathy; Suneeta, Mittal; Anjana, Sharma; Niranjan, Nayak; Sujata, Mohanty; Pandey, R M

2011-01-01

Presence of Chlamydia trachomatis in endocervix was determined in 2466 women attending a tertiary care hospital in New Delhi, India over a period of 16 years, using a monoclonal-based direct immunofluorescence assay, tissue culture isolation, and a conventional PCR assay. Chlamydia antigen could be detected in 391 out of 2466 (15.85%) of patients studied; in 27.27% women with PID, 16.74% women with cervicitis, 16.03% women with infertility, and 12.06% women with adverse pregnancy outcomes, respectively. There was a statistically significant decreasing trend in Chlamydia antigen positivity between the years 1994-1999 and 2000-2004; the apparent decline in antigen positivity between the years 2000-2004 and 2005-2010 was not statistically significant. Antigen detection assay detected equal number of positives as the PCR assay; tissue culture isolation demonstrated lower positivity. In a few representative specimens from cervicitis patients, genotyping was done using RFLP pattern analysis of C. trachomatis MOMP gene amplified by PCR assay, all of these belonged to Chlamydia trachomatis serovar E.

C. trachomatis in Female Reproductive Tract Infections and RFLP-Based Genotyping: A 16-Year Study from a Tertiary Care Hospital

PubMed Central

Gita, Satpathy; Suneeta, Mittal; Anjana, Sharma; Niranjan, Nayak; Sujata, Mohanty; Pandey, R. M.

2011-01-01

Presence of Chlamydia trachomatis in endocervix was determined in 2466 women attending a tertiary care hospital in New Delhi, India over a period of 16 years, using a monoclonal-based direct immunofluorescence assay, tissue culture isolation, and a conventional PCR assay. Chlamydia antigen could be detected in 391 out of 2466 (15.85%) of patients studied; in 27.27% women with PID, 16.74% women with cervicitis, 16.03% women with infertility, and 12.06% women with adverse pregnancy outcomes, respectively. There was a statistically significant decreasing trend in Chlamydia antigen positivity between the years 1994–1999 and 2000–2004; the apparent decline in antigen positivity between the years 2000–2004 and 2005–2010 was not statistically significant. Antigen detection assay detected equal number of positives as the PCR assay; tissue culture isolation demonstrated lower positivity. In a few representative specimens from cervicitis patients, genotyping was done using RFLP pattern analysis of C. trachomatis MOMP gene amplified by PCR assay, all of these belonged to Chlamydia trachomatis serovar E. PMID:21747643

Pros and Cons of Ion-Torrent Next Generation Sequencing versus Terminal Restriction Fragment Length Polymorphism T-RFLP for Studying the Rumen Bacterial Community

PubMed Central

de la Fuente, Gabriel; Belanche, Alejandro; Girwood, Susan E.; Pinloche, Eric; Wilkinson, Toby; Newbold, C. Jamie

2014-01-01

The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T-RFLP). Although absolute number differed, there was a high correlation between NGS and T-RFLP in terms of richness and diversity with R values of 0.836 and 0.781 for richness and Shannon-Wiener index, respectively. Dendrograms for both datasets were also highly correlated (Mantel test = 0.742). Eighteen OTUs and ten genera were significantly impacted by the addition of rumen protozoa, with an increase in the relative abundance of Prevotella, Bacteroides and Ruminobacter, related to an increase in free ammonia levels in the rumen. Our findings suggest that classic fingerprinting methods are still valuable tools to study microbial diversity and structure in complex environments but that NGS techniques now provide cost effect alternatives that provide a far greater level of information on the individual members of the microbial population. PMID:25051490

Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece

PubMed Central

Kizis, Dimosthenis; Natskoulis, Pantelis; Nychas, George-John E.; Panagou, Efstathios Z.

2014-01-01

A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS). Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP), four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immuno Sorbent Assay (ELISA), resulting in significant interspecies differences in OTA production. PMID:24710283

DYZ1 arrays show sequence variation between the monozygotic males

PubMed Central

2014-01-01

Background Monozygotic twins (MZT) are an important resource for genetical studies in the context of normal and diseased genomes. In the present study we used DYZ1, a satellite fraction present in the form of tandem arrays on the long arm of the human Y chromosome, as a tool to uncover sequence variations between the monozygotic males. Results We detected copy number variation, frequent insertions and deletions within the sequences of DYZ1 arrays amongst all the three sets of twins used in the present study. MZT1b showed loss of 35 bp compared to that in 1a, whereas 2a showed loss of 31 bp compared to that in 2b. Similarly, 3b showed 10 bp insertion compared to that in 3a. MZT1a germline DNA showed loss of 5 bp and 1b blood DNA showed loss of 26 bp compared to that of 1a blood and 1b germline DNA, respectively. Of the 69 restriction sites detected in DYZ1 arrays, MboII, BsrI, TspEI and TaqI enzymes showed frequent loss and or gain amongst all the 3 pairs studied. MZT1 pair showed loss/gain of VspI, BsrDI, AgsI, PleI, TspDTI, TspEI, TfiI and TaqI restriction sites in both blood and germline DNA. All the three sets of MZT showed differences in the number of DYZ1 copies. FISH signals reflected somatic mosaicism of the DYZ1 copies across the cells. Conclusions DYZ1 showed both sequence and copy number variation between the MZT males. Sequence variation was also noticed between germline and blood DNA samples of the same individual as we observed at least in one set of sample. The result suggests that DYZ1 faithfully records all the genetical changes occurring after the twining which may be ascribed to the environmental factors. PMID:24495361

Genetic variants of neurotransmitter-related genes and miRNAs in Egyptian autistic patients.

PubMed

Salem, Ahmed M; Ismail, Samira; Zarouk, Waheba A; Abdul Baky, Olwya; Sayed, Ahmed A; Abd El-Hamid, Sawsan; Salem, Sohair

2013-01-01

Autism is a neurodevelopmental disorder with indisputable evidence for a genetic component. This work studied the association of autism with genetic variations in neurotransmitter-related genes, including MAOA uVNTR, MAOB rs1799836, and DRD2 TaqI A in 53 autistic patients and 30 healthy individuals. The study also analyzed sequence variations of miR-431 and miR-21. MAOA uVNTR was genotyped by PCR, MAOB and DRD2 polymorphisms were analyzed by PCR-based RFLP, and miR-431 and miR-21 were sequenced. Low expressing allele of MAOA uVNTR was frequently higher in female patients compared to that in controls (OR = 2.25). MAOB G allele frequency was more significantly increased in autistic patients than in controls (P < 0.001 for both males and females). DRD2 A1+ genotype increased autism risk (OR = 5.1). Severity of autism tends to be slightly affected by MAOA/B genotype. Plasma MAOB activity was significantly reduced in G than in A allele carrying males. There was no significant difference in patients and maternal plasma MAOA/B activity compared to controls. Neither mutations nor SNPs in miR-431 and miR-21 were found among studied patients. This study threw light on some neurotransmitter-related genes suggesting their potential role in Autism pathogenesis that warrants further studies and much consideration.

Differentiation of strains from the Bacillus cereus group by RFLP-PFGE genomic fingerprinting.

PubMed

Otlewska, Anna; Oltuszak-Walczak, Elzbieta; Walczak, Piotr

2013-11-01

Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus belong to the B. cereus group. The last three species are characterized by different phenotype features and pathogenicity spectrum, but it has been shown that these species are genetically closely related. The macrorestriction analysis of the genomic DNA with the NotI enzyme was used to generate polymorphism of restriction profiles for 39 food-borne isolates (B. cereus, B. mycoides) and seven reference strains (B. mycoides, B. thuringiensis, B. weihenstephanensis, and B. cereus). The PFGE method was applied to differentiate the examined strains of the B. cereus group. On the basis of the unweighted pair group method with the arithmetic mean method and Dice coefficient, the strains were divided into five clusters (types A-E), and the most numerous group was group A (25 strains). A total of 21 distinct pulsotypes were observed. The RFLP-PFGE analysis was successfully used for the differentiation and characterization of B. cereus and B. mycoides strains isolated from different food products. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Giardia duodenalis genotypes in domestic and wild animals from Romania identified by PCR-RFLP targeting the gdh gene.

PubMed

Adriana, Gyӧrke; Zsuzsa, Kalmár; Mirabela Oana, Dumitrache; Mircea, Gherman Călin; Viorica, Mircean

2016-02-15

Sixty Giardia duodenalis isolates from domestic (n=49) and wild (n=11) animals (dogs, cats, deers, wolves, raccoon dog and muskrat) were analysed by PCR-RFLP at glutamate dehydrogenase locus (gdh). The isolates were obtained from positive feces samples for Giardia cysts analysed by flotation technique with saturated sodium chloride solution (specific gravity 1.28). Three G. duodenalis genotypes were identified: C (10/60; 16.7%); D (42/60; 70.0%); and E (7/60; 11.7%). In dogs all three genotypes were found, with the following prevalences: 76.9% genotype D (30/39); 23.1% C (9/39); 2.6% genotype E (1/39). One dog was co-infected with C and D genotypes. In cats we identified only G. duodenalis genotype D. Wolves and raccoon dog harbored infection with G. duodenalis genotype D, deers with E type and muskrat C type. This is the first study regarding genotyping of G. duodenalis in cats and wild animals from Romania. To the best of our knowledge, this is the first report of assemblages E in roe deers; assemblage C in wolves and muskrat; and assemblage D in raccoon dog. Copyright © 2016. Published by Elsevier B.V.

A Simple Restriction Fragment Length Polymorphism-Based Strategy That Can Distinguish the Internal Genes of Human H1N1, H3N2, and H5N1 Influenza A Viruses

PubMed Central

Cooper, Lynn A.; Subbarao, Kanta

2000-01-01

A simple molecular technique for rapid genotyping was developed to monitor the internal gene composition of currently circulating influenza A viruses. Sequence information from recent H1N1, H3N2, and H5N1 human virus isolates was used to identify conserved regions within each internal gene, and gene-specific PCR primers capable of amplifying all three virus subtypes were designed. Subtyping was based on subtype-specific restriction fragment length polymorphism (RFLP) patterns within the amplified regions. The strategy was tested in a blinded fashion using 10 control viruses of each subtype (total, 30) and was found to be very effective. Once standardized, the genotyping method was used to identify the origin of the internal genes of 51 influenza A viruses isolated from humans in Hong Kong during and immediately following the 1997–1998 H5N1 outbreak. No avian-human or H1-H3 reassortants were detected. Less than 2% (6 of 486) of the RFLP analyses were inconclusive; all were due to point mutations within a restriction site. The technique was also used to characterize the internal genes of two avian H9N2 viruses isolated from children in Hong Kong during 1999. PMID:10878047

Polymorphism in exon 2 encoding the putative ligand binding pocket of the bovine insulin-like growth factor 1 receptor affects milk traits in four different cattle breeds.

PubMed

Szewczuk, M

2017-02-01

As a member of the somatotropic axis, insulin-like growth factor I receptor (IGF1R) seems to be a promising candidate gene. Two silent polymorphisms, identified by MspI and TaqI restriction enzymes, were selected within exon 2, encoding the majority of the putative ligand binding pocket. A total of 1169 cows of four pure breeds (Polish Holstein Friesian, Montbeliarde, Jersey and Holstein Friesian) were genotyped. The T (IGF1R/e2/MspI) and G (IGF1R/e2/TaqI) alleles were found to be prevalent. Three combinations of genotypes (TT/GG, TT/AG and CT/GG) were associated with the highest productivity (milk, protein and fat yields) among all breeds under study, as opposed to individuals carrying the worst CC/AA combination. In view of the specific structure of the ligand binding pocket and the significance of insulin-like growth factor I signalling promoting the development and differentiation in a variety of tissues (not only limited to mammary gland), the existence of missense mutation is unlikely. Potential mutations are likely limited to mRNA transcription and further post-transcriptional modifications. Further investigations should follow searching for the most useful IGF1R haplotypes, associated with higher milk production traits, exerting at the same time positive or neutral impact on health and welfare of individuals. © 2016 Blackwell Verlag GmbH.

MELAS syndrome in a patient with a point mutation in MTTS1.

PubMed

Lindberg, C; Moslemi, A-R; Oldfors, A

2008-02-01

BACKGROUND, OBJECTIVE AND METHODS: We describe a female patient with a mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome. As a child, she developed epilepsy and stroke-like episodes giving cognitive impairment and ataxia but no hearing impairment. At the age of 44 years, she suffered a cerebral sinus thrombosis which was warfarin treated. One month later, she developed an episode of severe acidosis associated with encephalopathy and myelopathy. She was found to harbour a 7512T>C mutation in the mitochondrial encoded tRNA(Ser(UCN)) gene (MTTS1). The mutation load was 91% in muscle and 24% in blood. Enzyme histochemical analysis of the muscle tissue showed numerous cytochrome c oxidase (COX)-negative fibres. Restriction fragment length polymorphism (RFLP) analysis of single muscle fibres showed significantly higher level (median 97%, range: 94-99%) of the mutation in the COX-negative fibres compared with COX-positive fibres (median 36%, range: 12-91%), demonstrating the pathogenic effect of the mutation. Different levels of heteroplasmy (range 34-61%) were detected in hair shafts analysed by RFLP. This case adds to the spectrum of clinical presentations, i.e. sinus thrombosis, in patients having MTTS1 mutations.

A comprehensive experiment for molecular biology: Determination of single nucleotide polymorphism in human REV3 gene using PCR-RFLP.

PubMed

Zhang, Xu; Shao, Meng; Gao, Lu; Zhao, Yuanyuan; Sun, Zixuan; Zhou, Liping; Yan, Yongmin; Shao, Qixiang; Xu, Wenrong; Qian, Hui

2017-07-08

Laboratory exercise is helpful for medical students to understand the basic principles of molecular biology and to learn about the practical applications of molecular biology. We have designed a lab course on molecular biology about the determination of single nucleotide polymorphism (SNP) in human REV3 gene, the product of which is a subunit of DNA polymerase ζ and SNPs in this gene are associated with altered susceptibility to cancer. This newly designed experiment is composed of three parts, including genomic DNA extraction, gene amplification by PCR, and genotyping by RFLP. By combining these activities, the students are not only able to learn a series of biotechniques in molecular biology, but also acquire the ability to link the learned knowledge with practical applications. This comprehensive experiment will help the medical students improve the conceptual understanding of SNP and the technical understanding of SNP detection. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):299-304, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Differentiation of Cannabis subspecies by THCA synthase gene analysis using RFLP.

PubMed

Cirovic, Natasa; Kecmanovic, Miljana; Keckarevic, Dusan; Keckarevic Markovic, Milica

2017-10-01

Cannabis sativa subspecies, known as industrial hemp (C. sativa sativa) and marijuana (C. sativa indica) show no evident morphological distinctions, but they contain different levels of psychoactive Δ-9-tetrahidrocanabinol (THC), with considerably higher concentration in marijuana than in hemp. C. sativa subspecies differ in sequence of tetrahydrocannabinolic acid (THCA) synthase gene, responsible for THC production, and only one active copy of the gene, distinctive for marijuana, is capable of producing THC in concentration more then 0,3% in dried plants, usually punishable by the law. Twenty different samples of marijuana that contain THC in concentration more then 0,3% and three varieties of industrial hemp were analyzed for presence of an active copy of THCA synthase gene using in-house developed restriction fragment length polymorphism (RFLP) method All twenty samples of marijuana were positive for the active copy of THCA synthase gene, 16 of them heterozygous. All three varieties of industrial hemp were homozygous for inactive copy. An algorithm for the fast and accurate forensic analysis of samples suspected to be marijuana was constructed, answering the question if an analyzed sample is capable of producing THC in concentrations higher than 0.3%. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

RFLP-facilitated investigation of the quantitative resistance of rice to brown planthopper ( Nilaparvata lugens).

PubMed

Xu, X. F.; Mei, H. W.; Luo, L. J.; Cheng, X. N.; Li, Z. K.

2002-02-01

Quantitative trait loci (QTLs), conferring quantitative resistance to rice brown planthopper (BPH), were investigated using 160 F(11) recombinant inbred lines (RILs) from the Lemont/Teqing cross, a complete RFLP map, and replicated phenotyping of seedbox inoculation. The paternal indica parent, Teqing, was more-resistant to BPH than the maternal japonica parent, Lemont. The RILs showed transgressive segregation for resistance to BPH. Seven main-effect QTLs and many epistatic QTL pairs were identified and mapped on the 12 rice chromosomes. Collectively, the main-effect and epistatic QTLs accounted for over 70% of the total variation in damage scores. Teqing has the resistance allele at four main-effect QTLs, and the Lemont allele resulted in resistance at the other three. Of the main-effect QTLs identified, QBphr5b was mapped to the vicinity of gl1, a major gene controlling leaf and stem pubescence. The Teqing allele controlling leaf and stem pubescence was associated with resistance, while the Lemont allele for glabrous stem and leaves was associated with susceptibility, indicating that this gene may have contributed to resistance through antixenosis. Similar to the reported BPH resistance genes, the other six detected main-effect QTLs were all mapped to regions where major disease resistance genes locate, suggesting they might have contributed either to antibiosis or tolerance. Our results indicated that marker-aided pyramiding of major resistance genes and QTLs should provide effective and stable control over this devastating pest.

Common rs5918 (PlA1/A2) polymorphism in the ITGB3 gene and risk of coronary artery disease

PubMed Central

Heidari, Mohammad Mehdi; Soheilyfar, Sorour

2016-01-01

Introduction The T to C transition at nucleotide 1565 of the human glycoprotein IIIa (ITGB3) gene represents a genetic polymorphism (PlA1/A2) that can influence both platelet activation and aggregation and that has been associated with many types of disease. Here, we present a newly designed multiplex tetra-primer amplification refractory mutation system – polymerase chain reaction (T-ARMS-PCR) for genotyping a single nucleotide polymorphism (SNP) (dbSNP ID: rs5918) in the human ITGB3 gene. Material and methods We set up T-ARMS-PCR for the rs5918 SNP in a single-step PCR and the results were validated by the PCR-RFLP method in 132 coronary artery disease (CAD) patients and 122 unrelated healthy individuals. Results Full accordance was found for genotype determination by the PCR-RFLP method. The multiple logistic regression analysis showed a significant association of the rs5918 polymorphism and CAD according to dominant and recessive models (dominant model OR: 2.40, 95% CI: 1.33–4.35; p = 0.003, recessive model OR: 4.71, 95% CI: 1.32–16.80; p = 0.0067). Conclusions Our T-ARMS-PCR in comparison with RFLP and allele-specific PCR is more advantageous because this PCR method allows the evaluation of both the wild type and the mutant allele in the same tube. Our results suggest that the rs5918 (PlA1/A2) polymorphism in the ITGB3 gene may contribute to the susceptibility of sporadic Iranian coronary artery disease (CAD) patients. PMID:28905013

Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy.

PubMed

Neela, Venkata Sanjeev Kumar; Suryadevara, Naveen Chandra; Shinde, Vidya Gouri; Pydi, Satya Sudheer; Jain, Suman; Jonnalagada, Subbanna; Singh, Surya Satyanarayana; Valluri, Vijaya Lakshmi; Anandaraj, M P J S

2015-06-01

Vitamin D Receptor (VDR) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive immune responses through its ligand and polymorphisms in VDR gene may affect its regulatory function. To investigate the association of three VDR gene polymorphisms (TaqI rs731236, FokI rs2228570 and ApaI rs7975232) with leprosy. The study group includes 404 participants of which 222 were leprosy patients (paucibacillary=87, multibacillary=135) and 182 healthy controls. Genotyping was done using PCR-RFLP technique. Statistical analysis was performed using SNP Stats and PLINK software. The VDR FokI (rs2228570) ff genotype, ApaI (rs7975232) AA, Aa genotype and haplotype T-f-a, T-F-A were positively associated with leprosy when compared to healthy controls. The two variants at Fok and Apa positions in VDR gene are significantly associated with leprosy. Genotypes at FokI (ff), ApaI (aa) and haplotype (T-F-a, T-f-a) may contribute to the risk of developing leprosy by altering VDR phenotype/levels subsequently modulation of immune response. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Polymorphisms in VDR gene in Tunisian postmenopausal women are associated with osteopenia phenotype.

PubMed

Sassi, R; Sahli, H; Souissi, C; Sellami, S; Ben Ammar El Gaaied, A

2015-01-01

Osteopenia is characterized by intermediate values of bone mineral density (BMD) as compared to normal and osteoporotic subjects. BMD, a surrogate phenotype for osteoporosis, is influenced in part by genetic factors. Among the genes associated with BMD, the vitamin D receptor (VDR) was the first gene studied as a potential candidate associated with BMD in adult and postmenopausal bone loss. However, results are controversial. To determine whether VDR polymorphisms ApaI and TaqI are associated with BMD, osteopenia, osteoporosis and low-impact fracture risk in North Africans, these genotypes were analyzed in 566 postmenopausal Tunisian women. In postmenopausal Tunisian women, the GT ApaI genotype seems to be protective against osteoporosis development (p = 0.02; odds ratio = 0.54). Moreover, the presence of the combined GT/TT genotype of ApaI and TaqI polymorphisms is more frequent in normal BMD women than in osteoporotic women (p = 0.00; odds ratio = 0.41). Interestingly, the GG ApaI genotype is associated with osteopenia development (p = 0.02; odds ratio = 1.86) and also the TT TaqI polymorphism (p = 0.02; odds ratio = 1.53). The GG ApaI genotype is associated with a three times risk of vertebral fracture. The ApaI polymorphism showed an association with osteopenia and low-impact vertebral fracture incidence but not with osteoporosis. The TaqI polymorphism is associated specifically with the osteopenia phenotype. The presence of the two polymorphisms increases the risk to develop osteopenia in postmenopausal Tunisian women. Osteopenia seems to be genetically determined. However, osteoporosis is the result of interaction between genetic and environmental factors.

Novel compound heterozygous mutations in MYO7A in a Chinese family with Usher syndrome type 1

PubMed Central

Liu, Fei; Li, Pengcheng; Liu, Ying; Li, Weirong; Wong, Fulton; Du, Rong; Wang, Lei; Li, Chang; Jiang, Fagang; Tang, Zhaohui

2013-01-01

Purpose To identify the disease-causing mutation(s) in a Chinese family with autosomal recessive Usher syndrome type 1 (USH1). Methods An ophthalmic examination and an audiometric test were conducted to ascertain the phenotype of two affected siblings. The microsatellite marker D11S937, which is close to the candidate gene MYO7A (USH1B locus), was selected for genotyping. From the DNA of the proband, all coding exons and exon-intron boundaries of MYO7A were sequenced to identify the disease-causing mutation(s). Restriction fragment length polymorphism (RFLP) analysis was performed to exclude the alternative conclusion that the mutations are non-pathogenic rare polymorphisms. Results Based on severe hearing impairment, unintelligible speech, and retinitis pigmentosa, a clinical diagnosis of Usher syndrome type 1 was made. The genotyping results did not exclude the USH1B locus, which suggested that the MYO7A gene was likely the gene associated with the disease-causing mutation(s) in the family. With direct DNA sequencing of MYO7A, two novel compound heterozygous mutations (c.3742G>A and c.6051+1G>A) of MYO7A were identified in the proband. DNA sequence analysis and RFLP analysis of other family members showed that the mutations cosegregated with the disease. Unaffected members, including the parents, uncle, and sister of the proband, carry only one of the two mutations. The mutations were not present in the controls (100 normal Chinese subjects=200 chromosomes) according to the RFLP analysis. Conclusions In this study, we identified two novel mutations, c.3742G>A (p.E1248K) and c.6051+1G>A (donor splice site mutation in intron 44), of MYO7A in a Chinese non-consanguineous family with USH1. The mutations cosegregated with the disease and most likely cause the phenotype in the two affected siblings who carry these mutations compound heterozygously. Our finding expands the mutational spectrum of MYO7A. PMID:23559863

Novel compound heterozygous mutations in MYO7A in a Chinese family with Usher syndrome type 1.

PubMed

Liu, Fei; Li, Pengcheng; Liu, Ying; Li, Weirong; Wong, Fulton; Du, Rong; Wang, Lei; Li, Chang; Jiang, Fagang; Tang, Zhaohui; Liu, Mugen

2013-01-01

To identify the disease-causing mutation(s) in a Chinese family with autosomal recessive Usher syndrome type 1 (USH1). An ophthalmic examination and an audiometric test were conducted to ascertain the phenotype of two affected siblings. The microsatellite marker D11S937, which is close to the candidate gene MYO7A (USH1B locus), was selected for genotyping. From the DNA of the proband, all coding exons and exon-intron boundaries of MYO7A were sequenced to identify the disease-causing mutation(s). Restriction fragment length polymorphism (RFLP) analysis was performed to exclude the alternative conclusion that the mutations are non-pathogenic rare polymorphisms. Based on severe hearing impairment, unintelligible speech, and retinitis pigmentosa, a clinical diagnosis of Usher syndrome type 1 was made. The genotyping results did not exclude the USH1B locus, which suggested that the MYO7A gene was likely the gene associated with the disease-causing mutation(s) in the family. With direct DNA sequencing of MYO7A, two novel compound heterozygous mutations (c.3742G>A and c.6051+1G>A) of MYO7A were identified in the proband. DNA sequence analysis and RFLP analysis of other family members showed that the mutations cosegregated with the disease. Unaffected members, including the parents, uncle, and sister of the proband, carry only one of the two mutations. The mutations were not present in the controls (100 normal Chinese subjects=200 chromosomes) according to the RFLP analysis. In this study, we identified two novel mutations, c.3742G>A (p.E1248K) and c.6051+1G>A (donor splice site mutation in intron 44), of MYO7A in a Chinese non-consanguineous family with USH1. The mutations cosegregated with the disease and most likely cause the phenotype in the two affected siblings who carry these mutations compound heterozygously. Our finding expands the mutational spectrum of MYO7A.

Mitochondrial DNA diversity of orchid bee Euglossa fimbriata (Hymenoptera: Apidae) populations assessed by PCR-RFLP.

PubMed

Suzuki, Karen M; Arias, Maria C; Giangarelli, Douglas C; Freiria, Gabriele A; Sofia, Silvia H

2010-04-01

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085-0.289) and nucleotide diversity (0.000-0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (phi(st) = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (phi(st) = 0.161-0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.

Microbial structural diversity estimated by dilution-extinction of phenotypic traits and T-RFLP analysis along a land-use intensification gradient

NASA Technical Reports Server (NTRS)

Gomez, Elena del V.; Garland, Jay L.; Roberts, Michael S.

2004-01-01

The present work tested whether the relationship between functional traits and inoculum density reflected structural diversity in bacterial communities from a land-use intensification gradient applying a mathematical model. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also performed to provide an independent assessment of species richness. Successive 10-fold dilutions of a soil suspension were inoculated onto Biolog GN(R) microplates. Soil bacterial density was determined by total cell and plate counts. The relationship between phenotypic traits and inoculum density fit the model, allowing the estimation of maximal phenotypic potential (Rmax) and inoculum density (KI) at which Rmax will be half-reduced. Though Rmax decreased with time elapsed since clearing of native vegetation, KI remained high in two of the disturbed sites. The genetic pool of bacterial community did not experience a significant reduction, but the active fraction responding in the Biolog assay was adversely affected, suggesting a reduction in the functional potential. c2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonella koehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats

PubMed Central

Chomel, Bruno B.; Molia, Sophie; Kasten, Rickie W.; Borgo, Gina M.; Stuckey, Matthew J.; Maruyama, Soichi; Chang, Chao-chin; Haddad, Nadia; Koehler, Jane E.

2016-01-01

Domestic cats are the natural reservoir of Bartonella henselae, B. clarridgeiae and B. koehlerae. To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions (Puma concolor) and 19 bobcats (Lynx rufus). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B. henselae genotype II. All other isolates were distinguished from B. henselae and B. koehlerae by PCR-RFLP of the gltA gene using endonucleases HhaI, TaqI and AciI, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B. henselae, B. koehlerae and B. clarridgeiae. Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B. henselae and B. koehlerae. Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B. koehlerae. We propose naming the mountain lion isolates B. koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B. koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B. koehlerae as B. koehlerae subsp. koehlerae. The mode of transmission and the zoonotic potential of these new Bartonella subspecies remain to be determined. PMID:26981874

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR-RFLP in Iran.

PubMed

Momeni, Zohreh; Sadraei, Javid; Kazemi, Bahram; Dalimi, Abdolhossein

2015-12-01

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral, parasitic sexually transmitted infection in the world. At present, little is known regarding the degree of strain variability of T. vaginalis. A classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, drug resistance, pathogenesis and transmission of T. vaginalis. Eight different types of actin genes have been identified by PCR-RFLP in T. vaginalis; the purpose of this study is to determine the genotypes of this parasite in Karaj city, Iran. Forty-five clinical T. vaginalis isolates from vaginal secretions and urine sediment were collected from Karaj city from 2012 through 2014. DNA was extracted and the actin gene was amplified by nested-PCR; all samples were positive. To determine the genetic differences, sequencing on seven samples was conducted. Then, all PCR products were digested with HindII, MseI, and RsaI restriction enzymes. Of 45 isolates, 23 samples (51.1%) were of actin genotype G, 11 samples (24.4%) of genotype E, six samples (13.3%) of genotype H, three samples (6.6%) of genotype I, and two samples (4.4%) were mixed genotypes of G and E. Genetic diversity of T. vaginalis isolates is notable. The actin genotype G may be the dominant genotype in Karaj city, Iran. Copyright © 2015 Elsevier Inc. All rights reserved.

Interaction between ALDH2*1*1 and DRD2/ANKK1 TaqI A1A1 genes may be associated with antisocial personality disorder not co-morbid with alcoholism.

PubMed

Lu, Ru-Band; Lee, Jia-Fu; Huang, San-Yuan; Lee, Sheng-Yu; Chang, Yun-Hsuan; Kuo, Po-Hsiu; Chen, Shiou-Lan; Chen, Shih-Heng; Chu, Chun-Hsien; Lin, Wei-Wen; Wu, Pei-Lin; Ko, Huei-Chen

2012-09-01

Previous studies on acetaldehyde dehydrogenase 2 (ALDH2) focused on drinking behavior or alcoholism because the ALDH2*2 allele protects against the risk of developing alcoholism. The mechanism provides that the ALDH2 gene's protective effect is also involved in dopamine metabolism. The interaction of the ALDH2 gene with neurotransmitters, such as dopamine, is suggested to be related to alcoholism. Because alcoholism is often co-morbid with antisocial personality disorder (ASPD), previous association studies on antisocial alcoholism cannot differentiate whether those genes relate to ASPD with alcoholism or ASPD only. This study examined the influence of the interaction effect of the ALDH2*1*1, *1*2 or *2*2 polymorphisms with the dopamine 2 receptor (DRD2) Taq I polymorphism on ASPD. Our 541 Han Chinese male participants were classified into three groups: antisocial alcoholism (ASPD co-morbid with alcohol dependence, antisocial ALC; n = 133), ASPD without alcoholism (ASPD not co-morbid with alcohol dependence, antisocial non-ALC; n = 164) and community controls (healthy volunteers from the community; n = 244). Compared with healthy controls, individuals with the DRD2 A1/A1 and the ALDH2*1/*1 genotypes were at a 5.39 times greater risk for antisocial non-ALC than were those with other genotypes. Our results suggest that the DRD2/ANKK1 and ALDH2 genes interacted in the antisocial non-ALC group; a connection neglected in previous studies caused by not separating antisocial ALC from ASPD. Our study made this distinction and showed that these two genes may be associated ASPD without co-morbid alcoholism. © 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction.

RFLP Mapping of Genes Conferring Complete and Partial Resistance to Blast in a Durably Resistant Rice Cultivar

PubMed Central

Wang, G. L.; Mackill, D. J.; Bonman, J. M.; McCouch, S. R.; Champoux, M. C.; Nelson, R. J.

1994-01-01

Moroberekan, a japonica rice cultivar with durable resistance to blast disease in Asia, was crossed to the highly susceptible indica cultivar, CO39, and 281 F(7) recombinant inbred (RI) lines were produced by single seed descent. The population was evaluated for blast resistance in the greenhouse and the field, and was analyzed with 127 restriction fragment length polymorphism (RFLP) markers. Two dominant loci associated with qualitative resistance to five isolates of the fungus were tentatively named Pi-5(t) and Pi-7(t). They were mapped on chromosomes 4 and 11, respectively. To identify quantitative trait loci (QTLs) affecting partial resistance, RI lines were inoculated with isolate PO6-6 of Pyricularia oryzae in polycyclic tests. Ten chromosomal segments were found to be associated with effects on lesion number (P < 0.0001 and LOD > 6.0). Three of the markers associated with QTLs for partial resistance had been reported to be linked to complete blast resistance in previous studies. QTLs identified in greenhouse tests were good predictors of blast resistance at two field sites. This study illustrates the usefulness of RI lines for mapping a complex trait such as blast resistance and suggests that durable resistance in the traditional variety, Moroberekan, involves a complex of genes associated with both partial and complete resistance. PMID:7912216

LD2SNPing: linkage disequilibrium plotter and RFLP enzyme mining for tag SNPs

PubMed Central

Chang, Hsueh-Wei; Chuang, Li-Yeh; Chang, Yan-Jhu; Cheng, Yu-Huei; Hung, Yu-Chen; Chen, Hsiang-Chi; Yang, Cheng-Hong

2009-01-01

Background Linkage disequilibrium (LD) mapping is commonly used to evaluate markers for genome-wide association studies. Most types of LD software focus strictly on LD analysis and visualization, but lack supporting services for genotyping. Results We developed a freeware called LD2SNPing, which provides a complete package of mining tools for genotyping and LD analysis environments. The software provides SNP ID- and gene-centric online retrievals for SNP information and tag SNP selection from dbSNP/NCBI and HapMap, respectively. Restriction fragment length polymorphism (RFLP) enzyme information for SNP genotype is available to all SNP IDs and tag SNPs. Single and multiple SNP inputs are possible in order to perform LD analysis by online retrieval from HapMap and NCBI. An LD statistics section provides D, D', r2, δQ, ρ, and the P values of the Hardy-Weinberg Equilibrium for each SNP marker, and Chi-square and likelihood-ratio tests for the pair-wise association of two SNPs in LD calculation. Finally, 2D and 3D plots, as well as plain-text output of the results, can be selected. Conclusion LD2SNPing thus provides a novel visualization environment for multiple SNP input, which facilitates SNP association studies. The software, user manual, and tutorial are freely available at . PMID:19500380

Cutaneous leishmaniasis in the central provinces of Hama and Edlib in Syria: Vector identification and parasite typing.

PubMed

Haddad, Nabil; Saliba, Hanadi; Altawil, Atef; Villinsky, Jeffrey; Al-Nahhas, Samar

2015-10-12

Cutaneous leishmaniasis is a disease transmitted by sand fly bites. This disease is highly prevalent in Syria where Leishmania major and Leishmania tropica are the known aetiological agents. In 2011, more than 58,000 cases were reported in the country by the Ministry of Health. The central region of the country harbors 20 % of the reported cases. However, the epidemiology of the disease in this area is not well understood. An epidemiological survey was conducted in 2010 to identity the circulating parasite and the sand fly vector in the central provinces of Edlib and Hama. Sand fly specimens were collected using CDC light traps and identified morphologically. Total DNA was extracted from the abdomens of female specimens and from Giemsa-stained skin lesion smears of 80 patients. Leishmania parasites were first identified by sequencing the ITS1 gene amplicons. Then polymorphism analysis was performed using the RFLP technique. A total of 2142 sand flies were collected. They belonged to eight species, among which Phlebotomus sergenti and Phlebotomus papatasi were the most predominant. L. tropica ITS1 gene was amplified from two pools of P. sergenti specimens and from skin smears of cutaneous leishmaniasis patients. This suggests that P. sergenti is the potential vector species in the study area. The digestion profiles of the obtained amplicons by TaqI restriction enzyme were identical for all analysed L. tropica parasites. Moreover, L. infantum ITS1 gene was amplified from two pools of Phlebotomus tobbi in the relatively humid zone of Edlib. L. tropica is confirmed to be the aetiological agent of cutaneous leishmaniasis cases in the central provinces. RFLP technique failed to show any genetic heterogeneity in the ITS1 gene among the tested parasites. The molecular detection of this parasite in human skin smears and in P. sergenti supports the vector status of this species in the study area. The detection of L. infantum in P. tobbi specimens indicates a potential

Specific detection of benzimidazole resistance in Colletotrichum gloeosporioides from fruit crops by PCR-RFLP.

PubMed

Chung, Wen-Hsin; Chung, Wen-Chuan; Peng, Mun-Tsu; Yang, Hong-Ren; Huang, Jenn-Wen

2010-02-28

Anthracnose diseases, caused by Colletotrichum gloeosporioides, are a worldwide problem and are especially important in Taiwan owing to the severe economic damage they cause to tropical fruits that are grown for local consumption and export. Benzimidazoles are systemic fungicides widely used for controlling these diseases in Taiwan. Thirty-one isolates of C. gloeosporioides from mango and strawberry grown in Taiwan were examined for their sensitivity to benzimidazole fungicides. The responses of the isolates grown on benzimidazole-amended culture media were characterized as sensitive, moderately resistant, resistant or highly resistant. Analysis of point mutations in the beta-tubulin gene by DNA sequencing of PCR-amplified fragments revealed a substitution of GCG for GAG at codon 198 in resistant and highly resistant isolates and a substitution of TAC for TTC at codon 200 in moderately resistant isolates. A set of specific primers, TubGF1 and TubGR, was designed to amplify a portion of the beta-tubulin gene for the detection of benzimidazole-resistant C. gloeosporioides. Bsh1236I restriction maps of the amplified beta-tubulin gene showed that the resistant isolate sequence, but not the sensitive isolate sequence, was cut. The PCR restriction fragment length polymorphism (PCR-RFLP) was validated to detect benzimidazole-resistant and benzimidazole-sensitive C. gloeosporioides isolates recovered from avocado, banana, carambola, dragon fruit, grape, guava, jujube, lychee, papaya, passion fruit and wax apple. This method has the potential to become a valuable tool for monitoring the occurrence of benzimidazole-resistant C. gloeosporioides and for assessment of the need for alternative management practices. Copyright 2009 Elsevier B.V. All rights reserved.

D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.

PubMed

Dagar, Sumit S; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K

2011-09-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing

PubMed Central

Green-Engert, Rebecca; Hoelzle, Katharina; Zeller, Ellen; Seifert, Jana; Hoelzle, Ludwig E.; Rodehutscord, Markus

2015-01-01

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information. PMID:26588075

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing.

PubMed

Witzig, Maren; Carminha-Silva, Amelia; Camarinha da Silva, Amelia; Green-Engert, Rebecca; Hoelzle, Katharina; Zeller, Ellen; Seifert, Jana; Hoelzle, Ludwig E; Rodehutscord, Markus

2015-01-01

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.

D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp.▿

PubMed Central

Dagar, Sumit S.; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K.

2011-01-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation. PMID:21784906

Prevalence of renal lesions in slaughtered cattle in Shiraz, Iran, and detection of Leptospira in them by nested PCR-RFLP.

PubMed

Taghadosi, Vahideh; Hosseinzadeh, Saeid; Shekarforoush, Seyed Shahram; Samiei, Azadeh

2016-12-01

Renal diseases in cattle are frequently not recognized due to the subclinical conditions. Some species of Leptospira are the main cause of infectious agents that damage the kidneys and lead to abortion and economic losses in cattle and are also of major concern in the public health. This study was aimed to assess the prevalence of renal lesions of slaughtered cattle in the Shiraz abattoir and to determine the correlation between rejected kidneys and infection with Leptospira using nested PCR-restriction fragment length polymorphism (RFLP) techniques. Out of 1000 inspected animals, 205 (20.5 %) revealed the renal lesions. Chronic nephritis (7.5 %), white-spotted kidney (7.3 %), and petechial hemorrhage (3.5 %) were the most prevalent forms of the lesions. A direct correlation between increasing the age and significant increase in the rate of lesions was also observed (P = 0.03). Using nested PCR-RFLP assay, 40.8 % of the tested kidneys were turned to be infected to the pathogenic species of Leptospira. The risk of infection of the kidneys with white spot to pathogenic species of Leptospira (53.8 %) was more than that of the kidneys with other lesions (25.0 %) (P = 0.014). The odd ratio indicates that the kidneys with white spot lesions are likely to be infected with pathogenic species of Leptospira, five times greater than other lesions. This study showed that renal lesions especially white-spotted kidney, which were considerably associated with Leptospira in slaughtered cattle in Shiraz, were very high. This is important in terms of public health and in particular, increases the risk of transmission of disease to human specially in the high-risk careers including farmers, veterinarians, and abattoir workers.

Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1.

PubMed

Noutsios, Georgios T; Papi, Rigini M; Ekateriniadou, Loukia V; Minas, Anastasios; Kyriakidis, Dimitrios A

2012-03-01

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele's clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.

PCR-Restriction Fragment Length Polymorphism Analysis of the Phospholipase B (PLB1) Gene for Subtyping of Cryptococcus neoformans Isolates

PubMed Central

Latouche, G. Nicolas; Huynh, Matthew; Sorrell, Tania C.; Meyer, Wieland

2003-01-01

Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report describes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) as a simple tool to differentiate between C. neoformans subgroups. A PLB1 fragment, 1,970 bp, was amplified and digested with either AvaI or HindIII. Both sets of profiles grouped the isolates into their respective varieties, but only the AvaI profiles allowed for the identification of the eight molecular types via the corresponding RFLP profiles A1 to A8. Digestion of the same fragments with HindIII resulted in RFLP profiles H1 to H5, which distinguished only between serotype A, AD, D, and B/C. Neither enzyme distinguished serotype B from serotype C. The serotype AD profile was a composite of the serotype A and D profiles. Further investigation showed that the serotype AD isolates used in this study are heterozygous, with one allele of PLB1 originating from a serotype A parent and the other from a serotype D parent. PMID:12676686

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro

2010-08-27

Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonucleasemore » I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.« less

Genotyping of friesian horses to detect a hydrocephalus-associated c.1423C>T mutation in B3GALNT2 using PCR-RFLP and PCR-PIRA methods: Frequency in stallion horses in México.

PubMed

Ayala-Valdovinos, Miguel Angel; Galindo-García, Jorge; Sánchez-Chiprés, David; Duifhuis-Rivera, Theodor

2017-04-01

Hydrocephalus in Friesian horses is an autosomal recessive hereditary disease that can result in an abortion, a stillbirth, or euthanization of a newborn foal. Here, the hydrocephalus-associated c.1423C > T mutation in B3GALNT2 gene was detected with PCR-RFLP and PCR-PIRA methods for horse genotyping. A preliminary genotyping survey was performed on 83 randomly selected Friesian stallion horses to determine the current allele frequency in Mexico. The frequency of the mutant T allele was 9.6%. Copyright © 2016 Elsevier Ltd. All rights reserved.

Physical and genetic mapping of the dipeptidase gene DPEP1 to 16q24. 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Austruy, E.; Jeanpierre, C.; Junien, C.

1993-03-01

The authors report the subregional physical and genetic mapping on chromosome 16q of a cDNA clone selected as a potential tumor/growth suppressor sequence. By DNA sequencing and RNA expression pattern, this clone was identified as part of the renal dipeptidase gene (DPEP1). Using somatic cell hybrids carrying either different human chromosomes or chromosome 16 segments, they confirm and refine the physical mapping of DPEP1 to the chromosome 16 subregion q24.3. Two RFLPs, a biallelic polymorphism detected by TaqI and a VNTR detected by BamHI, EcoRI, and BglII, are described. Using the VNTR polymorphism, DPEP1 was shown to be linked tomore » D16S7 with a maximum lod score of 5.8 at a recombination fraction of 0.03. 14 refs., 2 figs., 2 tabs.« less

[Methodological study for detecting gene mutation of family with genotyping of compound heterogenicity of SEA alpha-thalassemia 1 and HbCS].

PubMed

Chen, Jian; Luo, Bi; Qi, Zhu; Huo, Pei-Dan; Zhang, Quan-Sheng; Wang, Hong

2010-06-01

This study was aimed to establish a method of PCR combination with PCR-RFLP for detecting the South-East Asian (SEA) deletion type alpha-thalassemia 1 and non-deletion mutation of Hb Constant Spring (CS), and to investigate the application value of this method. For the members of the families with alpha-thalassemia, SEA deletion mutation was detected by PCR, then the HbCS point mutation was screened by PCR-RFLP. The results indicated that 15 carriers with alpha-thalassemia (--(SEA)/) were found in 19 members from 7 families, and 2 families with genotype of --(SEA)/alpha(CS)alpha were screened out successfully. It is concluded that the PCR combination with PCR-RFLP is a simple, rapid, and reliable method for screening HbH disease with genotype of --(SEA)/alpha(CS)alpha.

Genetic analysis of eight loci tightly linked to neurofibromatosis 1

PubMed Central

Stephens, Karen; Green, Philip; Riccardi, Vincent M.; Ng, Siu; Rising, Marcia; Barker, David; Darby, John K.; Falls, Kathleen M.; Collins, Francis S.; Willard, Huntington F.; Donis-Keller, Helen

1989-01-01

The genetic locus for neurofibromatosis 1 (NF1) has recently been mapped to the pericentromeric region of chromosome 17. We have genotyped eight previously identified RFLP probes on 50 NF1 families to determine the placement of the NF1 locus relative to the RFLP loci. Thirty-eight recombination events in the pericentromeric region were identified, eight involving crossovers between NF1 and loci on either chromosomal arm. Multipoint linkage analysis resulted in the unique placement of six loci at odds >100:1 in the order of pter–A10-41–EW301–NF1–EW207–CRI-L581–CRI-L946–qter. Owing to insufficient crossovers, three loci–D17Z1, EW206, and EW203–could not be uniquely localized. In this region female recombination rates were significantly higher than those of males. These data were part of a joint study aimed at the localization of both NF1 and tightly linked pericentromeric markers for chromosome 17. PMID:2491775

Vitamin D receptor polymorphisms and the risk of cutaneous melanoma: a systematic review and meta-analysis.

PubMed

Mocellin, Simone; Nitti, Donato

2008-11-01

It has been hypothesized that polymorphisms in the vitamin D receptor (VDR) gene affect the risk of developing melanoma. However, results often are conflicting, and no meta-analysis has been performed to date on published data. Six studies (cases, 2152; controls, 2410) that investigated the association between 5 VDR polymorphisms (TaqI, FokI, BsmI, EcoRV, and Cdx2) and the risk of melanoma were retrieved and analyzed. The model-free approach was applied to meta-analyze these molecular association studies. Available data suggested a significant association between the BsmI VDR polymorphism and melanoma risk (pooled odds ratio [OR], 1.30; 95% confidence interval [CI], 1.11-1.53; P= .002; heterogeneity Cochran Q test, P> .1), and the population-attributable risk was 9.2%. In contrast, the FokI polymorphism did not appear to be associated with such risk (OR, 1.09; 95% CI, 0.99-1.21; P= .07; heterogeneity Cochran Q test, P> .1). For the TaqI and the EcoRV polymorphisms, significant between-study heterogeneity did not support genotype data pooling. Only 1 study investigated the Cdx2 variant, and the findings were negative. Current evidence is in favor of an association between 1 VDR gene polymorphism (BsmI) and the risk of developing melanoma. The current findings prompt further investigation on this subject and indirectly support the hypothesis that sun exposure may have an antimelanoma effect through activation of the vitamin D system.

The TaqIA RFLP is associated with attenuated intervention-induced body weight loss and increased carbohydrate intake in post-menopausal obese women.

PubMed

Cameron, Jameason D; Riou, Marie-Ève; Tesson, Frédérique; Goldfield, Gary S; Rabasa-Lhoret, Rémi; Brochu, Martin; Doucet, Éric

2013-01-01

Polymorphisms of the dopamine receptor D2 (DRD2) gene have been associated with obesity phenotypes. Our aim was to examine if the genotype of TaqIA Restriction Fragment Length Polymorphism (RFPL) was related to an attenuated weight loss response or to changes in energy expenditure (EE) and food preference before and after weight loss. methods: Obese post-menopausal women (age=57.1 ± 4.6 yr, weight=85.4 ± 15.4 kg and BMI=32.8 ± 4.5 kg/m(2)) were genotyped for TaqIA (n=127) by using PCR-RFLP analysis and categorized as possessing at least one copy of the A1 allele (A1(+)) or no copy (A1(-)). Women were randomized into two groups, caloric restriction (CR) and caloric restriction+resistance training (CRRT) and in this study were further classified as follows: A1(+)CR, A1(+)CRRT, A1-(-)CR and (-)A1(-)CRRT. Body composition, total daily EE, physical activity EE, Resting EE (REE), and energy intake were obtained at baseline and post-intervention using DXA, doubly-labeled water, indirect calorimetry, and 3-day dietary records, respectively. Overall, all of the anthropometric variables and REE significantly decreased post-intervention (p<0.001). Women in the CRRT group lost significantly more fat mass (FM) than the CR women (p<0.05). There were significant time by group by allele interactions for attenuated body weight (BW), BMI, and FM loss for A1(+) (vs. A1(-)) in CRRT (p<0.05) and for increased % carbohydrate intake (p<0.01). TaqIA genotype was associated with body weight loss post-intervention; more specifically, carriers of the A1 allele lost significantly less BW and FM than the A1(-) and had increased carbohydrate intake in the CRRT group. Copyright © 2012 Elsevier Ltd. All rights reserved.

Distinguishing Heterodera filipjevi and H. avenae using polymerase chain reaction-restriction fragment length polymorphism and cyst morphology.

PubMed

Yan, Guiping; Smiley, Richard W

2010-03-01

The cereal cyst nematodes Heterodera filipjevi and H. avenae impede wheat production in the Pacific Northwest (PNW). Accurate identification of cyst nematode species and awareness of high population density in affected fields are essential for designing effective control measures. Morphological methods for differentiating these species are laborious. These species were differentiated using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS)-ribosomal (r)DNA with up to six restriction endonucleases (TaqI, HinfI, PstI, HaeIII, RsaI, and AluI). The method was validated by inspecting underbridge structures of cyst vulval cones. Grid soil sampling of an Oregon field infested by both species revealed that H. filipjevi was present at most of the infested grid sites but mixtures of H. avenae and H. filipjevi also occurred. These procedures also detected and differentiated H. filipjevi and H. avenae in soil samples from nearby fields in Oregon and H. avenae in samples from Idaho and Washington. Intraspecific polymorphism was not observed within H. filipjevi or PNW H. avenae populations based on the ITS-rDNA. However, intraspecific variation was observed between H. avenae populations occurring in the PNW and France. Methods described here will improve detection and identification efficiencies for cereal cyst nematodes in wheat fields.

The potential of SNP-based PCR-RFLP capillary electrophoresis analysis to authenticate and detect admixtures of Mediterranean olive oils.

PubMed

Bazakos, Christos; Khanfir, Emna; Aoun, Mariem; Spano, Thodhoraq; Zein, Zeina El; Chalak, Lamis; Riachy, Milad El; Abou-Sleymane, Gretta; Ali, Sihem Ben; Grati Kammoun, Naziha; Kalaitzis, Panagiotis

2016-07-01

Authentication and traceability of extra virgin olive oil is a challenging research task due to the complexity of fraudulent practices. In this context, the monovarietal olive oils of Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) require new tests and cutting edge analytical technologies to detect mislabeling and misleading origin. Toward this direction, DNA-based technologies could serve as a complementary to the analytical techniques assay. Single nucleotide polymorphisms are ideal molecular markers since they require short PCR analytical targets which are a prerequisite for forensic applications in olive oil sector. In the present study, a small number of polymorphic SNPs were used with an SNP-based PCR-RFLP capillary electrophoresis platform to discriminate six out of 13 monovarietal olive oils of Mediterranean origin from three different countries, Greece, Tunisia, and Lebanon. Moreover, the high sensitivity of capillary electrophoresis in combination with the DNA extraction protocol lowered the limit of detection to 10% in an admixture of Tsounati in a Koroneiki olive oil matrix. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human T-lymphotropic virus type 1 (HTLV-1) genetic typing in Kakeroma Island, an island at the crossroads of the ryukyuans and Wajin in Japan, providing further insights into the origin of the virus in Japan.

PubMed

Eguchi, Katsuyuki; Fujii, Hidefumi; Oshima, Kengo; Otani, Masashi; Matsuo, Toshiaki; Yamamoto, Taro

2009-08-01

Peripheral blood samples were collected from 23 human T-lymphotropic virus type-1 (HTLV-1) carriers residing in Kakeroma Island, Japan (Kagoshima Prefecture, Oshima County, Setouchi Town), one of the most highly endemic areas in Japan. The samples were subjected to amplification by PCR and sequencing of the Long Terminal Repeat in order to reconstruct a phylogenetic tree of HTLV-1 isolates. Restriction Fragment Length Polymorphism (RFLP) analysis of env region was also conducted for subgrouping of HTLV-1. Although one sample could not be amplified by PCR, and three more could not be sequenced due to the existence of conspicuous nonspecific bands or repeated sequences, the phylogenetic analysis revealed that the remaining 19 isolates obtained from Kakeroma Island belonged to either the Transcontinental or the Japanese subgroups of the Cosmopolitan subtype, one of the three major subtypes. The RFLP data corresponded closely with the typing data throughout the sequencing. The proportion of the Transcontinental subgroup among the isolates was 26.3% (5 of 19) by sequence analysis and 27.3% (6 of 22) by RFLP. Unlike in Taiwan, China and Okinawa, the Japanese subgroup was dominant in Kakeroma Island. The analysis would also suggest that the Japanese subgroup seems not to have derived from the Transcontinental subgroup, but rather that the Transcontinental subgroup came to Japan first and was followed later by the Japanese one. 2009 Wiley-Liss, Inc.

Near East/South Asia Report

DTIC Science & Technology

1985-12-24

gas’ ,;frorn bio- digestion and use of. digested i sludge as manure . The consensus was that the available technologies In India in the’ .held of...nine months of 1985 TUrkey bought 4.6 million tonnes of oil ■ from Iran and 4.3 million from Iraq, its two biggest suppliers. Mr Taqi had returned...try cooperation and exchange of oil expertise between the two countries. During his week-long visit, Mr Taqi also had talks with officials of the

Lab-on-a-chip-based PCR-RFLP assay for the confirmed detection of short-length feline DNA in food.

PubMed

Ali, Md Eaqub; Al Amin, Md; Hamid, Sharifah Bee Abd; Hossain, M A Motalib; Mustafa, Shuhaimi

2015-01-01

Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.

Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

PubMed Central

Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H. M.; Gurjar, Suraj K.; Gupta, I. D.; Verma, Archana

2017-01-01

Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible. PMID:28344410

Evaluation of Semiautomated IS6110-Based Restriction Fragment Length Polymorphism Typing for Mycobacterium tuberculosis in a High-Burden Setting.

PubMed

Said, Halima M; Krishnamani, Keshav; Omar, Shaheed V; Dreyer, Andries W; Sansom, Bianca; Fallows, Dorothy; Ismail, Nazir A

2016-10-01

The manual IS6110-based restriction fragment length polymorphism (RFLP) typing method is highly discriminatory; however, it is laborious and technically demanding, and data exchange remains a challenge. In an effort to improve IS6110-based RFLP to make it a faster format, DuPont Molecular Diagnostics recently introduced the IS6110-PvuII kit for semiautomated typing of Mycobacterium tuberculosis using the RiboPrinter microbial characterization system. This study aimed to evaluate the semiautomated RFLP typing against the standard manual method. A total of 112 isolates collected between 2013 and 2014 were included. All isolates were genotyped using manual and semiautomated RFLP typing methods. Clustering rates and discriminatory indexes were compared between methods. The overall performance of semiautomated RFLP compared to manual typing was excellent, with high discriminatory index (0.990 versus 0.995, respectively) and similar numbers of unique profiles (72 versus 74, respectively), numbers of clustered isolates (33 versus 31, respectively), cluster sizes (2 to 6 and 2 to 5 isolates, respectively), and clustering rates (21.9% and 17.1%, respectively). The semiautomated RFLP system is technically simple and significantly faster than the manual RFLP method (8 h versus 5 days). The analysis is fully automated and generates easily manageable databases of standardized fingerprints that can be easily exchanged between laboratories. Based on its high-throughput processing with minimal human effort, the semiautomated RFLP can be a very useful tool as a first-line method for routine typing of M. tuberculosis isolates, especially where Beijing strains are highly prevalent, followed by manual RFLP typing if resolution is not achieved, thereby saving time and labor. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Association analysis of clinical aspects and vitamin D receptor gene polymorphism with external apical root resorption in orthodontic patients.

PubMed

Fontana, Maria Luiza S Simas Netta; de Souza, Cleber Machado; Bernardino, José Fabio; Hoette, Felix; Hoette, Maura Levi; Thum, Lotario; Ozawa, Terumi O; Capelozza Filho, Leopoldino; Olandoski, Marcia; Trevilatto, Paula Cristina

2012-09-01

Vitamin D is responsible for the regulation of certain genes at the transcription level, via interaction with the vitamin D receptor, and influences host immune responses and aspects of bone development, growth, and homeostasis. Our aim was to investigate the association of TaqI vitamin D receptor gene polymorphism with external apical root resorption during orthodontic treatment. Our subjects were 377 patients with Class II Division 1 malocclusion, divided into 3 groups: (1) 160 with external apical root resorption ≤1.43 mm, (2) 179 with external apical root resorption >1.43 mm), and (3) 38 untreated subjects. External apical root resorption of the maxillary incisors was evaluated on periapical radiographs taken before and after 6 months of treatment. After DNA collection and purification, vitamin D receptor TaqI polymorphism analysis was performed by polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate analyses were performed to verify the association of clinical and genetic variables with external apical root resorption (P <0.05). There was a higher proportion of external apical root resorption in orthodontically treated patients compared with the untreated subjects. In patients orthodontically treated, age higher than 14 years old, initial size of the maxillary incisor root superior to 30 mm, and premolar extraction were associated with increased external apical root resorption. Genotypes containing the C allele were weakly associated with protection against external apical root resorption (CC + CT × TT [odds ratio, 0.29; 95% confidence interval, 0.07-1.23; P = 0.091]) when treated orthodontic patients were compared to untreated individuals. Clinical factors and vitamin D receptor TaqI polymorphism were associated with external apical root resorption in orthodontic patients. Copyright © 2012 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Associations of a HinfI PCR-RFLP of POU1F1 gene with growth traits in Qinchuan cattle.

PubMed

Zhang, Chunlei; Liu, Bo; Chen, Hong; Lan, Xianyong; Lei, Chuzhao; Zhang, Zhiqing; Zhang, Runfeng

2009-01-01

The objectives of the present study were to estimate the allele and genotype frequencies of the POU1F1/HinfI polymorphisms in beef cattle belonging to four different genetic groups and to determine the effects of these polymorphisms on growth traits in cattle. The 451-bp PCR products of POU1F1 gene digested with HinfI exhibited three genotypes and two alleles, which were at Hardy-Weinberg equilibrium (P > 0.05). Genotype BB was the predominant genotype and B the predominant allele in the studied populations. There was significant difference between Limousin x Qinchuan and Qinchuan in the distribution of genotypes (P < 0.0001). The association of the polymorphism of the POU1F1 gene with growth traits among Qinchuan, Limousin x Qinchuan, Angus x Qinchuan and Germany Yellow x Qinchuan crosses was analyzed. Body weight and wither height of individuals with genotypes AB were higher (P < 0.05)than that of individuals with genotype BB in the Germany Yellow x Qinchua cross, but were not in the other three populations (P > 0.05).

Assessing Subsurface Bioaugmentation of a Mixed Culture Capable of Chlorinated Solvent Cometabolism via Molecular Methods

NASA Astrophysics Data System (ADS)

Dolan, M. E.; Lim, H. K.; Semprini, L.; Giovanonni, S.; Vergin, K.; McCarty, P. L.; Hopkins, G. D.

2001-12-01

The goal of this project is the successful bioaugmentation of a mixed culture capable of aerobic cometabolism of chlorinated solvent mixtures into an aquifer test zone at Moffett Federal Airfield, CA (Moffett). The test zone consists of two parallel well legs both fed butane and oxygen. One leg will be bioaugmented and the other will serve as an indigenous control. Injection and extraction wells and six (3 per leg) intermediately placed groundwater monitoring points will be frequently monitored for chlorinated solvents, butane, dissolved oxygen, and pH. Groundwater will also be periodically analyzed for microbial content using terminal restriction fragment length polymorphism (T-RFLP) and fluorescence in-situ hybridization (FISH) analyses. In each well leg, two fully-penetrating wells containing solid media will be periodically analyzed for microbial colonization (T-RFLP). The mixed bioaugmentation culture originated from environmental samples taken from Hanford, WA. The culture was enriched on butane and tested for viability in Moffett groundwater and aquifer solids. A clone library was created from the 16S rDNA in the mixed culture and 86 clones were sorted based on RFLP patterns. Complete sequencing of the 16S rDNA gene from the three most prevalent clones revealed 45 clones similar to Acidovorax or Hydrogenophaga, gram negative proteobacterium, and 12 clones similar to Rhodococcus, a gram positive filamentous organism. Fluorescently-labeled rRNA probes were designed for FISH analyses and appropriate restriction enzymes were chosen for T-RFLP analyses based upon the sequence information. Microcosm tests were conducted prior to the initiation of the field study to evaluate butane, 1,1-dichloroethylene (1,1-DCE), and 1,1,1-trichloroethane (TCA) degradation kinetics and microbial community composition. Bioaugmented microcosms began butane utilization sooner than non-bioaugmented ones in the presence and absence of 1,1-DCE, and were able to degrade more 1,1-DCE (up

A novel nonsense mutation in CRYBB1 associated with autosomal dominant congenital cataract

PubMed Central

Yang, Juhua; Zhu, Yihua; Gu, Feng; He, Xiang; Cao, Zongfu; Li, Xuexi; Tong, Yi

2008-01-01

Purpose To identify the molecular defect underlying an autosomal dominant congenital nuclear cataract in a Chinese family. Methods Twenty-two members of a three-generation pedigree were recruited, clinical examinations were performed, and genomic DNA was extracted from peripheral blood leukocytes. All members were genotyped with polymorphic microsatellite markers adjacent to each of the known cataract-related genes. Linkage analysis was performed after genotyping. Candidate genes were screened for mutation using direct sequencing. Individuals were screened for presence of a mutation by restriction fragment length polymorphism (RFLP) analysis. Results Linkage analysis identified a maximum LOD score of 3.31 (recombination fraction [θ]=0.0) with marker D22S1167 on chromosome 22, which flanks the β-crystallin gene cluster (CRYBB3, CRYBB2, CRYBB1, and CRYBA4). Sequencing the coding regions and the flanking intronic sequences of these four candidate genes identified a novel, heterozygous C→T transition in exon 6 of CRYBB1 in the affected individuals of the family. This single nucleotide change introduced a novel BfaI site and was predicted to result in a nonsense mutation at codon 223 that changed a phylogenetically conserved amino acid to a stop codon (p.Q223X). RFLP analysis confirmed that this mutation co-segregated with the disease phenotype in all available family members and was not found in 100 normal unrelated individuals from the same ethnic background. Conclusions This study has identified a novel nonsense mutation in CRYBB1 (p.Q223X) associated with autosomal dominant congenital nuclear cataract. PMID:18432316

PCR-RFLP assays to distinguish the Western and Eastern phylogroups in wild and cultured tench Tinca tinca.

PubMed

Lajbner, Z; Kotlík, P

2011-03-01

The tench Tinca tinca is a valued table fish native to Europe and Asia, but which is now widely distributed in many temperate freshwater regions of the world as the result of human-mediated translocations. Fish are currently being transplanted between watersheds without concern for genetic similarity to wild populations or local adaptation, and efficient phylogeographic markers are therefore urgently needed to rapidly distinguish genetically distinct geographical populations and to assess their contribution to the hatchery breeds and to the stocked wild populations. Here, we present a new method to distinguish recently discovered and morphologically undistinguishable Western and Eastern phylogroups of the tench. The method relies on PCR-RFLP assays of two independent nuclear-encoded exon-primed intron-crossing (EPIC) markers and of one mitochondrial DNA (mDNA) marker and allows the rapid identification of the Western and Eastern phylogroup and also of three geographical mtDNA clades within the Eastern phylogroup. Our method will enable researchers and fishery practitioners to rapidly distinguish genetically divergent geographical populations of the tench and will be useful for monitoring the introduction and human-mediated spread of the phylogroups in wild populations, for characterization of cultured strains and in breeding experiments. © 2010 Blackwell Publishing Ltd.

Cytochrome P2A13 and P1A1 gene polymorphisms are associated with the occurrence of uterine leiomyoma.

PubMed

Herr, D; Bettendorf, H; Denschlag, D; Keck, C; Pietrowski, D

2006-10-01

To investigate the association between the occurrence of uterine leiomyoma and two SNPs of the CYP 2A13 and CYP 1A1 genes. Prospective case control study with 132 women with clinically and surgically diagnosed uterine leiomyoma and 260 controls. Genotyping was performed by polymerase chain reaction (PCR) based amplification of CYP 2A13 and CYP 1A1 genes, and restriction fragment length polymorphism (RFLP) analysis. Comparing women with uterine leiomyoma and controls, we demonstrate statistical significant differences of allele frequency and genotype distribution for the CYP 1A1 polymorphism (P = 0.025 and P = 0.046, respectively). Furthermore, for the CYP 2A13 polymorphism we found a significant difference concerning allele frequency (P = 0.033). However, for the genotype distribution, only borderline significance was observed (P = 0.064). The CYP 2A13 and CYP 1A1 SNPs are associated with uterine leiomyoma in a Caucasian population and may contribute to the understanding of the pathogenic mechanisms of uterine leiomyoma.

A PCR technique based on the Hip1 interspersed repetitive sequence distinguishes cyanobacterial species and strains.

PubMed

Smith, J K; Parry, J D; Day, J G; Smith, R J

1998-10-01

The use of primers based on the Hip1 sequence as a typing technique for cyanobacteria has been investigated. The discovery of short repetitive sequence structures in bacterial DNA during the last decade has led to the development of PCR-based methods for typing, i.e., distinguishing and identifying, bacterial species and strains. An octameric palindromic sequence known as Hip1 has been shown to be present in the chromosomal DNA of many species of cyanobacteria as a highly repetitious interspersed sequence. PCR primers were constructed that extended the Hip1 sequence at the 3' end by two bases. Five of the 16 possible extended primers were tested. Each of the five primers produced a different set of products when used to prime PCR from cyanobacterial genomic DNA. Each primer produced a distinct set of products for each of the 15 cyanobacterial species tested. The ability of Hip1-based PCR to resolve taxonomic differences was assessed by analysis of independent isolates of Anabaena flos-aquae and Nostoc ellipsosporum obtained from the CCAP (Culture Collection of Algae and Protozoa, IFE, Cumbria, UK). A PCR-based RFLP analysis of products amplified from the 23S-16S rDNA intergenic region was used to characterize the isolates and to compare with the Hip1 typing data. The RFLP and Hip1 typing yielded similar results and both techniques were able to distinguish different strains. On the basis of these results it is suggested that the Hip1 PCR technique may assist in distinguishing cyanobacterial species and strains.

Chloroplast and nuclear DNA studies in a few members of the Brassica oleracea L. group using PCR-RFLP and ISSR-PCR markers: a population genetic analysis.

PubMed

Panda, S; Martín, J P; Aguinagalde, I

2003-04-01

A population genetic analysis of chloroplast and nuclear DNA was performed covering nine wild populations of Brassica oleracea. Three members of the n = 9 group, all close to B. oleracea, Brassica alboglabra Bailey, Brassica bourgeaui (Webb) O. Kuntze and Brassica montana Pourret, were also studied to better understand their relationship with B. oleracea. Chloroplast DNA was analysed using the PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) method. The ISSR-PCR (inter-simple sequence repeat - polymerase chain reaction) technique was adopted to study nuclear DNA. Twelve primer pairs of chloroplast DNA showed very good amplification. The amplified product of each primer pair, digested by three restriction enzymes, revealed no variation of cpDNA among the taxa studied. This indicates they may have the same chloroplast genotype. Seven selected ISSR primers have detected genetic variation, both within and among the populations/taxa surveyed. The information obtained on the intra- and inter-populational genetic diversity of wild populations of B. oleracea neatly defined the individual plants. It could provide important guidelines for backing management and conservation strategies in this species. The study confirms a close relationship between B. alboglabra, B. bourgeaui and B. montana, which is parallel to their morphological similitude.

Lack of Association between ESR1 and CYP1A1 Gene Polymorphisms and Susceptibility to Uterine Leiomyoma in Female Patients of Iranian Descent.

PubMed

Taghizade Mortezaee, Fatemeh; Tabatabaiefar, Mohammad Amin; Hashemzadeh Chaleshtori, Morteza; Miraj, Sepideh

2014-01-01

Uterine leiomyoma (UL) is the most common benign smooth muscle cell tumor with as yet unknown etiology and pathogenesis. This study was carried out to investigate the association of ESR1-351 A>G, ESR1 -397 T>C and CYP1A1 (Ile462Val) polymorphisms with UL in female patients of Iranian origin. In this case-control study, 276 patients with UL and 156 healthy women were recruited. The genetic polymorphisms ESR1-351 A>G, ESR1-397 T>C and CYP1A1 (Ile462Val) were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). No significant difference were found in frequencies of both genotypes and alleles of ESR1-351 A>G, ESR1-397 T>C and CYP1A1 (Ile462Val) polymorphisms between the two groups (p>0.05). Our findings indicated that these ESR1 and CYP1A1 polymorphisms were not associated with the development of UL in the cases reported here.

Comparative Study of IS6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands, Based on a 5-Year Nationwide Survey

PubMed Central

de Beer, Jessica L.; van Ingen, Jakko; de Vries, Gerard; Erkens, Connie; Sebek, Maruschka; Mulder, Arnout; Sloot, Rosa; van den Brandt, Anne-Marie; Enaimi, Mimount; Kremer, Kristin; Supply, Philip

2013-01-01

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n = 3,123). Of the remaining 855 cases, 12% (n = 479) of the cases were clustered only by VNTR, 7.7% (n = 305) only by RFLP typing, and 1.8% (n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems. PMID:23363841

A Molecular Epidemiological Survey of Clinically Important Dermatophytes in Iran Based on Specific RFLP Profiles of Beta-tubulin Gene

PubMed Central

ABASTABAR, Mahdi; REZAEI-MATEHKOLAEI, Ali; SHIDFAR, Mohammad Reza; KORDBACHEH, Parivash; MOHAMMADI, Rasoul; SHOKOOHI, Tahereh; HEDAYATI, Mohammad Taghi; JALALIZAND, Nilufar; MIRHENDI, Hossein

2013-01-01

Abstract Background Surveillance of dermatophytosis is essential to determine the likely changes in etiological trends and distribution profile of this infection. In this study beta tubulin gene (BT2), was used as the first time in a PCR-RFLP format to clarify the distribution of dermatophytosis agents in some parts of Iran. Methods A total of 603 clinical isolates was obtained from 500 patients in Tehran, Isfahan, Mazandaran and Guilan provinces. The isolates were identified using macro/micro-morphological criteria and electrophoretic patterns of PCR amplicons of BT2after digestion with each of the restriction enzymes FatI, HpyCH4V, MwoI and Alw21I. Results Among the patients, 59.2% were male and 40.8% female. The most prevalent clinical form was tinea pedis (42.4%), followed by tinea cruris (24.2%), tinea unguium (12.3%), tinea corporis (10.8%), tinea faciei (4%), tinea manuum (3.14%), tinea capitis (3%) and tinea barbae (0.16%), respectively. Trichophyton interdigitale ranked the first, followed by T. rubrum, Epidermophyton floccosum, Microsporum canis, T. tonsurans, T. erinacei and T. violaceum (each 0.49%) and the less frequent species were T. schoenleinii, M. gypseum and T.anamorph of Arthroderma benhamiae (each 0.16%). A case of scalp infection by E. floccosum was an exceptional event in the study. No case of T. verrucosum was found. Conclusion Trichophyton species and E. floccosum are yet the predominant agents of infection in Iran, while Microsporum species are decreasing. T. interdigitale and Tinea pedis remain as the most causal agent and clinical form of dermatophytosis, respectively. It seems that BT2 can be a useful genetic marker for epidemiological survey of common pathogenic dermatophytes. PMID:26060667

Association of CYP1A1 gene polymorphism with chronic kidney disease: a case control study.

PubMed

Siddarth, Manushi; Datta, Sudip K; Ahmed, Rafat S; Banerjee, Basu D; Kalra, Om P; Tripathi, Ashok K

2013-07-01

CYP1A1 is an important xenobiotic metabolizing enzyme, present in liver and kidney. Expression of CYP1A1 enzyme increases manifold when kidney cells are exposed to nephrotoxins/chemicals leading to oxidative stress-induced cell damage. To study the association of CYP1A1 gene polymorphism in patients of chronic kidney disease with unknown etiology (CKDU), we recruited 334 CKDU patients and 334 age and sex matched healthy controls. CYP1A1*2A and *2C polymorphisms were studied by PCR-RFLP and allele specific-PCR respectively. Subjects carrying at least one mutant allele of CYP1A1*2A (TC, CC) and *2C (AG, GG) were shown to be associated with 1.4-2-fold increased risk of CKDU. Also, genotypic combinations of hetero-/homozygous mutants of CYP1A1*2A (TC, CC) with hetero-/homozygous mutant genotypes of CYP1A1*2C (AG, GG) i.e. TC/AG (p<0.01), TC/GG (p<0.05), CC/AG (p<0.05) and CC/GG (p<0.01) were associated with CKDU with an odd ratio ranging 1.8-3.3 times approximately. This study demonstrates association of CYP1A1 polymorphisms with CKDU. Copyright © 2013 Elsevier B.V. All rights reserved.

Plasmodium vivax merozoite surface protein-3 alpha: a high-resolution marker for genetic diversity studies.

PubMed

Prajapati, Surendra Kumar; Joshi, Hema; Valecha, Neena

2010-06-01

Malaria, an ancient human infectious disease caused by five species of Plasmodium, among them Plasmodium vivax is the most widespread human malaria species and causes huge morbidity to its host. Identification of genetic marker to resolve higher genetic diversity for an ancient origin organism is a crucial task. We have analyzed genetic diversity of P. vivax field isolates using highly polymorphic antigen gene merozoite surface protein-3 alpha (msp-3 alpha) and assessed its suitability as high-resolution genetic marker for population genetic studies. 27 P. vivax field isolates collected during chloroquine therapeutic efficacy study at Chennai were analyzed for genetic diversity. PCR-RFLP was employed to assess the genetic variations using highly polymorphic antigen gene msp-3 alpha. We observed three distinct PCR alleles at msp-3 alpha, and among them allele A showed significantly high frequency (53%, chi2 = 8.22, p = 0.001). PCR-RFLP analysis revealed 14 and 17 distinct RFLP patterns for Hha1 and Alu1 enzymes respectively. Further, RFLP analysis revealed that allele A at msp-3 alpha is more diverse in the population compared with allele B and C. Combining Hha1 and Alu1 RFLP patterns revealed 21 distinct genotypes among 22 isolates reflects higher diversity resolution power of msp-3 alpha in the field isolates. P. vivax isolates from Chennai region revealed substantial amount of genetic diversity and comparison of allelic diversity with other antigen genes and microsatellites suggesting that msp-3 alpha could be a high-resolution marker for genetic diversity studies among P. vivax field isolates.

Analysis of vitamin D receptor gene polymorphisms in patients with chronic periodontitis.

PubMed

Gunes, Sezgin; Sumer, A Pinar; Keles, Gonca Cayir; Kara, Nurten; Koprulu, Hulya; Bagci, Hasan; Bek, Yuksel

2008-01-01

Genetic polymorphisms in the vitamin D receptor (VDR) gene are related to bone mineral density, bone turnover, and diseases with bone loss. Alveolar bone loss is a key feature in periodontitis. The aim of this study was to determine whether severe generalized chronic periodontitis (CP) in a Turkish population was associated with polymorphisms in the VDR gene. Samples of venous blood and DNA were obtained from 72 patients with severe generalized chronic periodontitis and 102 healthy controls. The polymorphic regions were amplified using PCR followed by digestion with restriction enzymes BsmI A/G(rs1544410), ApaI G/T(rs11168271), TaqI T/C(rs731236), and analyzed electrophoretically. Genotype and allele frequencies were calculated. There were no statistically significant differences in the frequencies of VDR BsmI, ApaI, TaqI genotypes between the CP patients and healthy controls. The GTT haplotype, constructed from the three adjacent restriction fragment length polymorphisms was found to be over-represented among CP cases. This corresponded an OR of 2.4 (95% confidence interval, 1.12-5.18) for heterozygous carriers and 2.27 (95% confidence interval, 0.95-5.4) for homozygous carrier of the risk haplotype. The present findings indicated that BsmI, ApaI, TaqI polymorphisms of the VDR gene were not associated with the severe generalized CP in the studied Turkish patients. Moreover, the VDR genotypes based on haplotype analysis may be associated with chronic periodontitis. In the future, diagnostic periodontal risk assessments like polymorphisms may be useful in detection of individuals susceptible for periodontitis.

Petroleum degradation by Pseudomonas sp. ZS1 is impeded in the presence of antagonist Alcaligenes sp. CT10.

PubMed

Liang, Jibei; Cheng, Tao; Huang, Yi; Liu, Jianhua

2018-05-28

Enhanced bioremediation is a favorable approach for petroleum pollutant cleanup, which depends on the growth of oil-eating microorganisms. In this study, we show that, by using the modified T-RFLP (mT-RFLP) methodology, one of the four major microbial populations derived from oil sludge has failed to propagate in MS medium supplemented with 2% yeast extract (YE). rDNA sequence-based analysis indicated that the four populations were Donghicola sp. CT5, Bacillus sp. CT6, Alcaligenes sp. CT10, and Pseudomonas sp. ZS1. Four purified strains grow well individually in MS medium supplemented with 2% YE, suggesting that ZS1 growth is antagonized by other strains. Co-growth analysis using mT-RFLP methodology and plate inhibitory assay indicated that ZS1 exhibited antagonistic effect against CT5 and CT6. On the other hand, co-growth analysis and plate inhibition assay showed that CT10 antagonized against ZS1. To investigate the potential compounds responsible for the antagonism, supernatant of CT10 culture was subjected to GC-MS analysis. Analysis indicated that CT10 produced a number of antimicrobial compounds including cyclodipeptide c-(L-Pro-L-Phe), which was known to inhibit the growth of Pseudomonas sp. Growth test using the purified c-(L-Pro-L-Phe) from CT10 confirmed its inhibitory activity. We further showed that, using both gravimetric and GC analysis, CT10 antagonism against the oil-eating ZS1 led to the diminishing of crude oil degradation. Together, our results indicate that bioremediation can be affected by environmental antagonists.

Epidemiologic Study of Malassezia Yeasts in Patients with Malassezia Folliculitis by 26S rDNA PCR-RFLP Analysis

PubMed Central

Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

2011-01-01

Background So far, studies on the inter-relationship between Malassezia and Malassezia folliculitis have been rather scarce. Objective We sought to analyze the differences in body sites, gender and age groups, and to determine whether there is a relationship between certain types of Malassezia species and Malassezia folliculitis. Methods Specimens were taken from the forehead, cheek and chest of 60 patients with Malassezia folliculitis and from the normal skin of 60 age- and gender-matched healthy controls by 26S rDNA PCR-RFLP. Results M. restricta was dominant in the patients with Malassezia folliculitis (20.6%), while M. globosa was the most common species (26.7%) in the controls. The rate of identification was the highest in the teens for the patient group, whereas it was the highest in the thirties for the control group. M. globosa was the most predominant species on the chest with 13 cases (21.7%), and M. restricta was the most commonly identified species, with 17 (28.3%) and 12 (20%) cases on the forehead and cheek, respectively, for the patient group. Conclusion Statistically significant differences were observed between the patient and control groups for the people in their teens and twenties, and in terms of the body site, on the forehead only. PMID:21747616

VDR gene methylation as a molecular adaption to light exposure: Historic, recent and genetic influences.

PubMed

Beckett, Emma L; Jones, Patrice; Veysey, Martin; Duesing, Konsta; Martin, Charlotte; Furst, John; Yates, Zoe; Jablonski, Nina G; Chaplin, George; Lucock, Mark

2017-09-10

The vitamin D receptor (VDR) is a member of the nuclear receptor family of transcription factors. We examined whether degree of VDR gene methylation acts as a molecular adaptation to light exposure. We explored this in the context of photoperiod at conception, recent UV irradiance at 305 nm, and gene-latitude effects. Eighty subjects were examined for VDR gene-CpG island methylation density. VDR gene variants were also examined by PCR-RFLP. Photoperiod at conception was significantly positively related to VDR methylation density, explaining 17% of the variance in methylation (r 2  = 0.17; P = .001). Within this model, photoperiod at conception and plasma 25(OH)D independently predicted methylation density at the VDR-CpG island. Recent UV exposure at 305 nm led to a fivefold increase in mean methylation density (P = .02). Again, UV exposure and plasma 25(OH)D independently predicted methylation density at the VDR-CpG island. In the presence of the BsmI mutant allele, methylation density was increased (P = .01), and in the presence of the TaqI or FokI mutant allele, methylation density was decreased (P = .007 and .04 respectively). Multivariate modelling suggests plasma 25(OH)D, photoperiod at conception, recent solar irradiance, and VDR genotype combine as independent predictors of methylation at the VDR-CpG island, explaining 34% of the variance in methylation (R 2  = 0.34, P < .0001). Duration of early-life light exposure and strength of recent irradiance, along with latitudinal genetic factors, influence degree of VDR gene methylation consistent with this epigenetic phenomenon being a molecular adaptation to variation in ambient light exposure. Findings contribute to our understanding of human biology. © 2017 Wiley Periodicals, Inc.

Prolonged P300 latency in children with the D[sub 2] dopamine receptor A1 allele

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noble, E.P.; Berman, S.M.; Ozkaragoz, T.Z.

1994-04-01

Previous studies have indicated the presence of a hereditary component in the generation of the P300, or P3, a late positive component of the event-related potential. Moreover, the dopaminergic system has been implicated in the P3. In the present study, 98 healthy Caucasian boys, mean age of 12.5 years and of above-average intelligence, were studied. The sample was composed of 32 sons of active alcoholic (SAA) fathers, 36 sons of recovering alcoholic (SRA) fathers, and 30 sons of social drinker (SSD) fathers, with none of them having yet begun to consume alcohol or other drugs. TaqI A D[sub 2] dopaminemore » receptor alleles (A1 and A2) were determined. A significant difference in the frequency of the A1 allele was found among these three groups of boys, with the SAA group having the highest A1 allele frequency (.313), followed by the SRA (.139) and the SSD (.133) groups. The relationship of the A1 and A2 alleles to P3 amplitude and latency was also determined. The results showed no significant difference in P3 amplitude between boys with the A1 and A2 allele. However, P3 latency was significantly longer in the total sample of boys with the A1 allele compared with those carrying the A2 allele. These findings suggest that polymorphism of the D[sub 2] dopamine receptor gene is an important determinant of P3 latency. 84 refs., 2 figs., 3 tabs.« less

Genetic profiling of GSTP1, DPYD, FCGR2A, FCGR3A and CCND1 genes in an Argentinian population.

PubMed

Galván, Cristian A; Elbarcha, Osvaldo C; Fernández, Eduardo J; Beltramo, Dante M; Soria, Néstor W

2011-09-01

To determine the frequencies of relevant allelic variants in oncology for the GSTP1, DPYD, FCGR2A, FCGR3A and CCND1 genes in a population from Central Argentina. To compare the allelic distribution found with the frequencies reported for other ethnic groups. Genotyping was carried out in a total of 102 unrelated Argentinian subjects. FCGR3A (rs396991) was detected using allele specific polymerase chain reaction (PCR) assay, while GSTP1 (rs1695), DPYD (rs3918290), FCGR2A (rs1801274) and CCND1 (rs9344) variants were assessed by PCR-restriction fragment length polymorphism (PCR-RFLP). The allele frequencies for GSTP*1B, DPYD*2A, FCGR2A (131R), FCGR3A (158F) and CCND1 (870G) in Argentinians were 0.35, 0.005, 0.41, 0.77 and 0.47, respectively. We found that the Argentinian population tested resembles other Caucasians populations, especially Spaniards; yet the differences in allele distribution with other Caucasian groups, uncover population admixture with native Amerindian and other ethnic groups, consistent with the well documented immigration flows landing Argentina from several countries. Copyright © 2011. Published by Elsevier Inc.

Association of CYP1A1 and CYP1B1 polymorphisms with bone mineral density variations in postmenopausal Mexican-Mestizo women.

PubMed

Chávez, Bertha; Vilchis, Felipe; Rojano-Mejía, David; Coral Vázquez, Ramón Mauricio; Aguirre-García, María Del Carmen; Canto, Patricia

2017-08-01

Herein, we investigated potential associations between polymorphisms of genes related to estrogen metabolism and bone mineral density (BMD) in postmenopausal women. This was a cross-sectional study, in which two hundred and ninety postmenopausal Mexican-Mestizo women were studied. The BMD of the lumbar spine (LS), total hip (TH), and femoral neck (FN) was measured. The distribution of the genetic polymorphisms, including rs1799814 and rs1048943 at CYP1A1 as well as rs1056836 at CYP1B1, were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-stranded conformational polymorphism (SSCP), and DNA sequencing. Deviations from Hardy-Weinberg equilibrium (HWE) were tested, and linkage disequilibrium (LD) was calculated by direct correlation (r 2 ). Moreover, haplotype analysis was performed. All polymorphisms were in HWE. The genotype and allele distributions of the three single nucleotide polymorphisms (SNPs) studied showed no significant differences. However, statistical significance was reached when constructing haplotypes. The CG haplotype in CYP1A1 was associated with variations in LS and FN BMD after adjustment for covariates (p = 0.021 and 0.045, respectively), but the association with TH BMD was not significant. These results suggested that the CG haplotype in CYP1A1 may play an important role in the mechanism of osteoporosis and may be useful as a genetic marker.

A proline-to-histidine mutation in POU1F1 is associated with production traits in dairy cattle.

PubMed

Huang, W; Maltecca, C; Khatib, H

2008-10-01

POU class 1 homeobox 1 (POU1F1) is a member of the tissue-specific POU-containing transcription factor family. The expression of POU1F1 in mammalian pituitary gland controls the transcription of the genes encoding growth hormone, prolactin (PRL) and the subunits of thyroid-stimulating hormone. In addition, some genes in the JAK/STAT signalling pathway downstream of POU1F1 have been shown to be associated with different production traits in dairy cattle. To investigate whether the POU1F1 gene is associated with economically important traits in dairy cattle, a pooled DNA sequencing approach was used to identify single nucleotide polymorphisms (SNPs) in the gene. An SNP in exon 3 of POU1F1 that changes a proline to a histidine was identified. A total of 2141 individuals from two North American Holstein cattle resource populations were genotyped for this SNP using a modified PCR-RFLP method. Statistical analyses revealed significant association of POU1F1 variants with milk yield and productive life, which makes POU1F1 a possible candidate for marker-assisted selection in dairy cattle breeding programmes.

Bio-Augmentation of Cupriavidus sp. CY-1 into 2,4-D Contaminated Soil: Microbial Community Analysis by Culture Dependent and Independent Techniques

PubMed Central

Chang, Young-Cheol; Reddy, M. Venkateswar; Umemoto, Honoka; Sato, Yuki; Kang, Mi-Hye; Yajima, Yuka; Kikuchi, Shintaro

2015-01-01

In the present study, a 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterial strain CY-1 was isolated from the forest soil. Based on physiological, biochemical and 16S rRNA gene sequence analysis it was identified as Cupriavidus sp. CY-1. Further 2,4-D degradation experiments at different concentrations (200 to 800 mg l-1) were carried out using CY-1. Effect of NaCl and KNO3 on 2,4-D degradation was also evaluated. Degradation of 2,4-D and the metabolites produced during degradation process were analyzed using high pressure liquid chromatography (HPLC) and GC-MS respectively. The amount of chloride ions produced during the 2,4-D degradation were analyzed by Ion chromatography (IC) and it is stoichiometric with 2,4-D dechlorination. Furthermore two different types of soils collected from two different sources were used for 2,4-D degradation studies. The isolated strain CY-1 was bio-augmented into 2,4-D contaminated soils to analyze its degradation ability. Culture independent methods like denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), and culture dependent methods like colony forming units (CFU) and most probable number (MPN) were used to analyze the survivability of strain CY-1 in contaminated soil. Results of T-RFLP were coincident with the DGGE analysis. From the DGGE, T-RFLP, MPN and HPLC results it was concluded that strain CY-1 effectively degraded 2,4-D without disturbing the ecosystem of soil indigenous microorganisms. PMID:26710231

Genetic Polymorphisms of Cytochrome P4501A1 (CYP1A1) and Glutathione S-Transferase P1 (GSTP1) and Risk of Hepatocellular Carcinoma Among Chronic Hepatitis C Patients in Egypt.

PubMed

Abo-Hashem, Ekbal M; El-Emshaty, Wafaa M; Farag, Raghda El Sayed; Zakaria, Sahar; Abd El-Aziz, Mohammed; Ghonaim, Azza

2016-10-01

Cytochrome P450 1A1 (CYP1A1) and Glutathione S-transferase P1 (GSTP1) genes are involved in the metabolism of many carcinogens. Polymorphisms in these genes with altered enzyme activity have been reported. The present study evaluated the synergistic effect between CYP1A1 and GSTP1 gene polymorphisms and smoking on development of HCV-related liver disease and hepatocellular carcinoma (HCC). The patients group comprised 40 patients with HCC and 40 patients with liver cirrhosis. The control group comprised 40 healthy subjects having no history of malignancy. The genetic polymorphisms were studied using polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) technique on blood samples. The number of current or former smoker among HCC and cirrhotic patients as well as the median Pack/year of cigarette smoked were significantly higher in HCC and liver cirrhotic patients than in control group. Subjects with CYP1A1 gene variants (m1 and m3) had no significant risk to develop cirrhosis or HCC compared to control group. Individuals carrying the Ile/Val genotype of GSTP1 had a significant increased risk of HCC (OR of 2.2, 95 % CI 1.143-4.261) and had larger tumor size. No significant risk was observed on combining both genes variants or on combining smoking with variants of both genes. In conclusion, the GSTP1 Ile/Val genotype and Val allele are associated with an increased risk of HCC. CYP1A1 and GSTP1 genes variants interaction did not increase the risk of HCC.

DGAT1 K232A polymorphism in Brazilian cattle breeds.

PubMed

Lacorte, G A; Machado, M A; Martinez, M L; Campos, A L; Maciel, R P; Verneque, R S; Teodoro, R L; Peixoto, M G C D; Carvalho, M R S; Fonseca, C G

2006-08-31

Recent reports identified DGAT1 (EC 2.3.1.20) harboring a lysine to alanine substitution (K232A) as a candidate gene with a strong effect on milk production traits. Our objective was to estimate the frequency of the DGAT1 K232A polymorphism in the main Zebu and Taurine breeds in Brazil as well as in Zebu x Taurine crossbreds as a potential QTL for marker-assisted selection. Samples of 331 animals from the main Brazilian breeds, Nellore, Guzerat, Red Sindhi, Gyr, Holstein, and Gyr x Holstein F1 were genotyped for DGAT1 K232A polymorphism (A and K alleles) using the PCR-RFLP technique. The highest frequency of the A allele was found in the Holstein sample (73%) followed by Gyr x Holstein F1 (39%). Gyr and Red Sindhi showed low frequencies of A alleles (4 and 2.5%, respectively). The A allele was not found in the Nellore and Guzerat samples. Our results could be used to guide association studies between this locus and milk traits in these breeds.

Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP.

PubMed

Oh, Byung Ho; Lee, Yang Won; Choe, Yong Beom; Ahn, Kyu Joong

2010-05-01

This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups.

Population structure of wild bananas, Musa balbisiana, in China determined by SSR fingerprinting and cpDNA PCR-RFLP.

PubMed

Ge, X J; Liu, M H; Wang, W K; Schaal, B A; Chiang, T Y

2005-04-01

Both demographic history and dispersal mechanisms influence the apportionment of genetic diversity among plant populations across geographical regions. In this study, phylogeography and population structure of wild banana, Musa balbisiana, one of the progenitors of cultivated bananas and plantains in China were investigated by an analysis of genetic diversity of simple sequence repeat (SSR) fingerprint markers and cpDNA PCR-RFLP. A chloroplast DNA (cpDNA) genealogy of 21 haplotypes identified two major clades, which correspond to two geographical regions separated by the Beijiang and Xijiang rivers, suggesting a history of vicariance. Significant genetic differentiation was detected among populations with cpDNA markers, a result consistent with limited seed dispersal in wild banana mediated by foraging of rodents. Nuclear SSR data also revealed significant geographical structuring in banana populations. In western China, however, there was no detected phylogeograpahical pattern, possibly due to frequent pollen flow via fruit bats. In contrast, populations east of the Beijiang River and the population of Hainan Island, where long-range soaring pollinators are absent, are genetically distinct. Colonization-extinction processes may have influenced the evolution of Musa populations, which have a metapopulation structure and are connected by migrating individuals. Effective gene flow via pollen, estimated from the nuclear SSR data, is 3.65 times greater than gene flow via seed, estimated from cpDNA data. Chloroplast and nuclear DNAs provide different insights into phylogeographical patterns of wild banana populations and, taken together, can inform conservation practices.

Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

PubMed

Vidal, Jorge E; Enríquez-Rincón, Fernando; Giono-Cerezo, Silvia; Ribas-Aparicio, Rosa María; Figueroa-Arredondo, Paula

2009-01-01

To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

A Comprehensive Genetic Study of Streptococcal Immunoglobulin A1 Proteases: Evidence for Recombination within and between Species

PubMed Central

Poulsen, Knud; Reinholdt, Jesper; Jespersgaard, Christina; Boye, Kit; Brown, Thomas A.; Hauge, Majbritt; Kilian, Mogens

1998-01-01

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of

-383 A/C tumor necrosis factor receptor 1 polymorphism and ankylosing spondylitis in Mexicans: a preliminary study.

PubMed

Corona-Sanchez, Esther Guadalupe; Muñoz-Valle, José Francisco; Gonzalez-Lopez, Laura; Sanchez-Hernandez, Julia Dolores; Vazquez-Del Mercado, Monica; Ontiveros-Mercado, Heriberto; Huerta, Miguel; Trujillo, Xochitl; Rocha-Muñoz, Alberto Daniel; Celis, Alfredo; Ortega-Flores, Ricardo; Gamez-Nava, Jorge Ivan

2012-08-01

The objective of this study was to evaluate the differences in allele and genotype frequencies of -383 tumor necrosis factor receptor 1 (TNFR1) polymorphism between ankylosing spondylitis (AS) and controls. Mexican Mestizos with AS were matched by gender, age, and ethnicity with healthy controls and compared in allele and genotype frequencies of the -383 TNFR1 polymorphism. Polymorphisms were genotyped using PCR-RFLP. The AA genotype occurred at a higher frequency in the AS group (92%) compared with controls (79%, P = 0.03). A allele was increased in AS (96% vs. 88%, P = 0.015) and was associated with genetic susceptibility for AS (odds ratio = 3.48, 95% CI = 1.23-10.61). This preliminary study is the first assessing the association of the -383 A/C TNFR1 polymorphism with AS, although it has the limitation of a small sample size. These data are of interest for the genetic epidemiology of AS in the Mexican population, requiring further investigation in other countries.

Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

PubMed Central

Oh, Byung Ho; Choe, Yong Beom; Ahn, Kyu Joong

2010-01-01

Background This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. Objective The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. Methods 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. Results The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. Conclusion According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups. PMID:20548904

Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis

PubMed Central

Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

2014-01-01

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment. PMID:25178301

Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis.

PubMed

Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

2014-10-01

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

Uterine leiomyoma is associated with a polymorphism in the interleukin 1-beta gene.

PubMed

Pietrowski, Detlef; Thewes, Roberta; Sator, Michael; Denschlag, Dominik; Keck, Christoph; Tempfer, Clemens

2009-08-01

To investigate whether polymorphisms in the interleukin-1beta (IL-1beta) gene are associated with uterine leiomyoma. Case-control study in a collective of 131 patients and 280 controls. Genotyping of the IL-1beta-511 and IL-1beta-3954 polymorphism was performed by PCR amplification and subsequent RFLP analysis. A significant difference in the allele frequencies of the IL-1beta-511 C1beta-511 C1% in the control group [P < 0.0002; odds ratio (OR) 1.81; 95% confidence interval (CI): 1.32-2.47]. The genotype distributions showed also differences using a dominant genotype model (C/C vs. C/T+T/T; P < 0.0002; OR 2.73; 95% CI: 1.77-4.2). No difference was found in the IL-1beta-3954 polymorphism. The IL-1beta-511 promoter polymorphism is related to an increased susceptibility to uterine leiomyoma, suggesting that this polymorphism does contribute to the development of this disease.

Association of hypoxia inducible factor-1 alpha gene polymorphism with both type 1 and type 2 diabetes in a Caucasian (Hungarian) sample

PubMed Central

Nagy, Geza; Kovacs-Nagy, Reka; Kereszturi, Eva; Somogyi, Aniko; Szekely, Anna; Nemeth, Nora; Hosszufalusi, Nora; Panczel, Pal; Ronai, Zsolt; Sasvari-Szekely, Maria

2009-01-01

Background Hypoxia inducible factor-1 alpha (HIF-1α) is a transcription factor that plays an important role in neo-vascularisation, embryonic pancreas beta-cell mass development, and beta cell protection. Recently a non synonymous single nucleotide polymorphism (g.C45035T SNP, rs11549465) of HIF-1α gene, resulting in the p.P582S amino acid change has been shown to be associated with type 2 diabetes (T2DM) in a Japanese population. Our aim was to replicate these findings on a Caucasian (Hungarian) population, as well as to study whether this genetic effect is restricted to T2DM or can be expanded to diabetes in general. Methods A large Caucasian sample (N = 890) was recruited including 370 T2DM, 166 T1DM and 354 healthy subjects. Genotyping was validated by two independent methods: a restriction fragment analysis (RFLP) and a real time PCR using TaqMan probes. An overestimation of heterozygotes by RFLP was observed as a consequence of a nearby SNP (rs34005929). Therefore genotyping results of the justified TaqMan system were accepted. The measured genotype distribution corresponded to Hardy-Weinberg equilibrium (P = 0.740) Results As the TT genotype was extremely rare in the population (0.6% in clinical sample and 2.5% in controls), the genotypes were grouped as T absent (CC) and T present (CT and TT). Genotype-wise analysis showed a significant increase of T present group in controls (24.0%) as compared to patients (16.8%, P = 0.008). This genetic effect was demonstrated in the separated samples of type 1 (15.1%, P = 0.020), and also in type 2 (17.6%, P = 0.032) diabetes. Allele-wise analysis gave identical results showing a higher frequency of the T allele in the control sample (13.3%) than in the clinical sample (8.7%, P = 0.002) with similar results in type 1 (7.8%, P = 0.010) and type 2 (9.1%, P = 0.011) diabetes. The odds ratio for diabetes (either type 1 or 2) was 1.56 in the presence of the C allele. Conclusion We confirmed the protective effect of a rare

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

PubMed

Fong, Wai-Ying; Ho, Chi-Chun; Poon, Wing-Tat

2017-05-12

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice.

PubMed

Gerstner, Arpad; DeFord, James H; Papaconstantinou, John

2003-07-25

Ames dwarfism is caused by a homozygous single nucleotide mutation in the pituitary specific prop-1 gene, resulting in combined pituitary hormone deficiency, reduced growth and extended lifespan. Thus, these mice serve as an important model system for endocrinological, aging and longevity studies. Because the phenotype of wild type and heterozygous mice is undistinguishable, it is imperative for successful breeding to accurately genotype these animals. Here we report a novel, yet simple, approach for prop-1 genotyping using PCR-based allele-specific amplification (PCR-ASA). We also compare this method to other potential genotyping techniques, i.e. PCR-based restriction fragment length polymorphism analysis (PCR-RFLP) and fluorescence automated DNA sequencing. We demonstrate that the single-step PCR-ASA has several advantages over the classical PCR-RFLP because the procedure is simple, less expensive and rapid. To further increase the specificity and sensitivity of the PCR-ASA, we introduced a single-base mismatch at the 3' penultimate position of the mutant primer. Our results also reveal that the fluorescence automated DNA sequencing has limitations for detecting a single nucleotide polymorphism in the prop-1 gene, particularly in heterozygotes.

Association of vitamin D receptor gene polymorphisms with susceptibility to childhood asthma: A meta-analysis.

PubMed

Zhao, Dong-Dong; Yu, Dan-Dan; Ren, Qiong-Qiong; Dong, Bao; Zhao, Feng; Sun, Ye-Huan

2017-04-01

As for the association of vitamin D receptor (VDR) gene polymorphisms with susceptibility to pediatric asthma, results of published studies yielded conflicts. A systematic review was conducted on the relationship between childhood asthma and VDR gene polymorphisms, including ApaI (rs7975232), BsmI (rs1544410), FokI (rs2228570), and TaqI (rs731236). PubMed, Web of Science, CBM (Chinese Biomedical Database), CNKI (China National Knowledge Infrastructure), and Wanfang (Chinese) database were searched for relevant studies. Pooled odds ratios (OR) with 95% confidence interval (CI) were calculated. Overall results suggested that there was a statistically significant association between ApaI polymorphism and childhood asthma in homozygote model (OR = 1.674, 95%CI = 1.269-2.208, P < 0.001) and allele model (OR = 1.221, 95%CI = 1.084-1.375, P = 0.001). Stratification by ethnicity revealed a statistical association in Asians (OR = 1.389, 95%CI = 1.178-1.638, P < 0.001). There was some evidence of an association between BsmI polymorphism and childhood asthma in the homozygote (OR = 1.462, 95%CI = 1.016-2.105, P = 0.041) and allele models (OR = 1.181, 95%CI = 1.006-1.386, P = 0.042). This association reached significance only in the Caucasian group (OR = 1.236, 95%CI = 1.029-1.485, P = 0.023). For FokI, a statistical association was detected in dominant model (OR = 1.281, 95%CI = 1.055-1.555, P = 0.012); this association was significant in allele model (OR = 1.591, 95%CI = 1.052-2.405, P = 0.028) in Caucasian. ApaI polymorphism plays a particular role in childhood asthma in Asians. FokI polymorphism may be connected with pediatric asthma in Caucasian population. And BsmI polymorphism marginally contributes to childhood asthma susceptibility, while there might be no association between TaqI polymorphism and childhood asthma risk. Pediatr Pulmonol. 2017;52:423-429. © 2016 Wiley

[Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study].

PubMed

Ovalle-Bracho, Clemencia; Camargo, Carolina; Díaz-Toro, Yira; Parra-Muñoz, Marcela

2018-03-15

Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. To establish the concordance between the two tests as identifying methods for circulating species in Colombia. A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

Polymorphisms in exons 1B and 1C of the type I interleukin-1 receptor gene in patients with endometriosis.

PubMed

D'Amora, Paulo; Sato, Hélio; Girão, Manoel J B C; Silva, Ismael D C G; Schor, Eduardo

2006-09-01

To study possible correlation between the prevalence of polymorphisms in the type I interleukin-1 receptor gene and pelvic endometriosis. Genotypes of 223 women were analyzed: 109 women with surgically and histologically confirmed endometriosis and 114 healthy women. Distributions of two single-base polymorphisms of the human interleukin-1 receptor type I (IL-1RI) gene were evaluated: PstI, due to a C-->T transition in exon 1B and BsrBI a C-->A transition at position 52 in exon 1C. Polymorphisms were detected by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP) resolved on 3% agarose gels stained with ethidium bromide. Genotypes for PstI polymorphisms did not differ significantly among control and endometriosis (P = 0.058). However, in relation to BsrBI polymorphism, protective risk was observed for the development of endometriosis [OR 0.39-IC 95% (0.2-0.9)]. BsrBI heterozygote genotype (C/A) showed protective effect against endometriosis development.

Molecular Analysis of Bacterial Communities in Biofilms of a Drinking Water Clearwell

PubMed Central

Zhang, Minglu; Liu, Wenjun; Nie, Xuebiao; Li, Cuiping; Gu, Junnong; Zhang, Can

2012-01-01

Microbial community structures in biofilms of a clearwell in a drinking water supply system in Beijing, China were examined by clone library, terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing of the amplified 16S rRNA gene. Six biofilm samples (designated R1–R6) collected from six locations (upper and lower sites of the inlet, middle and outlet) of the clearwell revealed similar bacterial patterns by T-RFLP analysis. With respect to the dominant groups, the phylotypes detected by clone library and T-RFLP generally matched each other. A total of 9,543 reads were obtained from samples located at the lower inlet and the lower outlet sites by pyrosequencing. The bacterial diversity of the two samples was compared at phylum and genus levels. Alphaproteobacteria dominated the communities in both samples and the genus of Sphingomonas constituted 75.1%–99.6% of this phylum. A high level of Sphingomonas sp. was first observed in the drinking water biofilms with 0.6–1.0 mg L−1 of chlorine residual. Disinfectant-resistant microorganisms deserve special attention in drinking water management. This study provides novel insights into the microbial populations in drinking water systems and highlights the important role of Sphingomonas species in biofilm formation. PMID:23059725

Covariance of bacterioplankton composition and environmental variables in a temperate delta system

USGS Publications Warehouse

Stepanauskas, R.; Moran, M.A.; Bergamaschi, B.A.; Hollibaugh, J.T.

2003-01-01

We examined seasonal and spatial variation in bacterioplankton composition in the Sacramento-San Joaquin River Delta (CA) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Cloned 16S rRNA genes from this system were used for putative identification of taxa dominating the T-RFLP profiles. Both cloning and T-RFLP analysis indicated that Actinobacteria, Verrucomicrobia, Cytophaga-Flavobacterium and Proteobacteria were the most abundant bacterioplankton groups in the Delta. Despite the broad variety of sampled habitats (deep water channels, lakes, marshes, agricultural drains, freshwater and brackish areas), and the spatial and temporal differences in hydrology, temperature and water chemistry among the sampling campaigns, T-RFLP electropherograms from all samples were similar, indicating that the same bacterioplankton phylotypes dominated in the various habitats of the Delta throughout the year. However, principal component analysis (PCA) and partial least-squares regression (PLS) of T-RFLP profiles revealed consistent grouping of samples on a seasonal, but not a spatial, basis. ??-Proteobacteria related to Ralstonia, Actinobacteria related to Microthrix, and ??-Proteobacteria identical to the environmental Clone LD12 had the highest relative abundance in summer/fall T-RFLP profiles and were associated with low river flow, high pH, and a number of optical and chemical characteristics of dissolved organic carbon (DOC) indicative of an increased proportion of phytoplankton-produced organic material as opposed to allochthonous, terrestrially derived organic material. On the other hand, Geobacter-related ??-Proteobacteria showed a relative increase in abundance in T-RFLP analysis during winter/spring, and probably were washed out from watershed soils or sediment. Various phylotypes associated with the same phylogenetic division, based on tentative identification of T-RFLP fragments, exhibited diverse seasonal patterns, suggesting that ecological

Genotyping of β-Lactoglobulin gene by PCR-RFLP in Sahiwal and Tharparkar cattle breeds

PubMed Central

Rachagani, Satyanarayana; Gupta, Ishwar Dayal; Gupta, Neelam; Gupta, SC

2006-01-01

Background Improvement of efficiency and economic returns is an important goal in dairy farming, as in any agricultural enterprise. The primary goal of dairy industry has been to identify an efficient and economical way of increasing milk production and its constituents without increasing the size of the dairy herd. Selection of animals with desirable genotypes and mating them to produce the next generation has been the basis of livestock improvement and this would continue to remain the same in the coming years. The use of polymorphic genes as detectable molecular markers is a promising alternative to the current methods of trait selection once these genes are proven to be associated with traits of interest in animals. The point mutations in exon IV of bovine β-Lactoglobulin gene determine two allelic variants A and B. These variants were distinguished by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) analysis in two indigenous Bos indicus breeds viz. Sahiwal and Tharparkar cattle. DNA samples (228 in Sahiwal and 86 in Tharparkar) were analyzed for allelic variants of β-Lactoglobulin gene. Polymorphism was detected by digestion of PCR amplified products with Hae III enzyme, and separation on 12% non-denaturing gels and resolved by silver staining. Results The allele B of β-Lactoglobulin occurred at a higher frequency than the allele A in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in Sahiwal and Tharparkar breeds were 0.031, 0.276, 0.693 and 0.023, 0.733, 0.244 respectively. Frequencies of A and B alleles were 0.17 and 0.83, and 0.39 and 0.61 in Sahiwal and Tharparkar breeds respectively. The Chi-square test results (at one degree of freedom at one per cent level) revealed that the Tharparkar population was not in Hardy-Weinberg equilibrium as there was a continuous migration of animals in the herd studied, where as, the results are not significant for the Sahiwal population. Conclusion

Preliminary Identification and Typing of Pathogenic and Toxigenic Fusarium Species Using Restriction Digestion of ITS1-5.8S rDNA-ITS2 Region.

PubMed

Mirhendi, H; Ghiasian, A; Vismer, Hf; Asgary, Mr; Jalalizand, N; Arendrup, Mc; Makimura, K

2010-01-01

Fusarium species are capable of causing a wide range of crop plants infections as well as uncommon human infections. Many species of the genus produce mycotoxins, which are responsible for acute or chronic diseases in animals and humans. Identification of Fusaria to the species level is necessary for biological, epidemiological, pathological, and toxicological purposes. In this study, we undertook a computer-based analysis of ITS1-5.8SrDNA-ITS2 in 192 GenBank sequences from 36 Fusarium species to achieve data for establishing a molecular method for specie-specific identification. Sequence data and 610 restriction enzymes were analyzed for choosing RFLP profiles, and subsequently designed and validated a PCR-restriction enzyme system for identification and typing of species. DNA extracted from 32 reference strains of 16 species were amplified using ITS1 and ITS4 universal primers followed by sequencing and restriction enzyme digestion of PCR products. The following 3 restriction enzymes TasI, ItaI and CfoI provide the best discriminatory power. Using ITS1 and ITS4 primers a product of approximately 550bp was observed for all Fusarium strains, as expected regarding the sequence analyses. After RFLP of the PCR products, some species were definitely identified by the method and some strains had different patterns in same species. Our profile has potential not only for identification of species, but also for genotyping of strains. On the other hand, some Fusarium species were 100% identical in their ITS-5.8SrDNA-ITS2 sequences, therefore differentiation of these species is impossible regarding this target alone. ITS-PCR-RFLP method might be useful for preliminary differentiation and typing of most common Fusarium species.

A link between osteomyelitis and IL1RN and IL1B polymorphisms—a study in patients from Northeast Brazil

PubMed Central

Alves De Souza, Clinio; Queiroz Alves De Souza, Argos; Queiroz Alves De Souza, Maria do Socorro; Dias Leite, José Alberto; Silva De Morais, Maíra; Barem Rabenhorst, Sílvia Helena

2017-01-01

Background and purpose Treatment failure of osteomyelitis can result from genetic susceptibility, highlighting polymorphisms of the interleukin-1 (IL-1) family members, central mediators of innate immunity and inflammation. Polymorphisms are DNA sequence variations that are common in the population (1% or more) and represent multiple forms of a single gene. We investigated the association of IL1RNVNTR (rs2234663) and IL1B-511C > T (rs16944) polymorphisms with osteomyelitis development in patients operated on because of bone trauma. Patients and methods 153 patients who fulfilled the inclusion criteria were enrolled from a referral public hospital for trauma. All the patients were followed up daily until hospital discharge and, after this, on an outpatient basis. Patients were treated with prophylactic antimicrobials and surgery according to traumatology service protocol. The IL1RNVNTR and the IL1B-511C > T polymorphisms were determined by PCR and PCR-RFLP, respectively. Results The IL1RN*2/*2 genotype was associated (OR: 7; p < 0.001) with a higher risk of osteomyelitis and was also significantly associated with Staphylococcus aureus infection. The haplotypes (combination of different markers) *2-C and *2-T were also associated with osteomyelitis development. Interpretation IL1B-511C > T and IL1RNVNTR polymorphisms were associated with osteomyelitis development, which may have implications for patients with bone traumas. These data may be relevant for new therapeutic strategies for this disease. PMID:28682145

A link between osteomyelitis and IL1RN and IL1B polymorphisms-a study in patients from Northeast Brazil.

PubMed

Alves De Souza, Clinio; Queiroz Alves De Souza, Argos; Queiroz Alves De Souza, Maria do Socorro; Dias Leite, José Alberto; Silva De Morais, Maíra; Barem Rabenhorst, Sílvia Helena

2017-10-01

Background and purpose - Treatment failure of osteomyelitis can result from genetic susceptibility, highlighting polymorphisms of the interleukin-1 (IL-1) family members, central mediators of innate immunity and inflammation. Polymorphisms are DNA sequence variations that are common in the population (1% or more) and represent multiple forms of a single gene. We investigated the association of IL1RNVNTR (rs2234663) and IL1B-511C > T (rs16944) polymorphisms with osteomyelitis development in patients operated on because of bone trauma. Patients and methods - 153 patients who fulfilled the inclusion criteria were enrolled from a referral public hospital for trauma. All the patients were followed up daily until hospital discharge and, after this, on an outpatient basis. Patients were treated with prophylactic antimicrobials and surgery according to traumatology service protocol. The IL1RNVNTR and the IL1B-511C > T polymorphisms were determined by PCR and PCR-RFLP, respectively. Results - The IL1RN*2/*2 genotype was associated (OR: 7; p < 0.001) with a higher risk of osteomyelitis and was also significantly associated with Staphylococcus aureus infection. The haplotypes (combination of different markers) *2-C and *2-T were also associated with osteomyelitis development. Interpretation - IL1B-511C > T and IL1RNVNTR polymorphisms were associated with osteomyelitis development, which may have implications for patients with bone traumas. These data may be relevant for new therapeutic strategies for this disease.

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

PubMed

Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

2017-12-18

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first

Primary DNA damage and genetic polymorphisms for CYP1A1, EPHX and GSTM1 in workers at a graphite electrode manufacturing plant

PubMed Central

Moretti, Massimo; Dell'Omo, Marco; Villarini, Milena; Pastorelli, Roberta; Muzi, Giacomo; Airoldi, Luisa; Pasquini, Rossana

2007-01-01

Background The results of a cross-sectional study aimed to evaluate whether genetic polymorphisms (biomarkers of susceptibility) for CYP1A1, EPHX and GSTM1 genes that affect polycyclic aromatic hydrocarbons (PAH) activation and detoxification might influence the extent of primary DNA damage (biomarker of biologically effective dose) in PAH exposed workers are presented. PAH-exposure of the study populations was assessed by determining the concentration of 1-hydroxypyrene (1OHP) in urine samples (biomarker of exposure dose). Methods The exposed group consisted of workers (n = 109) at a graphite electrode manufacturing plant, occupationally exposed to PAH. Urinary 1OHP was measured by HPLC. Primary DNA damage was evaluated by the alkaline comet assay in peripheral blood leukocytes. Genetic polymorphisms for CYP1A1, EPHX and GSTM1 were determined by PCR or PCR/RFLP analysis. Results 1OHP and primary DNA damage were significantly higher in electrode workers compared to reference subjects. Moreover, categorization of subjects as normal or outlier highlighted an increased genotoxic risk OR = 2.59 (CI95% 1.32–5.05) associated to exposure to PAH. Polymorphisms in EPHX exons 3 and 4 was associated to higher urinary concentrations of 1OHP, whereas none of the genotypes analyzed (CYP1A1, EPHX, and GSTM1) had any significant influence on primary DNA damage as evaluated by the comet assay. Conclusion The outcomes of the present study show that molecular epidemiology approaches (i.e. cross-sectional studies of genotoxicity biomarkers) can play a role in identifying common genetic risk factors, also attempting to associate the effects with measured exposure data. Moreover, categorization of subjects as normal or outlier allowed the evaluation of the association between occupational exposure to PAH and DNA damage highlighting an increased genotoxic risk. PMID:17908297

Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene.

PubMed

Hajjaran, Homa; Mohebali, Mehdi; Teimouri, Aref; Oshaghi, Mohammad Ali; Mirjalali, Hamed; Kazemi-Rad, Elham; Shiee, Mohammad Reza; Naddaf, Saied Reza

2014-08-01

The identity of Iranian Leishmania species has been resolved to some extent by some genetic markers. In this study, based on N-acetylglucosamine-1-phosphate transferase (nagt) gene, we further elucidated the identity and phylogeny of the prevalent species in this country. DNAs of 121 isolates belonging to cutaneous leishmaniasis (CL) patients, canine visceral leishmaniasis (CVL) cases, and Rhombomys opimus rodents were amplified by targeting a partial sequence of nagt gene. All the amplicons were analyzed with restriction fragment length polymorphism (RFLP) using Acc1 enzyme, and 49 amplicons representing different reservoir hosts were sequenced and aligned with similar sequences from GenBank database. The RFLP analysis revealed that 41 CL patients were infected Leishmania tropica and 36 with Leishmania major. Among 10 CVL isolates, 6 were identified as Leishmania infantum and 4 as L. tropica. Amongst 34 rodents' isolates, 11 and 23 isolates exhibited patterns similar to those of L. major, and L. tropica/Leishmania turanica, respectively. The sequencing results from all CL patients, CVL cases, and 4 reservoir rodents were in agreement with RFLP analysis and showed 99-100% homologies with the registered species of L. major, L. tropica, and L. infantum from Turkey, Tunisia, Iraq and Israel. Of the 7 rodent isolates exhibiting RFLP patterns similar to L. tropica/L. turanica, 3 exhibited the highest homologies (99-100%) with L. turanica and 4 with Leishmania gerbilli. The 49 nagt DNA sequences were grouped into five clusters representing L. major, L. tropica, L. infantum, L. turanica and L. gerbilli species, encompassing 19 haplotypes. No correlation was observed between intraspecies divergence and geographic distribution of haplotypes. The L. tropica haplotypes exhibited more homologies with those of L. infantum than L. major (97.2% vs. 96.9%), a probable indication to the potential ability of L. tropica to visceralize. Characterization of Iranian Leishmania isolates

Identification of the razor clam species Ensis arcuatus, E. siliqua, E. directus, E. macha, and Solen marginatus using PCR-RFLP analysis of the 5S rDNA region.

PubMed

Fernandez-Tajes, Juan; Méndez, Josefina

2007-09-05

Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the 5S ribosomal DNA region has been applied to the establishment of DNA-based molecular markers for the identification of five razor clam species: Ensis arcuatus, E. siliqua, E. directus, E. macha, and Solen marginatus. PCR amplifications were carried out using a pair of universal primers from the coding region of 5S rDNA. S. marginatus was simply distinguished by the different size of the amplicons obtained. Species-specific restriction endonuclease patterns were found with the enzymes Hae III for E. arcuatus, E. siliqua, and E. directus, and Acs I for E. macha, and when two enzymes were combined, the four species were also identified. Thus, this work provides a simple, reliable, and rapid protocol for the accurate identification of Ensis and Solen species in fresh and canned products, which is very useful for traceability and to enforce labeling regulations.

Genetic divergence and phylogenetic relationships in grey mullets (Teleostei: Mugilidae) based on PCR-RFLP analysis of mtDNA segments.

PubMed

Papasotiropoulos, V; Klossa-Kilia, E; Kilias, G; Alahiotis, S

2002-04-01

The genetic differentiation and phylogenetic relationships among five species of the Mugilidae family (Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens) were investigated at the mtDNA level, on samples taken from Messolongi lagoon-Greece. RFLP analysis of three PCR-amplified mtDNA gene segments (12s rRNA, 16s rRNA, and CO I) was used. Ten, eight, and nine restriction enzymes were found to have at least one recognition site at 12s rRNA, 16s rRNA, and CO I genes, respectively. Several fragment patterns were revealed to be species-specific, and thus they could be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. Seven different haplotypes were detected. The greatest amount of genetic differentiation was observed at the interspecific level, while little variation was revealed at the intraspecific level. The highest values of nucleotide sequence divergence were observed between M. cephalus and all the other species, while the lowest was found between C. labrosus and L. saliens. Dendrograms obtained by the three different methods (UPGMA, Neighbor-Joining, and Dollo parsimony), were found to exhibit in all cases the same topology. According to this, the most distinct species is M. cephalus, while the other species are clustered in two separate groups, thefirst one containing L. aurata and L. ramada, the other L. saliens and C. labrosus. This last clustering makes the monophyletic origin of the genus Liza questionable.

A common polymorphism of the MCT1 gene and athletic performance.

PubMed

Fedotovskaya, Olga N; Mustafina, Leysan J; Popov, Daniil V; Vinogradova, Olga L; Ahmetov, Ildus I

2014-01-01

In red skeletal muscle, monocarboxylate transporter 1 (MCT1) is required for lactate to enter the myocytes for oxidation. The A1470T polymorphism (rs1049434) in the MCT1 gene was shown to be associated with lactate transport rates in human skeletal muscles. The aim of the study was to compare genotype and allele frequencies of the MCT1 gene polymorphism in 323 Russian athletes and 467 nonathletic controls and to investigate the association of the MCT1 gene A1470T polymorphism with maximal oxygen consumption and maximal lactate concentration in rowers (n = 79). Genotyping for the A1470T MCT1 polymorphism was performed by PCR-RFLP method. Physiological measurements of 79 Russian rowers of national competitive standard were determined during an incremental test to exhaustion on a rowing ergometer. Frequencies of the A allele (71.8% vs 62.5%, P < .0001) and AA genotype (59.8% vs 39.4%, P < .0001) were significantly higher in endurance-oriented athletes (n = 142) than in the control group. Mean blood lactate concentration was higher in male rowers with the T allele (AT+TT 10.26 ± 1.89 mmol/L, AA 8.75 ± 1.69 mmol/L, P = .005). MCT1 gene A1470T polymorphism is associated with endurance athlete status and blood lactate level after intensive exercise.

The Vitamin D Receptor (VDR) Gene Polymorphisms in Turkish Brain Cancer Patients

PubMed Central

Toptaş, Bahar; Kafadar, Ali Metin; Cacina, Canan; Turan, Saime; Yurdum, Leman Melis; Yiğitbaşı, Nihal; Gökçe, Muhammed Oğuz; Zeybek, Ümit; Yaylım, Ilhan

2013-01-01

Objective. It has been stated that brain cancers are an increasingly serious issue in many parts of the world. The aim of our study was to determine a possible relationship between Vitamin D receptor (VDR) gene polymorphisms and the risk of glioma and meningioma. Methods. We investigated the VDR Taq-I and VDR Fok-I gene polymorphisms in 100 brain cancer patients (including 44 meningioma cases and 56 glioma cases) and 122 age-matched healthy control subjects. This study was performed by polymerase chain reaction-based restriction fragment length polymorphism (RF LP). Results. VDR Fok-I ff genotype was significantly increased in meningioma patients (15.9%) compared with controls (2.5%), and carriers of Fok-I ff genotype had a 6.47-fold increased risk for meningioma cases. There was no significant difference between patients and controls for VDR Taq-I genotypes and alleles. Conclusions. We suggest that VDR Fok-I genotypes might affect the development of meningioma. PMID:23691496

Genetic Heterogeneity in Mycobacterium tuberculosis Isolates Reflected in IS6110 Restriction Fragment Length Polymorphism Patterns as Low-Intensity Bands

PubMed Central

de Boer, Annette S.; Kremer, Kristin; Borgdorff, Martien W.; de Haas, Petra E. W.; Heersma, Herre F.; van Soolingen, Dick

2000-01-01

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS6110 RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple M. tuberculosis strains. We did not find any multiple LIBs, suggesting that multiple infections are rare in the Netherlands. However, we did observe a few LIBs in 94 patterns (7.4%) and examined the nature of this phenomenon. With single-colony cultures it was found that LIBs mostly represent mixed bacterial populations with slightly different RFLP patterns. Mixtures were expressed in RFLP patterns as LIBs when 10 to 30% of the DNA analyzed originated from a bacterial population with another RFLP pattern. Presumably, a part of the LIBs did not represent mixed bacterial populations, as in some clusters all strains exhibited LIBs in their RFLP patterns. The occurrence of LIBs was associated with increased age in patients. This may reflect either a gradual change of the bacterial population in the human body over time or IS6110-mediated genetic adaptation of M. tuberculosis to changes in the environmental conditions during the dormant state or reactivation thereafter. PMID:11101583

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

PubMed

Fadda, M E; Pisano, M B; Scaccabarozzi, L; Mossa, V; Deplano, M; Moroni, P; Liciardi, M; Cosentino, S

2013-01-01

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of

Effect of pharmacogenetic markers of vitamin D pathway on deferasirox pharmacokinetics in children.

PubMed

Allegra, Sarah; Cusato, Jessica; De Francia, Silvia; Longo, Filomena; Pirro, Elisa; Massano, Davide; Piga, Antonio; D'Avolio, Antonio

2018-01-01

Patients with β-thalassemia major have extremely low vitamin D levels, owing to reduced intestinal absorption, subicteric tint, and/or iron-induced higher pigmentation. We investigated whether some polymorphisms within the VDR, CYP24A1, CYP27B1, and GC genes could play a role in deferasirox pharmacokinetics in a cohort of pediatric patients. Eighteen children with β-thalassemia were enrolled. Drug plasma concentrations at the end of dosing interval (Ctrough) and after 0, 2, 4, 6, and 24 h of drug administration were measured by a HPLC-UV method. Allelic discrimination for VDR (TaqI, FokI, BsmI, Cdx2, and ApaI), CYP24A1 (22776, 3999 and 8620), CYP27B1 (2838 and -1260), and GC (1296) single nucleotide polymorphisms was performed by real-time PCR. CYP24A1 8620 AG/GG group negatively predicted Ctrough in regression analysis (P=0.012). ApaI AA genotype resulted as a negative predictor of Ctrough (P=0.025) and area under the concentration curve (P=0.007); FoKI CC genotype remained as area under the concentration curve positive predictor (P=0.008) and TC/CC group as half-life (t1/2) (P=0.003) and volume of distribution (Vd) (P=0.011) negative one; TaqI TC/CC was retained as a negative predictor of drug maximum concentration (Cmax) (P=0.004). Moreover, GC 1296 TG/GG seemed able to predict lower time to reach drug maximum concentration (Tmax) (P=0.033). Our preliminary experience suggested the potential usefulness of vitamin D pharmacogenetic to better understand deferasirox interindividual variability, also in pediatric patients.

alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases.

PubMed Central

Miller, P B; Wakarchuk, W W; Warren, R A

1985-01-01

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase. PMID:2987859

[Analysis of POU1F1 gene polymorphisms in Qinchuan cattle and Chinese Holstein cattle].

PubMed

Yan, Lin-Jun; Liu, Bo; Fang, Xin-Tang; Chen, Hong; Zhang, Run-Feng; Bao, Bin; Zhang, Hai-Jun

2006-11-01

PCR-RFLP was applied to analyze the polymorphisms of POU1F1 gene in 218 Qinchuan cattle (QQ) and Chinese Holstein cattle (HC). Results demonstrated Hinf I polymorphisms in the 451 bp PCR product in the two populations. The frequencies of alleles A/B in QQ and HC populations were 0.232/0.768 and 0.132/0.868, respectively. The frequencies of three genotypes AA, AB and BB were 0.030/0.403/0.567 and 0.007/0.251/0.742, respectively. Qinchuan cattle population was at Hardy-Weinberg equilibrium at this locus, but Chinese Holstein cattle population was not. The gene heterozygosity/effective allele gene number/Shannon information entropy/polymorphism information content of Qinchuan cattle and Chinese Holstein cattle populations were listed for 0.356/1.553/0.541/0.292 and 0.229/1.297/0.390/0.203, respectively. All indices were higher in the Qinchuan cattle population.

To Screen Inactivation Mutation of Exon 1 of FSHR Gene in Polycystic Ovarian Syndrome: A South Indian Cohort Study

NASA Astrophysics Data System (ADS)

Sekar, Nishu; Yeole, Samiksha; Pradeep, Rashmi; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

2017-11-01

Polycystic ovary syndrome is an endocrine disorder. Irregular menstrual cycle, acne, facial hair and elevated androgen levels are the most common signs for PCOS. PCOS has an estimated prevalence of 4-12% among reproductive age women, thus making it a forerunner in female infertility. FSHR plays an important role in FSH signaling pathway making it an important gene for PCOS. In this study, we aim to focus on any association between the FSHR gene and PCOS. Our study was to evaluate any polymorphism of exon 1 of FSHR gene associated with PCOS.PCR-RFLP technique was performed on the PCOS samples. Hormonal changes were found in the patients. Exon 1 inactivation mutation of FSHR gene was not observed in the patient sample. A study of this association needs to be done using large sample size.

High proportion of subgroup A' (genotype A) among Brazilian isolates of Hepatitis B virus.

PubMed

Araujo, N M; Mello, F C A; Yoshida, C F T; Niel, C; Gomes, S A

2004-07-01

Hepatitis B virus (HBV) genotype A has been divided recently into two subgroups, designated A-A' (genotype A excluding A') and A'. Isolates belonging to subgroup A' have been identified in Africa. A new genotyping method, based on PCR amplification of the pre-S/S genome region and subsequent restriction fragment length polymorphism (RFLP) analysis, was developed, that established a correlation between RFLP subtypes and subgroups within genotype A. To investigate the occurrence of subgroup A' in South America, 119 Brazilian HBV isolates were analyzed. Ninety-three (78%) of them belonged to genotype A, with three predominating RFLP subtypes: 44 (37%) isolates were classified as AI, 30 (25%) were AII, and 18 (15%) were AIII. Pre-S/S nucleotide sequences of 15 genotype A isolates were determined. Phylogenetic analysis performed with these 15 and an additional 41 sequences revealed that isolates AI and AII clustered in subgroup A', whereas isolates AIII were classified into subgroup A-A'. The correlation RFLP subtypes-subgroups was confirmed by the presence of amino acid residues specific for subgroup A' in the surface antigens and polymerase of isolates AI and AII. The high proportion (63%) of isolates from subgroup A' suggested an African origin for a large number of Brazilian HBVs.

Seasonal diversity of planktonic protists in Southwestern Alberta rivers over a 1-year period as revealed by terminal restriction fragment length polymorphism and 18S rRNA gene library analyses.

PubMed

Thomas, Matthew C; Selinger, L Brent; Inglis, G Douglas

2012-08-01

The temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure.

Seasonal Diversity of Planktonic Protists in Southwestern Alberta Rivers over a 1-Year Period as Revealed by Terminal Restriction Fragment Length Polymorphism and 18S rRNA Gene Library Analyses

PubMed Central

Thomas, Matthew C.; Selinger, L. Brent

2012-01-01

The temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure. PMID:22685143

Vitamin D receptor genetic polymorphisms and tuberculosis: updated systematic review and meta-analysis.

PubMed

Gao, L; Tao, Y; Zhang, L; Jin, Q

2010-01-01

Host genetic susceptibility has been suggested as one of the most important explanations for inter-individual differences in tuberculosis (TB) risk. The vitamin D receptor (VDR) gene has been studied as a candidate locus due to genetic polymorphisms that affects the activity of the receptor and subsequent downstream vitamin D-mediated effects. We reviewed published studies on VDR polymorphisms and TB susceptibility up to 15 April 2009 and quantitatively summarised associations of the most widely studied polymorphisms (FokI, TaqI, ApaI and BsmI) using meta-analysis. A total of 23 eligible studies were included in this review. Heterogeneous results were observed, which may be partly explained by the differences between populations. Among Asians, the FokI ff genotype showed a pronounced positive association (OR 2.0, 95%CI 1.3-3.2), a significant inverse association was observed for the BsmI bb genotype (OR 0.5, 95%CI 0.4-0.8), and marginal significant associations were found for TaqI and ApaI polymorphisms. However, none of the polymorphisms was significantly related to TB among Africans or South Americans. The association of VDR polymorphisms with risk of TB observed in our analyses supports the hypothesis that vitamin D deficiency might play a role as risk factor during the development of TB.

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

PubMed

Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

2010-04-01

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

Plasminogen activator inhibitor type 1 gene is located at region q21. 3-q22 of chromosome 7 and genetically linked with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klinger, K.W.; Winqvist, R.; Riccio, A.

1987-12-01

The regional chromosomal location of the human gene for plasminogen activator inhibitor type 1 (PAI1) was determined by three independent methods of gene mapping. PAI1 was localized first to 7cen-q32 and then to 7q21.3-q22 by Southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids with a PAI1 cDNA probe and in situ hybridization, respectively. The authors frequent HindIII restriction fragment length polymorphism (RFLP) of the PAI1 gene with an information content of 0.369. In family studies using this polymorphism, genetic linkage was found between PAI1 and the loci for erythropoietin (EPO), paraoxonase (PON), the metmore » protooncogene (MET), and cystic fibrosis (CF), all previously assigned to the middle part of the long arm of chromosome 7. The linkage with EPO was closest with an estimated genetic distance of 3 centimorgans, whereas that to CF was 20 centimorgans. A three-point genetic linkage analysis and data from previous studies showed that the most likely order of these loci is EPO, PAI1, PON, (MET, CF), with PAI1 being located centromeric to CF. The PAI1 RFLP may prove to be valuable in ordering genetic markers in the CF-linkage group and may also be valuable in genetic analysis of plasminogen activation-related diseases, such as certain thromboembolic disorders and cancer.« less

Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1.

PubMed

Loli, A; Tzouvelekis, L S; Tzelepi, E; Carattoli, A; Vatopoulos, A C; Tassios, P T; Miriagou, V

2006-09-01

To elucidate the mechanisms responsible for the diversity of beta-lactam resistance phenotypes among isolates of a VIM-1-producing Klebsiella pneumoniae (VPKP) strain that is endemic in Greek hospitals. Five VPKP clinical isolates were studied. MICs of beta-lactams were determined by agar dilution. PFGE of XbaI-digested genomic DNA was used for typing. Profiles of outer membrane proteins (OMPs) were determined by SDS-PAGE. Selected isolates were transformed with a plasmid encoding the Omp36K porin. beta-Lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding, self-transmissible plasmids were characterized by replicon typing, RFLP and hybridization with bla(VIM)- and IS26-specific probes. Characterization of integrons was performed by PCR, cloning and sequencing. Isolates exhibited highly similar PFGE patterns. Imipenem MICs were 2, 4, 16, 32 and 64 mg/L. The isolate with the highest imipenem MIC (Vipm-64) lacked a 36 kDa OMP. Expression of a cloned OmpK36 in this isolate reduced the imipenem MIC to susceptibility levels. Imipenem-hydrolysing activity was significantly higher in Vipm-16 as compared with the other isolates that expressed similar amounts of VIM-1. All isolates transferred beta-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla(VIM)- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of bla(VIM-1). Cloning and sequencing showed In-e541-like integrons truncated at the 5'CS by insertion of IS26 elements at two different positions. A VIM-1-producing strain of K. pneumoniae has evolved through OMP alterations and rearrangements in the bla(VIM-1)-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.

Effect of MLH1 -93G>A on gene expression in patients with colorectal cancer.

PubMed

Funck, Alexandre; Santos, Juliana C; Silva-Fernandes, Isabelle J L; Rabenhorst, Silvia H B; Martinez, Carlos A R; Ribeiro, Marcelo L

2014-09-01

The DNA repair machinery plays a key role in maintaining genomic stability by preventing the emergence of mutations. Furthermore, the -93G>A polymorphism in the MLH1 gene has been associated with an increased risk of developing colorectal cancer. Therefore, the aim of this study was to examine the expression pattern and effect of this polymorphism in normal and tumour samples from patients with colorectal cancer. The MLH1 -93G>A (rs1800734) polymorphism was detected by PCR-RFLP in 49 cases of colorectal cancer. MLH1 expression was investigated using real-time quantitative PCR. The results indicate a significant decrease in MLH1 expression in tumour samples compared to their normal counterparts. The MLH1 gene was also significantly repressed in samples from patients who had some degree of tumour invasion into other organs. Similarly, those patients who were in a more advanced tumour stage (TNM III and IV) exhibited a significant reduction in MLH1 gene expression. Finally, the mutant genotype AA of MLH1 was associated with a significant decrease in the expression of this gene. This finding suggests that this polymorphism could increase the risk of developing colorectal cancer by a defective mismatch repair system, particularly through the loss of MLH1 expression in an allele-specific manner.

Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lanser, J.A.; Doyle, R.; Sangster, N.

1990-09-01

The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis. Three radiolabeled probes were used in Southern hybridizations for the RFLP studies. They were Escherichia coli 16S and 23S rRNA and cloned fragments of L. longbeachae selected empirically from genomal banks in lambda and a cosmid. The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated form humans, 28 L. longbeachae serogroup 1 isolates from environmental sources, 3 L. longbeachae serogroup 2 environmental isolates. These were compared with the American Type Culture Collectionmore » reference strains of both serogroups and some other related Legionella species. Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L. longbeachae serogroup 1 isolates were closely related. They were also closely related to L. longbeachae serogroup 1 ATCC 33462. There was wider variation among the three L. longbeachae serogroup 2 environmental isolates. One of these was closely related to L. longbeachae serogroup 2 ATCC 33484. RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups.« less

GENETIC APPROACH TO THE STUDY OF EPIDEMIOLOGY AND PATHOGENESIS OF ACTINOBACILLUS ACTINOMYCETEMCOA4ITANS IN LOCALIZED JUVENILE PERIODONTITIS

PubMed Central

DiRienzo, J.M.; Slots, J.

2012-01-01

Summary Actinobacillus acrinomycetemcomirans isolates from periodontal pockets were examined for restriction fragment-length polymorphism using a characterized 4.7-kb DNA probe. A total of 6 patterns of RFLP was found in 133 isolates originating from 12 subjects. No relatedness was found between RFLP types and serotypes. Different periodontal sites within the same subject and different individuals within the same family sometimes showed only one type of A. actinomycetemcomitans RFLP. When members among the same family showed 2 RFLP types, children were always infected with the A. acfinomycefemcomitans strains found in at least one of the parents. These findings support the concept of familial spread of A. actinomycetemcomitans. A. actinomycetemcomitans RFLP type B, corresponding to reference strain JP2, seems to be particularly virulent, as indicated from the presence of RFLP type B in 3 subjects who converted from a healthy periodontal state to localized juvenile periodontitis. RFLP type B was not detected in any of the 21 A. acrinomycetemcomitans-infected patients with adult periodontitis. The RFLP method seems to be useful in determining the epidemiology and possibly the potential virulence of periodontal strains of A. actinomycetemcomitans. PMID:1982406

Expression of growth hormone and its transcription factor, Pit-1, in early bovine development.

PubMed

Joudrey, E M; Lechniak, D; Petrik, J; King, W A

2003-03-01

During bovine embryogenesis, bovine growth hormone (bGH) contributes to proliferation, differentiation, and modulation of embryo metabolism. Pituitary-specific transcription factor-1 (Pit-1) is a transcription factor that binds to promoters of GH, prolactin (PRL), and thyroid-stimulating hormone-beta (TSHbeta) encoding genes. A polymorphism in the fifth exon of the bGH gene resulting in a leucine (Leu) to valine (Val) substitution provides an Alu I restriction site when the Leu allele is present. To determine the onset of embryonic expression of the bGH gene, oocytes derived from ovaries homozygous for Leu alleles were fertilized in vitro with spermatozoa obtained from a Val homozygote. For each developmental stage examined, three separate pools of embryos composed of approximately 100 cell samples underwent RNA isolation, reverse transcription to cDNA, and amplification by nested PCR (nPCR). Bovine GH gene transcripts were identified at 2- to 4-cell (n = 162), 8- to 16-cell (n = 73), morulae (n = 51), and blastocyst (n = 15) stages. Likewise, transcripts for Pit-1 were detected at 2-cell (n = 125), 4-cell (n = 114), 8-cell (n = 56), 12-to-32-cell (n = 32), morulae (n = 68), and blastocyst (n = 14) stages. After digestion with Alu1, bGH cDNA was genotyped by restriction fragment length polymorphism (RFLP) analysis. Bovine GH mRNA was present in all pools of stages examined. Both Leu and Val alleles (maternal and paternal) were only detected in pools of embryos that had reached 8- to 16-cell stage. Results suggest that transcription of the bGH gene begins at the 8- to 16-cell stage in bovine embryos, possibly under control of the transcription factor, Pit-1, and that RFLP analysis of the bGH gene can be used to determine parental origin of transcripts in early embryonic development. Copyright 2003 Wiley-Liss, Inc.

Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities.

PubMed

Blackwood, Christopher B; Hudleston, Deborah; Zak, Donald R; Buyer, Jeffrey S

2007-08-01

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E(var)) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E(var)). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.

Association of single nucleotide polymorphisms in CACNA 1A/CACNA 1C/CACNA 1H calcium channel genes with diabetic peripheral neuropathy in Chinese population.

PubMed

Sun, Lin; Ma, Jun; Mao, Qian; Yang, Yun-Long; Ma, Lin-Lin; Niu, Ling; Liu, Li-Feng

2018-06-29

The present study was conducted to explore the correlations between single nucleotide polymorphisms (SNPs) in the calcium channel CACNA 1A, CACNA 1C, and CACNA 1H genes and diabetic peripheral neuropathy (DPN) amongst the Chinese population. In total, 281 patients diagnosed with type 2 diabetes participated in the present study. These patients were divided into the case group, which was subdivided into the DPN (143 cases) and the non-DPN groups (138 cases). Subsequently, 180 healthy individuals that had undergone routine health examinations were also recruited and assigned to the control group. PCR-restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotype and allele frequencies of CACNA 1A, CACNA 1C, and CACNA 1H genes; logistic regression analysis to investigate the association of gene polymorphisms with DNP. Gene-gene interactions were then detected by generalized multifactor dimensionality reduction (GMDR). The results revealed that CACNA 1A rs2248069 and rsl6030, CACNA 1C rs216008 and rs2239050, and CACNA 1H rs3794619, and rs7191246 SNPs were all associated with DPN, while rs2248069, rsl6030, rs2239050, and rs7191246 polymorphisms were attributed to the susceptibility to DPN. It was also observed that the optimal models were three-, four- and five-dimensional models with a prediction accuracy of 61.05% and the greatest consistency of cross-validation was 10/10. In summary, these findings demonstrated that the SNPs in the CACNA 1A, CACNA 1C, and CACNA 1H genes were involved in the pathophysiology of DPN. In addition, polymorphisms in the CACNA 1A, CACNA 1C, and CACNA 1H genes and their interactions also had effects on DPN. © 2018 The Author(s).

Oseltamivir-resistant influenza A(H1N1)pdm09 virus associated with high case fatality, India 2015.

PubMed

Tandel, Kundan; Sharma, Shashi; Dash, Paban Kumar; Parida, ManMohan

2018-05-01

Influenza A viruses has been associated with severe global pandemics of high morbidity and mortality with devastating impact on human health and global economy. India witnessed a major outbreak of influenza A(H1N1)pdm09 in 2015. This study comprises detailed investigation of cases died of influenza A(H1N1)pdm09 virus infection during explosive outbreak of 2015, in central part of India. To find out presence of drug resistant virus among patients who died of influenza A(H1N1)pdm09 virus infection and to find out presence of other mutations contributing to the morbidity and mortality. Twenty-two patients having confirmed influenza A(H1N1)pdm09 infection and subsequently died of this infection along with 20 non fatal cases with influenza A(H1N1)pdm09 infection were included in the study. Samples were investigated through RT-PCR/RFLP analysis, followed by nucleotide cycle sequencing of whole NA gene for detection of H275Y amino acid substitution in NA gene responsible for oseltamivir drug resistance. Out of 22 fatal cases, 6 (27.27%) were found to harbor oseltamivir resistant virus strains, whereas the H275Y mutation was not observed among the 20 non fatal cases. Amino acid substitution analysis of complete NA gene revealed V241I, N369K, N386K substitution in all strains playing synergistic role in oseltamivir drug resistance. High morbidity and mortality associated with influenza A(H1N1)pdm09 viruses can be explained by presence of drug resistant strains circulating in this outbreak. Presence of Oseltamivir resistant influenza A(H1N1)pdm09 viruses is a cause of great concern and warrants continuous screening for the circulation of drug resistant strains. © 2017 Wiley Periodicals, Inc.

Genotypic characterization of psittacid herpesvirus isolates from Brazil.

PubMed

Luppi, Marcela Miranda; Luiz, Ana Paula Moreira Franco; Coelho, Fabiana Magalhães; Ecco, Roselene; da Fonseca, Flávio Guimarães; Resende, Mauricio

2016-01-01

Thirty-six isolates of psittacid herpesvirus (PsHV), obtained from 12 different species of psittacids in Brazil, were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis and PCR amplification. RFLP analysis with the PstI enzyme revealed four distinct restriction patterns (A1, X, W and Y), of which only A1 (corresponding to PsHV-1) had previously been described. To study PCR amplification patterns, six pairs of primers were used. Using this method, six variants were identified, of which, variants 10, 8, and 9 (in this order) were most prevalent, followed by variants 1, 4, and 5. It was not possible to correlate the PCR and RFLP patterns. Twenty-nine of the 36 isolates were shown to contain a 419bp fragment of the UL16 gene, displaying high similarity to the PsHV-1 sequences available in GenBank. Comparison of the results with the literature data suggests that the 36 Brazilian isolates from this study belong to genotype 1 and serotype 1. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Paraoxonase 1 (PON1)-L55M among common variants in the coding region of the paraoxonase gene family may contribute to the glycemic control in type 2 diabetes.

PubMed

Mahrooz, Abdolkarim; Hashemi-Soteh, Mohammad Bagher; Heydari, Masoud; Boorank, Ruzbeh; Ramazani, Fatemeh; Mahmoudi, Ali; Kianmehr, Anvarsadat; Alizadeh, Ahad

2018-05-19

Genome studies have shown that the genes encoding paraoxonase 1 (PON1) and PON2 are associated with glucose metabolism. The goal of this study was to simultaneously evaluate the association between functional variants in PON1 and PON2 genes and susceptibility for type 2 diabetes (T2D) and determine whether they can affect glycemic control. We performed a case-control study with 145 newly diagnosed patients with T2D and 148 controls. The common variants including PON1-Q192R, PON1-L55M and PON2-S311C were genotyped by PCR-based RFLP. A mismatch-PCR/RFLP was applied for genotyping the PON2-A148G variant. The variant PON1-Q192R in males (OR = 2.55, 95%CI 1.16-5.69, p = 0.023) and PON2-A148G in females (OR = 1.56, 95%CI 1.00-2.44, p = 0.059) were associated with T2D. Compared with the LL genotypes of PON1-L55M, HbA1c levels were significantly lower in the LM genotypes (p = 0.01) and MM genotypes (p = 0.032) in patients. Multiple linear regression analyses showed that among the study variants only the PON1-L55M variant as an independent variable significantly associated with glycemic control. This variant significantly influenced glycemic control in patients with poor glycemic control so that it was better with the following order: LL < LM < MM. Based on gamma correlation, there was a significant inverse association between the number of M alleles of the PON1-L55M and HbA1c levels (r = -0.261, p = 0.001). Sex should be considered a confounding variable in association studies on the variants PON1-Q192R and PON2-A148G in T2D. Patients sharing the 55 M allele were prone to having good glycemic control. Our findings provide genetic evidence that the PON1-L55M variant may be a factor contributing to glycemic control. Copyright © 2018 Elsevier B.V. All rights reserved.

MSX1 gene variant - its presence in tooth absence - a case control genetic study.

PubMed

Reddy, Naveen Admala; Adusumilli, Gopinath; Devanna, Raghu; Pichai, Saravanan; Rohra, Mayur Gobindram; Arjunan, Sharmila

2013-10-01

Non Syndromic tooth agenesis is a congenital anomaly with significant medical, psychological and social ramifications. There is sufficient evidence to hypothesize that locus for this condition can be identified by candidate genes. The aim of this study was to test whether MSX1 671 T>C gene variant was involved in etiology of Non Syndromic tooth agenesis in Raichur Patients. Blood samples were collected with informed consent from 50 subjects having Non Syndromic tooth agenesis and 50 controls. Genomic DNA was extracted from the blood samples, Polymerase Chain Reaction was performed (PCR) and Restriction Fragment Length Polymorphism (RFLP) was performed for digestion products that were evaluated. The RESULTS showed positive correlation between MSX1671 T>C gene variant and Non Syndromic tooth agenesis in Raichur Patients. MSX1 671 T>C gene variant may be a good screening marker for Non Syndromic tooth agenesis in Raichur Patients . How to cite this article:Reddy NA, Adusumilli G, Devanna R, Pichai S, Rohra MG, Arjunan S. Msx1 Gene Variant - Its Presence in Tooth Absence - A Case Control Genetic Study. J Int Oral Health 2013; 5(5):20-6.

Msx1 Gene Variant - Its Presence in Tooth Absence - A Case Control Genetic Study

PubMed Central

Reddy, Naveen Admala; Adusumilli, Gopinath; Devanna, Raghu; Pichai, Saravanan; Rohra, Mayur Gobindram; Arjunan, Sharmila

2013-01-01

Background: Non Syndromic tooth agenesis is a congenital anomaly with significant medical, psychological and social ramifications. There is sufficient evidence to hypothesize that locus for this condition can be identified by candidate genes. The aim of this study was to test whether MSX1 671 T>C gene variant was involved in etiology of Non Syndromic tooth agenesis in Raichur Patients. Materials & Methods: Blood samples were collected with informed consent from 50 subjects having Non Syndromic tooth agenesis and 50 controls. Genomic DNA was extracted from the blood samples, Polymerase Chain Reaction was performed (PCR) and Restriction Fragment Length Polymorphism (RFLP) was performed for digestion products that were evaluated. Results: The Results showed positive correlation between MSX1671 T>C gene variant and Non Syndromic tooth agenesis in Raichur Patients. Conclusion: MSX1 671 T>C gene variant may be a good screening marker for Non Syndromic tooth agenesis in Raichur Patients . How to cite this article:Reddy NA, Adusumilli G, Devanna R, Pichai S, Rohra MG, Arjunan S. Msx1 Gene Variant - Its Presence in Tooth Absence - A Case Control Genetic Study. J Int Oral Health 2013; 5(5):20-6. PMID:24324300

Correlation between metabolic enzyme GSTP1 polymorphisms and susceptibility to lung cancer

PubMed Central

WANG, YUFEI; REN, BU; ZHANG, LEI; GUO, ZHANLIN

2015-01-01

The aim of the present study was to determine the frequency distribution and characteristics of polymorphic alleles and genotypes in glutathione S-transferase π 1 (GSTP1) exon 5, and to explore the correlation between GSTP1 exon 5 polymorphisms and susceptibility to lung cancer using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Patients were diagnosed with lung cancer from May 2006 to October 2008 by postoperative pathological examination. A total of 150 patients, including 115 males and 35 females, aged 31–76 years (mean, 57.1 years) were enrolled. The control group consisted of 152 healthy volunteers who received physical examination at outpatient clinics. Genomic DNA was extracted from the peripheral venous blood of the 302 subjects, and the GSTP1 genotype was determined by PCR-RFLP and restricted enzyme digestion of PCR products. GSTP1 polymorphisms were analyzed in the 302 subjects. The C and G allele frequencies of GSTP1 in the control and lung cancer groups showed no significant difference (P=0.135); the frequencies of three different genotypes, A/A, A/G and G/G, of GSTP1 in the control and lung cancer groups exhibited no significant differences between the two groups (P=0.223). GSTP1 genotype frequencies in the study population fitted the Hardy-Weinberg equilibrium, demonstrating that the genotype results of this study conform to this genetic law. Overall, 50.7% of the subjects in the lung cancer group carried the non-A/A genotype of GSTP1, which was higher than the 43.4% of the control group. The risk of lung cancer in subjects with the non-A/A genotype was 1.43-fold higher than that in those with the A/A genotype, but no statistical significance was found (P=0.138). GSTP1 exon 5 polymorphisms were demonstrated to be associated with lung cancer susceptibility on the whole. However, stratified analysis suggested the correlation of GSTP1 exon 5 polymorphisms with lung squamous cell carcinoma risk, and that

The Influence of ?afi? on Mu?ammad Taqi Bahar

ERIC Educational Resources Information Center

Solati, Bahman

2016-01-01

Looking through the eyes of Persian culture, we see that man is not all noetic; he is not driven by intellect or consistent in his use of reason. On the contrary, he is most moved by emotion. A perfect example: Persian philosophy is most commonly uttered in both poetry and prose. This is, I believe, where the brilliance of Persians rests, in the…

Cytochrome oxidase subunit 2 gene allows simultaneous detection and typing of Trypanosoma rangeli and Trypanosoma cruzi.

PubMed

de Sá, Amanda Regina Nichi; Steindel, Mário; Demeu, Lara Maria Kalempa; Lückemeyer, Débora Denardin; Grisard, Edmundo Carlos; Neto, Quirino Alves de Lima; de Araújo, Silvana Marques; Toledo, Max Jean de Ornelas; Gomes, Mônica Lúcia

2013-12-23

The parasites Trypanosoma rangeli and Trypanosoma cruzi share vectors and hosts over a wide geographical area in Latin America. In this study, we propose a single molecular approach for simultaneous detection and typing of T. rangeli and T. cruzi. A restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase II gene (COII-RFLP) using enzyme AluI and different amounts of DNA from the major genetic groups of T. rangeli and T. cruzi (KP1+/KP1- and DTU-I/DTU-II) was carried out. The same marker was tested on the other T. cruzi DTUs (DTU-III to DTU-VI) and on DNA extracted from gut contents of experimentally infected triatomines. The COII PCR generates a ~400 bp fragment, which after digestion with AluI (COII-RFLP) can be used to distinguish T. rangeli from T. cruzi and simultaneously differentiate the major genetic groups of T. rangeli (KP1+ and KP1-) and T. cruzi (DTU-I and DTU-II). The COII-RFLP generated bands of ~120 bp and ~280 bp for KP1+, whereas for KP1- no amplicon cleavage was observed. For T. cruzi, digestion of COII revealed a ~300 bp band for DTU-I and a ~250 bp band for DTU-II. For DTU-III to DTU-VI, COII-RFLP generated bands ranging from ~310 to ~330 bp, but the differentiation of these DTUs was not as clear as the separation between DTU-I and DTU-II. After AluI digestion, a species-specific fragment of ~80 bp was observed for all DTUs of T. cruzi. No cross-amplification was observed for Leishmania spp., T. vivax or T. evansi. The COII-RFLP allowed simultaneous detection and typing of T. rangeli and T. cruzi strains according to their major genetic groups (KP1+/KP1- and DTU-I/DTU-II) in vitro and in vivo, providing a reliable and sensitive tool for epidemiological studies in areas where T. rangeli and T. cruzi coexist.

Cytochrome oxidase subunit 2 gene allows simultaneous detection and typing of Trypanosoma rangeli and Trypanosoma cruzi

PubMed Central

2013-01-01

Background The parasites Trypanosoma rangeli and Trypanosoma cruzi share vectors and hosts over a wide geographical area in Latin America. In this study, we propose a single molecular approach for simultaneous detection and typing of T. rangeli and T. cruzi. Methods A restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase II gene (COII-RFLP) using enzyme AluI and different amounts of DNA from the major genetic groups of T. rangeli and T. cruzi (KP1+/KP1- and DTU-I/DTU-II) was carried out. The same marker was tested on the other T. cruzi DTUs (DTU-III to DTU-VI) and on DNA extracted from gut contents of experimentally infected triatomines. Results The COII PCR generates a ~400 bp fragment, which after digestion with AluI (COII-RFLP) can be used to distinguish T. rangeli from T. cruzi and simultaneously differentiate the major genetic groups of T. rangeli (KP1+ and KP1-) and T. cruzi (DTU-I and DTU-II). The COII-RFLP generated bands of ~120 bp and ~280 bp for KP1+, whereas for KP1- no amplicon cleavage was observed. For T. cruzi, digestion of COII revealed a ~300 bp band for DTU-I and a ~250 bp band for DTU-II. For DTU-III to DTU-VI, COII-RFLP generated bands ranging from ~310 to ~330 bp, but the differentiation of these DTUs was not as clear as the separation between DTU-I and DTU-II. After AluI digestion, a species-specific fragment of ~80 bp was observed for all DTUs of T. cruzi. No cross-amplification was observed for Leishmania spp., T. vivax or T. evansi. Conclusions The COII-RFLP allowed simultaneous detection and typing of T. rangeli and T. cruzi strains according to their major genetic groups (KP1+/KP1- and DTU-I/DTU-II) in vitro and in vivo, providing a reliable and sensitive tool for epidemiological studies in areas where T. rangeli and T. cruzi coexist. PMID:24360167

Selected vitamin D metabolic gene variants and risk for autism spectrum disorder in the CHARGE Study.

PubMed

Schmidt, Rebecca J; Hansen, Robin L; Hartiala, Jaana; Allayee, Hooman; Sconberg, Jaime L; Schmidt, Linda C; Volk, Heather E; Tassone, Flora

2015-08-01

Vitamin D is essential for proper neurodevelopment and cognitive and behavioral function. We examined associations between autism spectrum disorder (ASD) and common, functional polymorphisms in vitamin D pathways. Children aged 24-60 months enrolled from 2003 to 2009 in the population-based CHARGE case-control study were evaluated clinically and confirmed to have ASD (n=474) or typical development (TD, n=281). Maternal, paternal, and child DNA samples for 384 (81%) families of children with ASD and 234 (83%) families of TD children were genotyped for: TaqI, BsmI, FokI, and Cdx2 in the vitamin D receptor (VDR) gene, and CYP27B1 rs4646536, GC rs4588, and CYP2R1 rs10741657. Case-control logistic regression, family-based log-linear, and hybrid log-linear analyses were conducted to produce risk estimates and 95% confidence intervals (CI) for each allelic variant. Paternal VDR TaqI homozygous variant genotype was significantly associated with ASD in case-control analysis (odds ratio [OR] [CI]: 6.3 [1.9-20.7]) and there was a trend towards increased risk associated with VDR BsmI (OR [CI]: 4.7 [1.6-13.4]). Log-linear triad analyses detected parental imprinting, with greater effects of paternally-derived VDR alleles. Child GC AA-genotype/A-allele was associated with ASD in log-linear and ETDT analyses. A significant association between decreased ASD risk and child CYP2R1 AA-genotype was found in hybrid log-linear analysis. There were limitations of low statistical power for less common alleles due to missing paternal genotypes. This study provides preliminary evidence that paternal and child vitamin D metabolism could play a role in the etiology of ASD; further research in larger study populations is warranted. Copyright © 2015. Published by Elsevier Ireland Ltd.

Paraoxonase 1 192 and 55 polymorphisms in osteosarcoma.

PubMed

Ergen, Arzu; Kılıcoglu, Onder; Ozger, Harzem; Agachan, Bedia; Isbir, Turgay

2011-08-01

Paraoxonase is an HDL-associated enzyme that plays a preventive role against oxidative stres. Previous studies suggested that involved an amino acid substitution at position 192 gives rise to two alloenzymes with a low activity (Q allele) and a high activity (R allele) towards paraoxon. There also exists a second polymorphism of the human PON1 gene affecting amino acid 55, giving rise to a leucine (L-allele) substitution for methionine (M-allele). PON1 gene polymorphisms were studied in 50 patients with osteosarcoma and 50 healthy controls. Paraoxonase genotypes were determined by PCR-RFLP. We found a reduction in the frequency of PON1 192 R allele in patients (P=0.015). Besides, PON1 192 wild type QQ genotype (P=0.015) and PON1 55 wild type L allele (P=0.001) were higher in patients compared to healthy controls. PON1 192 QQ genotype was associated with osteosarcoma in multivariate logistic regression analysis. Our findings have suggested that PON1 192 wild type genotypes may be associated with a risk of developing osteosarcoma.

Association of polymorphic variants of PTPN22, TNF and VDR genes in children with lupus nephritis: A study in Colombian family triads.

PubMed

Garavito, Gloria; Egea, Eduardo; Fang, Luis; Malagón, Clara; Olmos, Carlos; González, Luz; Guarnizo, Pilar; Aroca, Gustavo; López, Guillermo; Iglesias, Antonio

2017-06-01

Systemic lupus erythematosus is an autoimmune disease in which the severity varies according to race, sex and age of onset. This variation is also observed in the genetic markers associated with the disease, including PTPN22, VDR and TNF genes. The genetic stratification in different populations worldwide can influence the variability. To analyze the heritability of PTPN22, VDR and TNF genetic variants and their association with pediatric lupus nephritis in Colombian families. We conducted a family-based study including 46 triads (case, father and mother). The variants rs2476601 of PTPN22; rs361525 and rs1800629 of TNF, and TaqI [rs731236], ApaI [rs7975232], BsmI [rs1544410] and FokI [rs2228570] of VDR were genotyped by qPCR. The effects of overtransmission of the risk allele from parents to children and linkage disequilibrium at the VDR and TNF loci were estimated. We found that allele A of rs2476601 in PTPN22 was distributed among 8.69 % (n=16) of the parents and 19.5 % (n=18) of the cases; this allele was overtransmitted from parents to children 17 times more often than the G allele (p=0.028). TNF and VDR polymorphisms did not exhibit transmission disequilibrium. VDR TaqI, ApaI and BsmI variants exhibited linkage disequilibrium. These findings showed an association between the PTPN22 rs2476601 polymorphism and pediatric lupus nephritis due to its overtransmission in the group of families studied.

Association study between XRCC1 gene polymorphisms and sporadic amyotrophic lateral sclerosis.

PubMed

Coppedè, Fabio; Migheli, Francesca; Lo Gerfo, Annalisa; Fabbrizi, Maria Rita; Carlesi, Cecilia; Mancuso, Michelangelo; Corti, Stefania; Mezzina, Nicoletta; del Bo, Roberto; Comi, Giacomo P; Siciliano, Gabriele; Migliore, Lucia

2010-01-01

The aim of the present study was to investigate the possible contribution of three common functional polymorphisms in the DNA repair protein X-ray repair cross-complementing group 1 (XRCC1), namely Arg194Trp (rs1799782), Arg280His (rs25489) and Arg399Gln (rs25487), to sporadic amyotrophic lateral sclerosis (SALS). We genotyped 206 Italian SALS patients and 203 matched controls for XRCC1 Arg194Trp, Arg280His and Arg399Gln polymorphisms by means of PCR/RFLP technique, searching for association between any of the studied polymorphisms and disease risk, age and site of onset. We observed a statistically significant difference in XRCC1 Gln399 allele frequencies between SALS cases and controls (0.39/0.28; p=0.001). The present study suggests that the XRCC1 Arg399Gln polymorphism might contribute to SALS risk.

DNA Carrier Testing and Newborn Screening for Maple Syrup Urine Disease in Old Order Mennonite Communities

PubMed Central

Carleton, Stephanie M.; Peck, Dawn S.; Grasela, Julie; Dietiker, Kristin L.

2010-01-01

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by mutations in the branched chain α-keto acid dehydrogenase complex. Worldwide incidence of MSUD is 1:225,000 live births. However, within Old Order Mennonite communities, the incidence is 1:150 live births and results from a common tyrosine to asparagine substitution (Y438N) in the E1α subunit of branched chain α-keto acid dehydrogenase. We developed a new DNA diagnostic assay utilizing TaqMan® technology and compared its efficacy, sensitivity, and duration with an existing polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. Carrier testing was performed by both TaqMan technology and PCR-RFLP on DNA isolated from buccal swabs of 160 individuals as well as from buccal swabs and blood spots of nine at-risk newborns; assay time, sensitivity, and reliability were also evaluated. The TaqMan assay, like the PCR-RFLP assay, accurately determined Y438N E1α allele status. However, the TaqMan assay appeared (1) more sensitive than the PCR-RFLP assay, requiring 10-fold less DNA (10 ng) to reliably determine genotype status and (2) faster, reducing the assay time required for diagnosis from ∼12 to 5 h. TaqMan technology allowed more rapid DNA diagnoses of MSUD in the neonate, thereby reducing the likelihood of neurological impairment while enhancing health and prognosis for affected infants. PMID:20136525

Impact of vitamin D receptor and binding protein gene polymorphisms in clinical and laboratory data of HCV patients

PubMed Central

Scalioni, Letícia de Paula; dos Santos, Betânia Rodrigues; Spritzer, Poli Mara; Villela-Nogueira, Cristiane Alves; Laura Lewis-Ximenez, Lia; Pollo-Flores, Priscila; Bordalo Cathalá Esberard, Eliane; Brandão-Mello, Carlos Eduardo; Lampe, Elisabeth; Villar, Livia Melo

2018-01-01

Abstract Potential relationship of vitamin D, vitamin D receptor (VDR), and vitamin D binding protein (DBP) have been suggested in the pathophysiology of hepatitis C virus (HCV) infection. The aim of this observational study is to determine vitamin D levels, and VDR and DBP genetic polymorphism according demographic and laboratory data in chronic HCV patients (CHC). A total of 148 CHC patients gave serum samples for testing 25-hydroxyvitamin D (25 (OH)D) level by immunochemiluminometric assay (<20 ng/mL defined as deficient) and donated blood samples to allelic discrimination analysis using TaqMan assays. Analyzed single nucleotide polymorphisms (SNPs) were: VDR-rs7975232 (ApaI) C>A, rs731236 A>G (TaqI), rs1544410 C>T (BsmI), rs10735810 T>C (FokI) and carrier globulin/binding protein (GC)-rs4588 and rs7041 and the haplotype bAt [CCA]. Hepatic fibrosis was assessed using Fib-4 and Forns index. Eighty-two (54.40%) patients demonstrated deficiency of vitamin D and this was associated to AST (P = .019 [CI: 1.003–1.034]), total cholesterol (P = .038 [CI: 1.004–1.164]), fibrosis grade (P < .001 [CI: 0.000–0.844]), and FokI (P = .028) allele T presence. Association was found between VDR polymorphism and fibrosis (BsmI andTaqI), triglycerides (TaqI), and HDL (FokI). DBP polymorphism was associated to HCV genotype (GC rs7041), previous HCV treatment, and GGT (GC rs4588). In conclusion, low frequency of vitamin D deficiency was found, but VDR polymorphisms were frequently associated to fibrosis grade suggesting that they could be used as disease evaluation markers to understand the mechanisms underlying the virus–host interaction. PMID:29465575

Molecular mapping and genetic analysis of a rice brown planthopper (Nilaparvata lugens Stål) resistance gene.

PubMed

Yang, Haiyuan; Ren, Xiang; Weng, Qingmei; Zhu, Lili; He, Guangcun

2002-01-01

The brown planthopper (BPH), Nilaparvata lugens Stål, is a serious insect pest of rice (Oryza saliva L.). We have determined the chromosomal location of a BPH resistance gene in rice using SSR and RFLP techniques. A rice line 'B14', derived from the wild rice Oryza latifolia, showed high resistance to BPH. For tagging the resistance gene in 'B14X', an F2 population and a recombinant inbred (RI) population from a cross between Taichung Native 1 and 'B14' were developed and evaluated for BPH resistance. The results showed that a single dominant gene controlled the resistance of 'B14' to BPH. Bulked segregant SSR analysis was employed for identification of DNA markers linked to the resistance gene. From the survey of 302 SSR primer pairs, three SSR (RM335, RM261, RM185) markers linked to the resistance gene were identified. The closest SSR marker RM261 was linked to the resistance gene at a distance of 1.8 cM. Regions surrounding the resistance gene and the SSR markers were examined with additional RFLP markers on chromosome 4 to define the location of the resistance gene. Linkage of RFLP markers C820, R288, C946 with the resistance gene further confirmed its location on the short arm of chromosome 4. Closely linked DNA markers will facilitate selection for resistant lines in breeding programs and provide the basis for map-based cloning of this resistance gene.

Assessment of Tools for Marker-Assisted Selection in a Marine Commercial Species: Significant Association between MSTN-1 Gene Polymorphism and Growth Traits

PubMed Central

Sánchez-Ramos, Irma; Cross, Ismael; Mácha, Jaroslav; Martínez-Rodríguez, Gonzalo; Krylov, Vladimir; Rebordinos, Laureana

2012-01-01

Growth is a priority trait from the point of view of genetic improvement. Molecular markers linked to quantitative trait loci (QTL) have been regarded as useful for marker-assisted selection in complex traits as growth. Polymorphisms have been studied in five candidate genes influencing growth in gilthead seabream (Sparus aurata): the growth hormone (GH), insulin-like growth factor-1 (IGF-1), myostatin (MSTN-1), prolactin (PRL), and somatolactin (SL) genes. Specimens evaluated were from a commercial broodstock comprising 131 breeders (from which 36 males and 44 females contributed to the progeny). In all samples eleven gene fragments, covering more than 13,000 bp, generated by PCR-RFLP, were analyzed; tests were made for significant associations between these markers and growth traits. ANOVA results showed a significant association between MSTN-1 gene polymorphism and growth traits. Pairwise tests revealed several RFLPs in the MSTN-1 gene with significant heterogeneity of genotypes among size groups. PRL and MSTN-1 genes presented linkage disequilibrium. The MSTN-1 gene was mapped in the centromeric region of a medium-size acrocentric chromosome pair. PMID:22666112

Tetrachloromethane-Degrading Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer.

PubMed

Penny, Christian; Gruffaz, Christelle; Nadalig, Thierry; Cauchie, Henry-Michel; Vuilleumier, Stéphane; Bringel, Françoise

2015-07-02

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl₄) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L(-1) CCl₄, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl₄ degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl₄ transformation. As estimated by T-RFLP, phylotypes of CCl₄-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community.

Tetrachloromethane-Degrading Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer

PubMed Central

Penny, Christian; Gruffaz, Christelle; Nadalig, Thierry; Cauchie, Henry-Michel; Vuilleumier, Stéphane; Bringel, Françoise

2015-01-01

Abstract: The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L−1 CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community. PMID:27682092

[Association between polymorphism in DVWA and IL-1beta and Kashin-Beck disease].

PubMed

Y U, Min; Guo, Xiong; Gao, Xiao-Yun; Lai, Jiang-Hua; Tu, Qian-Qian

2010-07-01

To investigate the association between IL-1beta and DVWA gene and Kashin-Beck disease (KBD). Peripheral genomic DNA were extracted from 105 patients with KBD and 98 healthy controls. PCR-RFLP were performed to detect SNP loci of IL-1beta gene and DVWA gene. The patients with KBD had significantly higher frequency of rs16944 (IL-1beta) locus (chi2 = 24.28, P < 0.001) and single allele frequency of rs16944 (chi2 = 5.683, P = 0.0171) than the healthy controls. There were no significant differences in genotype frequencies,single allele frequencies and haplotypes in rs4685241 and rs1143627 between the patients with KBD and the healthy controls. rs16944 (IL-1beta) is associated with KBD.

Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media.

PubMed Central

Taylor, T B; Patterson, C; Hale, Y; Safranek, W W

1997-01-01

A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species. These isolates were characterized by their morphologic and biochemical characteristics. Two of the isolates were identified as M. terrae complex and M. gordonae. The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp. most closely resembling M. chelonae. PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP. Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate. Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm. PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics. PMID:8968884

[Correlation analysis of G870A CCND1 gene polymorphism with digestive system tumors].

PubMed

Yang, Shu-Min; Shi, Ya-Lin

2016-11-20

To study the correlation of G870A CCND1 gene polymorphism and digestive system tumors. From August 2010 to August 2014, 164 digestive system cancer patients (including 82 patients with gastric cancer and 82 with colorectal cancer) and 82 healthy subjects (control group) were examined with PCR-restriction fragment length polymorphism (PCR-RFLP). The distribution of CCND1 gene G870A frequency in the 3 groups and its association with tumor staging and grading were analyzed. The frequencies of the GG, GA and AA genotypes in G870A CCND1 gene loci in patients with gastric cancer and colorectal cancer differed significantly from those in the control group (P<0.05). G870A CCND1 gene polymorphism was closely associated with an increased risk of digestive system tumors (P<0.05). The GA and AA genotypes were associated with a significantly higher risk of digestive system cancer risk than the GG genotype (P<0.05), and their frequencies were significantly higher in patients with tumors of higher pathological grade and in those in advanced tumor stages (P<0.05). G870A CCND1 gene polymorphism is associated with the risk of digestive system tumors. The allele A is associated with an increased risk of digestive system tumors and correlated with the tumor differentiation and staging of the tumor.

SLC11A1 polymorphisms and host susceptibility to cutaneous leishmaniasis in Pakistan.

PubMed

Sophie, Mariam; Hameed, Abdul; Muneer, Akhtar; Samdani, Azam J; Saleem, Saima; Azhar, Abid

2017-01-07

The vector-borne cutaneous leishmaniasis (CL) is endemic in several regions of Pakistan mainly affecting poor populations. Host genetic factors, particularly SLC11A1 (solute carrier transmembrane protein) within macrophages, play a crucial role in disease pathology and susceptibility. Association of SLC11A1 with cutaneous leishmaniasis, a neglected tropical disease, is not well established. Inconsistencies have been observed within different populations worldwide with respect to genetic susceptibility. This study was designed to investigate genetic variation(s) in SLC11A1 and to assess possible association with cutaneous leishmaniasis in Pakistan. Eight polymorphisms (rs2276631, rs3731864, rs2290708, rs2695342, rs201565523, rs17215556, rs17235409, rs17235416) were genotyped across SLC11A1 in 274 patients and 119 healthy controls. Six polymorphisms were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing. Two single nucleotide polymorphisms were analyzed with newly designed semi-nested PCR assays. Case-control analysis showed no association between selected polymorphisms in SLC11A1 and cutaneous leishmaniasis. No significant difference was observed in the distribution of alleles between leishmaniasis patients and healthy individuals. Strong pairwise linkage disequilibrium was observed between rs2276631 and rs2290708 (r 2  = 64); and rs17235409 and rs17235416 (r 2  = 78). This study shows that genetic variations in the candidate gene SLC11A1 do not affect susceptibility to cutaneous leishmaniasis in the sample population from Pakistan.

Mapping of the serotonin 5-HT{sub 1D{beta}} autoreceptor gene on chromosome 6 and direct analysis for sequence variants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lappalainen, J.; Dean, M.; Virkkunen, M.

1995-04-24

Abnormal brain serotonin function may be characteristic of several neuropsychiatric disorders. Thus, it is important to identify polymorphic genes and screen for functional variants at loci coding for genes that control normal serotonin functions. 5-HT{sub 1D{beta}} is a terminal serotonin autoreceptor which may play a role in regulating serotonin synthesis and release. Using an SSCP technique we screened for 5-HT{sub 1D{beta}} coding sequence variants in psychiatrically interviewed populations, which included controls, alcoholics, and alcoholic arsonists and alcoholic violent offenders with low CSF concentrations of the main serotonin metabolite 5-HIAA. A common polymorphism was identified in the 5-HT{sub 1D{beta}} gene withmore » allele frequencies of 0.72 and 0.28. The SSCP variant was caused by a silent G to C substitution at nucleotide 861 of the coding region. This polymorphism could also be detected as a HincII RFLP of amplified DNA. DNAs from informative CEPH families were typed for the HincII RFLP and analyzed with respect to 20 linked markers on chromosome 6. Multipoint analysis placed the 5-HT{sub 1D{beta}} receptor gene between markers D6S286 and D6S275. A maximum two-point lod score of 10.90 was obtained to D6S26, which had been previously localized on 6q14-15. Chromosomal aberrations involving this region have been previously shown to cause retinal anomalies, developmental delay, and abnormal brain development. This region also contains the gene for North Carolina-type macular dystrophy. 34 refs., 3 figs., 1 tab.« less

Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina.

PubMed

Consolo, Verónica F; Ortega, Leonel M; Salerno, Graciela; Astoreca, Andrea L; Alconada, Teresa M

2015-01-01

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Group 16SrXI phytoplasma strains, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D, are associated with sugar cane white leaf.

PubMed

Zhang, Rong-Yue; Li, Wen-Feng; Huang, Ying-Kun; Wang, Xiao-Yan; Shan, Hong-Li; Luo, Zhi-Ming; Yin, Jiong

2016-01-01

Sugar cane white leaf (SCWL) is a serious disease caused by phytoplasmas. In this study, we performed nested PCR with phytoplasma universal primer pairs (P1/P7 and R16F2n/R16R2) for the 16S rRNA gene to detect SCWL phytoplasmas in 31 SCWL samples collected from Baoshan and Lincang, Yunnan, China. We cloned and sequenced the nested PCR products, revealing that the 16S rRNA gene sequences from 31 SCWL samples were all 1247 bp in length and shared more than 99 % nucleotide sequence similarity with the 16S rRNA gene sequences of SCWL phytoplasmas from various countries. Based on the reported 16S rRNA gene sequence data from SCWL isolates of various countries, we conducted phylogenetic and virtual RFLP analysis. In the resulting phylogenetic tree, all SCWL isolates clustered into two branches, with the Lincang and Baoshan SCWL phytoplasma isolates belonging to different branches. The virtual RFLP patterns show that phytoplasmas of the Lincang branch belong to subgroup 16SrXI-B. However, the virtual RFLP patterns revealed by HaeIII digestion of phytoplasmas of the Baoshan branch differed from those of subgroup 16SrXI-B. According to the results of phylogenetic and virtual RFLP analysis, we propose that the phytoplasmas of the Baoshan branch represent a new subgroup, 16SrXI-D. These findings suggest that SCWL is caused by phytoplasmas from group 16SrXI, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D.

PCR and restriction fragment length polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato diseases.

PubMed Central

Darrasse, A; Priou, S; Kotoujansky, A; Bertheau, Y

1994-01-01

Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a 434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the whole set of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a genetic diversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed. Images PMID:7912502

Lack of an association of programmed cell death-1 PD1.3 polymorphism with risk of hepatocellular carcinoma susceptibility in Turkish population: a case-control study.

PubMed

Bayram, Süleyman; Akkız, Hikmet; Ülger, Yakup; Bekar, Aynur; Akgöllü, Ersin; Yıldırım, Selçuk

2012-12-15

The programmed cell death-1 (PD-1) is a potent immunoregulatory molecule which is responsible for the negative regulation of T-cell activation and peripheral tolerance. Recently, overexpression of PD-1 has been reported to contribute to immune system evasion and poor survival of hepatocellular carcinoma (HCC). A common single nucleotide polymorphism in intron 4 of PD-1 gene called PD-1.3 has been reported to influence PD-1 expression, but its association with HCC has yet to be investigated. The aim of the present study was to investigate whether this polymorphism could be involved in the risk of HCC susceptibility. The genotype frequency of PD-1.3 polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 236 subjects with HCC and 236 cancer-free control subjects matched on age, gender, smoking and alcohol status. No statistically significant differences were found in the genotype distributions of the PD-1.3 polymorphism among HCC and cancer-free control subjects (P=0.22). Our results demonstrate for the first time that the PD-1.3 polymorphism has not been in any major role in genetic susceptibility to hepatocellular carcinogenesis, at least in the population studied here. Independent studies are needed to validate our findings in a larger series, as well as in patients of different ethnic origins. Copyright © 2012 Elsevier B.V. All rights reserved.

SSU rDNA sequence diversity and seasonally differentiated distribution of nanoplanktonic ciliates in neritic Bohai and Yellow Seas as revealed by T-RFLP.

PubMed

Dong, Jun; Shi, Fei; Li, Han; Zhang, Xiaoming; Hu, Xiaozhong; Gong, Jun

2014-01-01

Nanociliates have been frequently found to be important players in the marine microbial loop, however, little is known about their diversity and distribution in coastal ecosystems. We investigated the molecular diversity and distribution patterns of nanoplanktonic oligotrich and choreotrich (OC) ciliates in surface water of three neritic basins of northern China, the South Yellow Sea (SYS), North Yellow Sea (NYS), and Bohai Sea (BS) in June and November 2011. SSU rRNA gene clone libraries generated from three summertime samples (sites B38, B4 and H8) were analyzed and revealed a large novel ribotype diversity, of which many were low-abundant phylotypes belonging to the subclass Oligotrichia, but divergent from described morphospecies. Based on the data of terminal-restriction fragment length polymorphism (T-RFLP) analysis of all 35 samples, we found that the T-RF richness was generally higher in the SYS than in the BS, and negatively correlated with the molar ratio of P to Si. Overall, multidimensional scaling and permutational multivariate analysis of variance of the community turnover demonstrated a distinct seasonal pattern but no basin-to-basin differentiation across all samples. Nevertheless, significant community differences among basins were recognized in the winter dataset. Mantel tests showed that the environmental factors, P:Si ratio, water temperature and concentration of dissolved oxygen (DO), determined the community across all samples. However, both biogeographic distance and environment shaped the community in winter, with DO being the most important physicochemical factor. Our results indicate that the stoichiometric ratio of P:Si is a key factor, through which the phytoplankton community may be shaped, resulting in a cascade effect on the diversity and community composition of OC nanociliates in the N-rich, Si-limited coastal surface waters, and that the Yellow Sea Warm Current drives the nanociliate community, and possibly the microbial food webs

Genetic diversity of Mycobacterium tuberculosis from Guadalajara, Mexico and identification of a rare multidrug resistant Beijing genotype.

PubMed

Flores-Treviño, Samantha; Morfín-Otero, Rayo; Rodríguez-Noriega, Eduardo; González-Díaz, Esteban; Pérez-Gómez, Héctor R; Bocanegra-García, Virgilio; Vera-Cabrera, Lucio; Garza-González, Elvira

2015-01-01

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.

Genetic Diversity of Mycobacterium tuberculosis from Guadalajara, Mexico and Identification of a Rare Multidrug Resistant Beijing Genotype

PubMed Central

Flores-Treviño, Samantha; Morfín-Otero, Rayo; Rodríguez-Noriega, Eduardo; González-Díaz, Esteban; Pérez-Gómez, Héctor R.; Bocanegra-García, Virgilio; Vera-Cabrera, Lucio; Garza-González, Elvira

2015-01-01

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America. PMID:25695431

Typing and Clustering of Yersinia pseudotuberculosis Isolates by Restriction Fragment Length Polymorphism Analysis Using Insertion Sequences

PubMed Central

Voskresenskaya, E.; Savin, C.; Leclercq, A.; Tseneva, G.

2014-01-01

Yersinia pseudotuberculosis is an enteropathogen that has an animal reservoir and causes human infections, mostly in temperate and cold countries. Most of the methods previously used to subdivide Y. pseudotuberculosis were performed on small numbers of isolates from a specific geographical area. One aim of this study was to evaluate the typing efficiency of restriction fragment length polymorphism of insertion sequence hybridization patterns (IS-RFLP) compared to other typing methods, such as serotyping, ribotyping, and multilocus sequence typing (MLST), on the same set of 80 strains of Y. pseudotuberculosis of global origin. We found that IS100 was not adequate for IS-RFLP but that both IS285 and IS1541 efficiently subtyped Y. pseudotuberculosis. The discriminatory index (DI) of IS1541-RFLP (0.980) was superior to those of IS285-RFLP (0.939), ribotyping (0.944), MLST (0.861), and serotyping (0.857). The combination of the two IS (2IS-RFLP) further increased the DI to 0.998. Thus, IS-RFLP is a powerful tool for the molecular typing of Y. pseudotuberculosis and has the advantage of exhibiting well-resolved banding patterns that allow for a reliable comparison of strains of worldwide origin. The other aim of this study was to assess the clustering power of IS-RFLP. We found that 2IS-RFLP had a remarkable capacity to group strains with similar genotypic and phenotypic markers, thus identifying robust populations within Y. pseudotuberculosis. Our study thus demonstrates that 2IS- and even IS1541-RFLP alone might be valuable tools for the molecular typing of global isolates of Y. pseudotuberculosis and for the analysis of the population structure of this species. PMID:24671793

Comparison of CHROMagar, polymerase chain reaction-restriction fragment length polymorphism, and polymerase chain reaction-fragment size for the identification of Candida species.

PubMed

Jafari, Zahra; Motamedi, Marjan; Jalalizand, Nilufar; Shokoohi, Gholam R; Charsizadeh, Arezu; Mirhendi, Hossein

2017-09-01

The epidemiological alteration in the distribution of Candida species, as well as the significantly increasing trend of either intrinsic or acquired resistance of some of these fungi highlights the need for a reliable method for the identification of the species. Polymerase chain reaction (PCR) is one of the methods facilitating the quick and precise identification of Candida species. The aim of this study was to compare the efficiency of CHROMagar, PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-fragment size polymorphism (PCR-FSP) assays in the identification of Candida species to determine the benefits and limitations of these methods. This study was conducted on 107 Candida strains, including 20 standard strains and 87 clinical isolates. The identification of the isolates was accomplished by using CHROMagar as a conventional method. The PCR-RFLP assay was performed on the entire internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), and the consequent enzymatic digestion was compared with PCR-FSP results in which ITS1 and ITS2 regions were separately PCR amplified. In both molecular assays, yeast identification was carried out through the specific electrophoretic profiles of the PCR products. According to the results, the utilization of CHROMagar resulted in the identification of 29 (33.3%) Candida isolates, while the PCR-RFLP and PCR-FSP facilitated the identification of 83 (95.4%) and 80 (91.9%) clinical isolates, respectively. The obtained concordances between CHROMagar and PCR-RFLP, between CHROMagar and PCR-FSP, as well as between PCR-RFLP and PCR-FSP were 0.23, 0.20, and 0.77, respectively. The recognition of the benefits and limitations of PCR methods allows for the selection of the most efficient technique for a fast and correct differentiation. The PCR-RFLP and PCR-FSP assays had satisfactory concordance. The PCR-FSP provides a rapid, technically simple, and cost-effective method for the identification of Candida species

Molecular identification of Fasciola spp. (Digenea: Fasciolidae) in Egypt

PubMed Central

Dar, Y.; Amer, S.; Mercier, A.; Courtioux, B.; Dreyfuss, G.

2012-01-01

A total of 134 Egyptian liver flukes were collected from different definitive hosts (cattle, sheep, and buffaloes) to identify them via the use of PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS1). Specimens of F. hepatica from France, as well as F. gigantica from Cameroon were included in the study for comparison. PCR products of ITS1 were subjected for digestion by RsaI restriction enzyme and visualized on agarose gel. According to RFLP pattern, Egyptian flukes were allocated into two categories. The first was identical to that of French hepatica flukes to have a pattern of 360, 100, and 60 (bp) band size, whereas the second resembled to that of Cameroonian gigantica worms to have a profile of 360, 170, and 60 bp in size. Results of RFLP analysis were confirmed by sequence analysis of representative ITS1 amplicons. No hybrid forms were detected in the present study. Taken together, this study concluded that both species of Fasciola are present in Egypt, whereas the hybrid form may be not very common. PMID:22550630

Vitamin D receptor gene polymorphisms and the risk of rickets among Asians: a meta-analysis.

PubMed

Mao, Song; Huang, Songming

2014-03-01

To evaluate the association between vitamin D receptor (VDR) gene polymorphisms and the risk of rickets among Asians. Eligible studies were included in our meta-analysis by searching PubMed, Embase, Cochrane and China National Knowledge Infrastructure databases according to a predefined criteria. A random effects model was used to calculate the combined ORs and its corresponding 95% CI. 16 studies were recruited for the analysis of the association between VDR BsmI (rs1544410), TaqI (rs731236), FokI (rs2228570) and ApaI (rs7975232) gene polymorphisms and the risk of rickets among Asians, most of whom were from China. B allele/BB genotype was associated with the susceptibility of rickets (p=0.017 and 0.044, respectively), and bb genotype was associated with lower risk of rickets (p=0.033). F allele/FF genotype was associated with the susceptibility of rickets (p<10(-4)), and ff genotype was associated with lower risk of rickets (p<10(-4)). AA genotype was associated with the onset of rickets (p=0.044). No significant association was observed between TaqI polymorphism the risk of rickets. A allele/aa genotype was not associated with the risk of rickets. No evidence of publication bias was observed. B allele/BB genotype at the BsmI site, F allele/FF genotype at the FokI site and AA genotype at the ApaI site may be risk factors for the onset of rickets among Asians; bb genotype at the BsmI site and ff genotype at the FokI site may be protective factors against the risk of rickets among Asians.

Association of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms with clinical response to imatinib mesylate treatment among Malaysian chronic myeloid leukaemia patients.

PubMed

Makhtar, Siti Maziras; Husin, Azlan; Baba, Abdul Aziz; Ankathil, Ravindran

2017-09-01

The detoxifying activity of glutathione S-transferases (GST) enzymes not only protect cells from the adverse effects of xenobiotics, but also alters the effectiveness of drugs in cancer cells, resulting in toxicity or drug resistance. In this study, we aimed to evaluate the association of GSTM1, GSTT1 and GSTP1 Ile105Val polymorphisms with treatment response among Malaysian chronic myeloid leukaemia (CML) patients who everyday undergo 400 mg of imatinib mesylate (IM) therapy. Multiplex polymerase chain reaction (multiplex-PCR) was performed to detect GSTM1 and GSTT1 polymorphisms simultaneously and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was conducted to detect the GSTP1 Ile195Val polymorphism. On evaluating the association of the variant genotype with treatment outcome, heterozygous variant (AG) and homozygous variant (GG) of GSTP1 Ile105Val showed significantly a higher risk for the development of resistance to IM with OR: 1.951 (95% CI: 1.186-3.209, P = 0.009) and OR: 3.540 (95% CI: 1.305-9.606, P = 0.013), respectively. Likewise, GSTT1 null genotype was also associated with a significantly higher risk for the development of resistance to IM with OR = 1.664 (95% CI: 1.011-2.739, P = 0.045). Our results indicate the potential usefulness of GST polymorphism genotyping in predicting the IM treatment response among CML patients.

Microbial Community Shifts Associated with RDX Loss in a Saturated and Well-Drained Surface Soil

DTIC Science & Technology

2005-03-01

community containing firmicutes (36%), proteobacteria (54%), actinobacteria (8%), and bacteroidetes (1%). The unsaturated soil contained a greater number of...genera (2.5 times that of the saturated soil) within similar phyla (19% firmicutes, 66% proteobacteria, 6% actinobacteria , 2% bacteroidetes, and 7...by the PLFA analysis. The T-RFLP analysis identified firmicutes (36%), proteobacteria (54%), actinobacteria (8%), and bacteroidetes (1%) in the

[Polymorphism of POU1F1 gene and PRL gene and their combined effects on milk performance traits in Chinese Holstein cattle].

PubMed

Jia, Xiang-Jie; Wang, Chang-Fa; Yang, Gui-Wen; Huang, Jin-Ming; Li, Qiu-Ling; Zhong, Ji-Feng

2011-12-01

Three novel SNPs were found by DNA sequencing, PCR-RFLP and CRS-PCR methods were used for genotyping in 979 Chinese Holstein cattle. One SNP, G1178C, was identified in exon 2 of POU1F1 gene. Two novel SNPs, A906G and A1134G, were identified in 5'-flanking regulatory region (5'-UTR) of PRL gene. The association between polymorphisms of the two genes and milk performance traits were analyzed with PROC GLM of SAS. The results showed that GC genotype at 1178 locus of POU1F1 gene was advantageous for milk yield, milk protein yield, and milk fat yield. AG genotype at 906 locus was advantageous for milk yield. There was no significant difference between 1134 locus and milk performance traits of 5'-UTR of PRL gene. Analysis of genotype combination effect on milk production traits showed that the effect of combined genotype was not simple sum of single genotypes and the effects of gene pyramiding seemed to be more important in molecular breeding.

Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides.

PubMed

Singh, Satyender; Kumar, Vivek; Vashisht, Kapil; Singh, Priyanka; Banerjee, Basu Dev; Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen; Jain, Sudhir Kumar; Rai, Arvind

2011-11-15

Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p<0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37±2.15 vs. 6.24±1.37 tail% DNA, p<0.001). Further, the workers with CYP2D6*3PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p<0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. Copyright © 2011 Elsevier Inc. All rights reserved.

Genetic polymorphisms in Plasmodium falciparum chloroquine resistance genes, pfcrt and pfmdr1, in North Sulawesi, Indonesia.

PubMed

Reteng, Patrick; Vrisca, Visia; Sukarno, Inka; Djarkoni, Ilham Habib; Kalangi, Jane Angela; Jacobs, George Eduardo; Runtuwene, Lucky Ronald; Eshita, Yuki; Maeda, Ryuichiro; Suzuki, Yutaka; Mongan, Arthur Elia; Warouw, Sarah Maria; Yamagishi, Junya; Tuda, Josef

2017-04-04

Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.

Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis.

PubMed

Hsieh, Ming-Kun; Shih, Pei-Yin; Wei, Chia-Fong; Vickroy, Thomas W; Chou, Chi-Chung

2016-03-30

The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols. © 2015 Society of Chemical Industry.

Specific personality traits and dopamine, serotonin genes in anxiety-depressive alcoholism among Han Chinese in Taiwan.

PubMed

Lin, Shu-Cheng; Wu, Pei-Lin; Ko, Huei-Chen; Wu, Jo Yung-Wei; Huang, San-Yuan; Lin, Wei-Wen; Lu, Ru-Band

2007-10-01

Cloninger [Cloninger CR. 1987. Neurogenetic adaptive mechanisms in alcoholism. Science 236: 410-416.] had proposed a psychobiological model suggesting that three main personality dimensions distinguish the alcoholism into two subtypes (type I and type II). However, the classification was equivocal for clinical diagnosis. Recently, anxiety-depressive alcohol dependence (ANX/DEP ALC) has been posited as a genetically specific subtype of alcoholism. Its clinical characteristics were similar to individuals with type I alcoholism [Cloninger, C.R. 1987. Neurogenetic adaptive mechanisms in alcoholism. Science 236: 410-6.] such as having a high comorbidity with mood disorder, late-onset and more anxious/depressed traits. We attempted to investigate whether the dopamine D2 receptor (DRD2) and the serotonin transporter promoter region (5-HTTLPR) genes were involved in Novelty Seeking (NS) and Harm Avoidance (HA) of ANX/DEP ALC. We recruited 46 pure alcohol dependents (Pure ALC) and 87 anxiety-depression alcohol dependents (ANX/DEP ALC). All participants were diagnosed by DSM-IV criteria, genotyped by the PCR method and assessed with Tridimensional Personality Questionnaire (TPQ). Both NS and HA were high in ANX/DEP ALC (p = 0.021; p = 0.001, respectively). The association between NS and ANX/DEP ALC only existed in subjects with DRD2 TaqI A1(+) allele (A1/A1 or A1/A2 genotypes) (p = 0.004) and in those with S/S genotype of 5-HTTLPR (p = 0.005). With the stratification of DRD2 TaqI A1(+) allele, high NS of ANX/DEP ALC existed only in carriers of 5-HTTLPR S/S genotype (p=0.001). Moreover, ANX/DEP ALC was related to high HA only in samples carrying 5-HTTLPR S/L or L/L genotype (p = 0.02). These findings provided the empirical genetic characterization of the specific personality traits in ANX/DEP ALC among Han Chinese population in Taiwan.

Molecular analyses of unrelated Charcot-Marie-Tooth (CMT) disease patients suggest a high frequency of the CMT1A duplication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wise, C.A.; Davis, S.N.; Heju, Z.

1993-10-01

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. One form of CMT, CMT type 1A, is characterized by uniformly decreased nerve conduction velocities, usually shows autosomal dominant inheritance, and is associated with a large submicroscopic duplication of the p11.2-p12 region of chromosome 17. A cohort of 75 unrelated patients diagnosed clinically with CMT and evaluated by electrophysiological methods were analyzed molecularly for the presence of the CMT1A DNA duplication. Three methodologies were used to assess the duplication: Measurement of dosage differences between RFLP alleles, analysis of polymorphic (GT)[sub n] repeats, and detection of a junction fragment by pulsed-fieldmore » gel electrophoresis. The CMT1A duplication was found in 68% of the 63 unrelated CMT patients with electrophysiological studies consistent with CMT type 1 (CMT1). The CMT1A duplication was detected as a de novo event in two CMT1 families. Twelve CMT patients who did not have decreased nerve conduction velocities consistent with a diagnosis of CMT type 2 (CMT2) were found not to have the CMT1A duplication. The most informative molecular method was the detection of the CMT1A duplication-specific junction fragment. Given the high frequency of the CMT1A duplication in CMT patients and the high frequency of new mutations, the authors conclude that a molecular test for the CMT1A DNA duplication is very useful in the differential diagnosis of patients with peripheral neuropathies. 61 refs., 4 figs.« less

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus.

PubMed

Mock, B; Krall, M; Blackwell, J; O'Brien, A; Schurr, E; Gros, P; Skamene, E; Potter, M

1990-05-01

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.

Genetic polymorphisms of vitamin D receptor (VDR) in Fabry disease.

PubMed

Teitcher, Michael; Weinerman, Sarah; Whybra, Catharina; Beck, Michael; Sharon, Nir; Elstein, Deborah; Altarescu, Gheona

2008-11-01

Fabry disease, an X-linked inborn error of metabolism, is characterized by multi-organ involvement including cardiac signs of left ventricular hypertrophy and abnormal intima-medial (IMT) thickening of arteries, progressive renal failure, neurological involvement, and more. The vitamin D receptor (VDR) and an enzyme producing vitamin D3 result in an autocrine loop with direct effects on blood vessels. The purpose of this study is to assess VDR polymorphisms (BsmI, FokI, ApaI, and TaqI) relative to clinically important disease parameters using a disease-specific severity score (MSSI) and haplotype analysis. There were statistically significant differences between females (43% of 74 patients) and males in MSSI total scores, and in general and neurologic sub-scores. There appears to be a protective effect of the TaqI tt genotype so that there were significantly lower scores in clinical categories between those with the tt genotype versus those with the TT genotype. Multivariate models of haplotypes with MSSI scores reveal that T-A-f-B and t-a-F-b haplotypes of the VDR gene polymorphisms are significantly associated with variation in the Fabry phenotype. Despite the limitations of using the MSSI score as a clinical correlate, these results are provocative and further studies in larger cohorts with more males are recommended.

Analysis of polymorphisms of the vitamin D receptor, estrogen receptor, and collagen Ialpha1 genes and their relationship with height in children with bone cancer.

PubMed

Ruza, Elena; Sotillo, Elena; Sierrasesúmaga, Luis; Azcona, Cristina; Patiño-García, Ana

2003-10-01

The authors' objectives were to compare height at diagnosis of children with bone tumors with that of Spanish reference children; to analyze the frequency of the genotypes for the polymorphisms of the vitamin D receptor (VDR), estrogen receptor (ER), and collagen Ialpha1 (COLIalpha1) genes in patients and in healthy controls; and to test the relationship between the genetic markers and height. Height and weight at diagnosis were measured in 58 osteosarcoma and 36 Ewing sarcoma patients and compared with standards published for Spanish reference children according to sex and age. For the molecular analysis, genetic polymorphisms of the VDR (Fok I, Apa I, and TaqI), ER (Pvu II and XbaI), and COLIalpha1 (Msc I) genes were characterized in 72 osteosarcoma and 53 Ewing sarcomas and in a group of 143 healthy matched children. Osteosarcoma and Ewing sarcoma patients were significantly taller than Spanish reference children. Osteosarcoma patients showed a significantly higher frequency of the Ff genotype for the Fok I polymorphism (VDR gene) than the control group. The odds ratio for this genotype was 1.78, with an increased relative risk of 78% for heterozygous Ff carriers. Among Ewing sarcoma patients, this same genotype was significantly associated with lower height than homozygotes (FF or ff). Children with bone cancer are significantly taller than the reference population, which may be influenced by the genotype for the Fok I polymorphism of the VDR gene.

Variability and distribution of COL1A2 (type I collagen) polymorphisms in the central-eastern Mediterranean Basin.

PubMed

Scorrano, Gabriele; Lelli, Roberta; Martínez-Labarga, Cristina; Scano, Giuseppina; Contini, Irene; Hafez, Hani S; Rudan, Pavao; Rickards, Olga

2016-01-01

The most abundant of the collagen protein family, type I collagen is encoded by the COL1A2 gene. The COL1A2 restriction fragment length polymorphisms (RFLPs) EcoRI, RsaI and MspI in samples from several different central-eastern Mediterranean populations were analysed and found to be potentially informative anthropogenetic markers. The objective was to define the genetic variability of COL1A2 in the central-eastern Mediterranean and to shed light on its genetic distribution in human groups over a wide geographic area. PCR-RFLP analysis of EcoRI, RsaI and MspI polymorphisms of the COL1A2 gene was performed on oral swab and blood samples from 308 individuals from the central-eastern Mediterranean Basin. The genetic similarities among these groups and other populations described in the literature were investigated through correspondence analysis. Single-marker data and haplotype frequencies seemed to suggest a genetic homogeneity within the European populations, whereas a certain degree of differentiation was noted for the Egyptians and the Turks. The genetic variability in the central-eastern Mediterranean area is probably a result of the geographical barrier of the Mediterranean Sea, which separated European and African populations over time.

MAP3K1 May be a Promising Susceptibility Gene for Type 2 Diabetes Mellitus in an Iranian Population

PubMed Central

Torkamandi, Shahram; Bastami, Milad; Ghaedi, Hamid; Moghadam, Fateme; Mirfakhraie, Reza; Omrani, Mir Davood

2016-01-01

Considering that MAPK (mitogen- activated protein kinase) signaling pathway has an important role in the progression of inflammatory cytokine secretion in type 2 diabetes mellitus (T2DM), we have recently investigated the reported genetic polymorphism from genome wide association study in MAP3K1 (mitogen-activated protein kinase kinase kinase 1) in diabetes as an important member of MAPK signaling. This study aimed to investigate the possible association of rs10461617 at the upstream of MAP3K1 gene in an Iranian case-control study with the risk of T2DM. The study population was comprised of 342 unrelated Iranian individuals including 177 patients with T2DM and 165 unrelated healthy control subjects. Genotyping was performed using PCR-RFLP and confirmed with sequencing. In a logistic regression analysis, the rs10461617A allele was associated with a significantly higher risk of T2DM assuming the log- additive model (OR: 1.44, 95% CI: 1.01-2.05, P = 0.039). In conclusion, we provided the first evidence for the association of rs10461617 at the upstream of MAP3K1 with the risk of T2DM in an Iranian population. PMID:27942499

Vitamin D Receptor Gene Polymorphisms Associated with Childhood Autism.

PubMed

Cieślińska, Anna; Kostyra, Elżbieta; Chwała, Barbara; Moszyńska-Dumara, Małgorzata; Fiedorowicz, Ewa; Teodorowicz, Małgorzata; Savelkoul, Huub F J

2017-09-09

Autism spectrum disorder (ASD) is a group of heterogeneous, behaviorally defined disorders whereby currently no biological markers are common to all affected individuals. A deregulated immune response may be contributing to the etiology of ASD. The active metabolite of vitamin D₃ has an immunoregulatory role mediated by binding to the vitamin D receptor (VDR) in monocyte, macrophages, and lymphocytes. The effects of vitamin D and interaction with the VDR may be influenced by polymorphism in the VDR gene. Genetic association of four different VDR polymorphisms (Apa-I, Bsm-I, Taq-I, Fok-I) associated with susceptibility to the development of autism in children was investigated. We uniquely found an association between the presence of the T allele at position Taq-I and presence of the a allele at position Apa-I of the VDR gene with decreased ASD incidence. There was also an association between female gender and the presence of the T allele. We found no statistical significant correlation between VDR single nucleotide polymorphisms (SNPs) and vitamin D₃ concentration in serum of ASD children. Genetic polymorphism in two SNP in VDR may be correlated with development of ASD symptoms by influencing functionality of vitamin D₃ metabolism, while vitamin D₃ levels were not significantly different between ASD and non-ASD children.

Association of vitamin D receptor gene polymorphisms with diabetic dyslipidemia in the elderly male population in North China.

PubMed

Xia, Zheng; Hu, Yazhuo; Han, Zhitao; Gao, Ya; Bai, Jie; He, Yao; Zhao, Hua; Zhang, Honghong

2017-01-01

The prevalence of dyslipidemia is rising alarmingly in elderly Han Chinese male patients with type 2 diabetes mellitus (T2DM). The genetic factors that contribute to the development of diabetic dyslipidemia remain incompletely identified. This study was conducted to assess the association between vitamin D receptor (VDR) polymorphisms and development of dyslipidemia in the Han elderly male population with T2DM in North China. A total of 242 T2DM patients with dyslipidemia (DH group, n=108) or without dyslipidemia (DO group, n=134) and 100 controls were genotyped for ApaI, TaqI and FokI single nucleotide polymorphisms (SNPs) of the VDR gene using polymerase chain reaction-restriction fragment length polymorphism and sequencing. The frequency and distribution of the SNPs were compared between cases and controls. The distribution of genotypes of VDR-FokI was significantly different between the control and DM group ( P =0.033), as well as between the control and DH subgroup ( P =0.011) but not DO subgroup ( P =0.111). The frequency of C allele and CC genotype of FokI was significantly higher in the DH patients than in the controls ( P =0.015 and P =0.003, respectively). Logistic regression analysis in a dominant model homozygous for the C allele of the FokI SNP showed that CC genotype was associated with DH patients (OR =1.797, 95% CI: 1.077-2.999, P =0.025). Significant associations of the ApaI and TaqI SNPs with either DO or DH subjects were not observed. These findings suggest that CC genotype of VDR-FokI is a risk factor for T2DM patients with dyslipidemia in elderly males in North China.

Association analysis of vitamin D receptor gene polymorphisms and bone mineral density in postmenopausal Mexican-Mestizo women.

PubMed

González-Mercado, A; Sánchez-López, J Y; Regla-Nava, J A; Gámez-Nava, J I; González-López, L; Duran-Gonzalez, J; Celis, A; Perea-Díaz, F J; Salazar-Páramo, M; Ibarra, B

2013-07-30

We investigated associations between vitamin D receptor (VDR) gene polymorphisms, FokI T>C (rs2228570), BsmI G>A (rs1544410), ApaI G>T (rs7975232), and TaqI T>C (rs731236), with bone mineral density (BMD) in postmenopausal Mexican-Mestizo women. Three hundred and twenty postmenopausal women participated, who were classified according to World Health Organization criteria as non-osteoporotic (Non-OP; N = 88), osteopenic (Opn; N = 144), and osteoporotic (OP; N = 88). BMD measurements at the lumbar (L1-L4) spine and at the left and right femoral neck were obtained by dual-energy X-ray absorptiometry. Single nucleotide polymorphisms (SNPs) were genotyped using real-time polymerase chain reaction and TaqMan probes. Genotype and allelic frequencies of the 4 VDR SNPs were similar among the 3 groups. Polymorphic allele frequencies were as follows: FokI (C) 0.53, 0.49, 0.56; BsmI (A) 0.26, 0.22, 0.23; ApaI (T) 0.43, 0.39, 0.44; TaqI (C) 0.27, 0.22, 0.23 for the Non-OP, Opn, and OP groups, respectively. Although no associations were found between the SNPs and BMD, based on the putative function of the FokI SNP, we constructed, for the first time, the haplotype with the 4 VDR SNPs, and found that the CGGT haplotype differed between the Non- OP and OP groups (21.8 vs 31.8%, P < 0.05). The risk analysis for this haplotype was nearly significant under the dominant model (OR = 1.783, 95%CI = 0.98-3.25, P = 0.058). This result suggests a possible susceptibility effect of the C allele of the FokI SNP for the development of osteoporosis in postmenopausal Mexican-Mestizo women.

Association of of IL-1 receptor antagonist (IL-1RN) and interleukin-1β genes (IL-1β) polymorphisms with recurrent pregnancy loss in Iranian Azeri women.

PubMed

Ali Rahmani, Seyyed; Paknejad, Zeynab; Mohammadkhanlou, Masoumeh; Daneshparvar, Marina

2017-12-27

Objective One of the most important problems in human reproduction is recurrent pregnancy loss (RPL). RPL is defined as three or more consecutive abortions in the first trimester of pregnancy. The association between the polymorphisms in the immunological factors and RPL was investigated. The aim of our study was to determine the association of interleukin receptor antagonist (IL-IRN) and interleukin-1β (IL-1β) polymorphisms with RPL in Iranian Azeri women. Materials and methods The study participants consisted of 100 women with RPL of Iranian Azeri origin. The control group comprised 100 age- and ethnically-matched healthy women of the same reproductive age. Genomic DNA was extracted from the whole blood and genotype determinations were performed using polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP) analysis. Results Our results showed no significant relationship between IL-1RN polymorphism and RPL. The homozygous state in -857 C/T variant was seen to be higher in RPL patients than in control subjects. Also frequency of wild type genotype was lower in RPL patients than in controls. However, this associations was not significant. Conclusion This study suggested that -511 C/T (rs16944) and -31 C/T (rs1143627) polymorphisms in IL-1β gene may not be involved in RPL in Iranian Azeri women. Also the promoter polymorphism of the IL-1RN gene may not play a role in the susceptibility to RPL.

Association between melatonin receptor 1A (MTNR1A) gene single-nucleotide polymorphisms and the velvet antler yield of Sika deer.

PubMed

Yang, Fei-Fei; Huo, Li-Jun; Yang, Li-Guo; Riaz, Hasan; Xiong, Li-Rong; Chen, Jian-Guo; Zhang, Shu-Jun; Xiong, Jia-Jun

2014-01-01

Melatonin, a secretion from pineal gland is ambiguously considered as the key hormone involved in regulation of the antler cycle in Sika deer. To find out more about the roles of melatonin and its receptor gene, we carried out current study to investigate the association between polymorphisms in melatonin receptor 1A (MTNR1A) gene and the antler yield from Sika deer. A total of 251 Sika deer were analyzed in this study, of which consisted of Wusan Sika deer (n = 163) and Dongfeng Sika deer (n = 88). MTNRA gene was amplified by PCR and genotyped by Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Three polymorphism loci (C518T, C629G and C635T) were detected in exon2 of MTNR1A gene. The restriction site Ecol881 was used for C518T while a C629G polymorphism locus was digested with Mval restriction endonucleases. In Wusan Sika deer the allele frequencies of C and T were 0.637 and 0.363 for C518T, Also C and G alleles in C629G locus were 0.206 and 0.794. Genotypic frequencies of allele CC, CT and TT were 33.7, 59.9 and 6.4 % respectively, It showed 1.8, 37.4 and 60.7 % for frequencies of genotypes CC, CG and GG. In Dongfeng Sika deer the allele frequencies of C and T were 0.518 and 0.482 for C518T, C and G alleles were 0.375 and 0.625 for C629G. Genotypic frequencies were 10.6, 82.4 and 7.1 % for genotypes CC, CT and TT respectively, and they were 1.1, 72.7 and 26.2 % for genotypes CC, CG and GG. Among three SNPs, only C629G showed significant association (P < 0.05) with average antler yield in Wusan Sika deer, while no SNP was significant in Dongfeng Sika deer. These preliminary results implied that the identified SNPs of MTNR1A gene might influence the antler yield in Wusan Sika deer.

Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism.

PubMed

Jernberg, Cecilia; Sullivan, Asa; Edlund, Charlotta; Jansson, Janet K

2005-01-01

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism

PubMed Central

Jernberg, Cecilia; Sullivan, Åsa; Edlund, Charlotta; Jansson, Janet K.

2005-01-01

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic. PMID:15640226

Genome-Wide Supported Risk Variants in MIR137, CACNA1C, CSMD1, DRD2, and GRM3 Contribute to Schizophrenia Susceptibility in Pakistani Population.

PubMed

Fatima, Ambrin; Farooq, Muhammad; Abdullah, Uzma; Tariq, Muhammad; Mustafa, Tanveer; Iqbal, Muhammad; Tommerup, Niels; Mahmood Baig, Shahid

2017-09-01

Schizophrenia is a chronic neuropsychiatric disease afflicting around 1.1% of the population worldwide. Recently, MIR137 , CACNA1C , CSMD1 , DRD2 , and GRM3 have been reported as the most robustly emerging candidates involved in the etiology of schizophrenia. In this case control study, we performed an association analysis of rs1625579 ( MIR137 ), rs1006737, rs4765905 ( CACNA1C ), rs10503253 ( CSMD1 ), rs1076560 ( DRD2 ), rs12704290, rs6465084, and rs148754219 ( GRM3 ) in Pakistani population. Schizophrenia was diagnosed on the basis of the Diagnostic and Statistical Manual of Mental Disorders 4th ed (DSM-IV). Detailed clinical information, family history of all patients and healthy controls were collected. RFLP based case control association study was performed in a Pakistani cohort of 508 schizophrenia patients and 300 healthy control subjects. Alleles and genotype frequencies were calculated using SPSS. A significant difference in the genotype and allele frequencies for rs4765905, rs1076560 and rs6465084 were found between the patients and controls (p=0.000). This study provides substantial evidence supporting the role of CACNA1C , GRM3 and DRD2 as schizophrenia susceptibility genes in Pakistani population.

Molecular identification key based on PCR/RFLP for three polychaete sibling species of the genus Marenzelleria, and the species' current distribution in the Baltic Sea

NASA Astrophysics Data System (ADS)

Blank, M.; Laine, A. O.; Jürss, K.; Bastrop, R.

2008-06-01

Studies of Marenzelleria species were often hampered by identification uncertainties when using morphological characters only. A newly developed PCR/RFLP protocol allows a more efficient discrimination of the three species Marenzelleria viridis, Marenzelleria neglecta and Marenzelleria arctia currently known for the Baltic Sea. The protocol is based on PCR amplification of two mitochondrial DNA gene segments (16S, COI) followed by digestion with restriction enzymes. As it is faster and cheaper than PCR/sequencing protocols used so far, the protocol is recommended for large-scale analyses. The markers allow an undoubted determination of species irrespective of life stage or condition of the worms in the samples. The protocol was validated on about 950 specimens sampled at more than 30 sites of the Baltic and the North Sea, and on specimens from populations of the North American east coast. Besides this test we used mitochondrial DNA sequences (16S, COI, Cytb) and starch gel electrophoresis to further investigate the distribution of the three Marenzelleria species in the Baltic Sea. The results show that M. viridis (formerly genetic type I or M. cf. wireni) occurred in the Öresund area, in the south western as well as in the eastern Baltic Sea, where it is found sympatric with M. neglecta. Allozyme electrophoresis indicated an introduction by range expansion from the North Sea. The second species, M. arctia, was only found in the northern Baltic Sea, where it sometimes occurred sympatric with M. neglecta or M. viridis. For Baltic M. arctia, the most probable way of introduction is by ship ballast water from the European Arctic. There is an urgent need for a new genetic analysis of all Marenzelleria populations of the Baltic Sea to unravel the current distribution of the three species.

Relation between HLA-DQA1 genes and genetic susceptibility to duodenal ulcer in Wuhan Hans

PubMed Central

Du, Yi-Ping; Deng, Chang-Sheng; Lu, De-Yin; Huang, Mei-Fang; Guo, Shu-Fang; Hou, Wei

2000-01-01

AIM: To study the genetic susceptibility of HLA-DQA1 alleles to duodenal ulcer in Wuhan Hans. METHODS: Seventy patients with duodenal ulcer and fifty health y controls were examined for HLA-DQA1 genotypes. HLA-DQA1 typing was carried out by digesting the locus specific polymerase chain reaction amplified products with alleles specific restriction enzymes (PCR-RFLP), i.e. Apal I, Bsaj I, Hph I, Fok I, Mbo II and Mnl I. RESULTS: The allele frequencies of DQA1*0301 and DQA1*0102 in patients with duodenal ulcer were significantly higher and lower respectivel y than those in healthy controls (0.40 vs 0.20, P = 0.003, Pc orret = 0.024) and (0.05 vs 0.14, P = 0.012, but P corret > 0.05), respectively. CONCLUSION: DQA1*0301 is a susceptible gene for duodenal ulcer in Wuhan Hans, and there are immunogenetic differences in HLA-DQA1 locus between duodenal ulcer patients and healthy controls. PMID:11819534

Molecular typing of Trichomonas vaginalis isolates by actin gene sequence analysis and carriage of T. vaginalis viruses.

PubMed

Masha, Simon C; Cools, Piet; Crucitti, Tania; Sanders, Eduard J; Vaneechoutte, Mario

2017-10-30

The protozoan parasite Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen. Although T. vaginalis is highly prevalent among women in Kenya, there is lack of data regarding genetic diversity of isolates currently in circulation in Kenya. Typing was performed on 22 clinical isolates of T. vaginalis collected from women attending the antenatal care clinic at Kilifi County Hospital, Kenya, in 2015. Genotyping followed a previously proposed restriction fragment length polymorphism (RFLP) scheme, which involved in silico cleavage of the amplified actin gene by HindII, MseI and RsaI restriction enzymes. Phylogenetic analysis of all the sequences was performed to confirm the results obtained by RFLP-analysis and to assess the diversity within the RFLP genotypes. Additionally, we determined carriage of the four different types of Trichomonas vaginalis viruses (TVVs) by polymerase chain reaction. In silico RFLP-analysis revealed five actin genotypes; 50.0% of the isolates were of actin genotype E, 27.3% of actin genotype N, 13.6% of actin genotype G and 4.5% of actin genotypes I and P. Phylogenetic analysis was in agreement with the RFLP-analysis, with the different actin genotypes clustering together. Prevalence of TVVs was 43.5% (95% confidence interval, CI: 23.2-65.5). TVV1 was the most prevalent, present in 39.1% of the strains and 90% of the T. vaginalis isolates which harbored TVVs had more than one type of TVV. None of the isolates of actin genotype E harbored any TVV. The presence of five actin genotypes in our study suggests notable diversity among T. vaginalis isolates occurring among pregnant women in Kilifi, Kenya. Isolates of the most prevalent actin genotype E lacked TVVs. We found no association between T. vaginalis genotype, carriage of TVVs and symptoms. Further studies with higher number of strains should be conducted in order to corroborate these results.

Ornithine aminotransferase (OAT): recombination between an X-linked OAT sequence (7.5 kb) and the Norrie disease locus.

PubMed

Ngo, J T; Bateman, J B; Spence, M A; Cortessis, V; Sparkes, R S; Kivlin, J D; Mohandas, T; Inana, G

1990-01-01

A human ornithine aminotransferase (OAT) locus has been mapped to the Xp11.2, as has the Norrie disease locus. We used a cDNA probe to investigate a 3-generation UCLA family with Norrie disease; a 4.2-kb RFLP was detected and a maximum lod score of 0.602 at zero recombination fraction was calculated. We used the same probe to study a second multigeneration family with Norrie disease from Utah. A different RFLP of 7.5 kb in size was identified and a recombinational event between the OAT locus represented by this RFLP and the disease loci was observed. Linkage analysis of these two loci in this family revealed a maximum load score of 1.88 at a recombination fraction of 0.10. Although both families have affected members with the same disease, the lod scores are reported separately because the 4.2- and 7.5-kb RFLPs may represent two different loci for the X-linked OAT.

[Presence of the double pfmdr1 mutation 86Tyr and 1246 Tyr in clones of a chloroquine-resistant west African isolate of Plasmodium falciparum].

PubMed

Pinheiro, L; Franco, S; Adagu, I S; Rosa, R; Rosário, V E; Warhurst, D C

2003-01-01

Isolates of Plasmodium falciparum from three areas of West Africa were recovered from cryopreservation and their chloroquine-sensitivity were determined in vitro. Of the 90 samples studied, 60 were from Guinea-Bissau (30Resistant/30Sensitive), 15 were from S. Tomé and Príncipe (11Resistant/4Sensitive) and 15 were from Angola (11Resistant/4Sensitive). All the isolates were sensitive to mefloquine. Using the polymerase chain reaction/restriction fragment length polymorphism technique (PCR/RFLP) it was possible to detect two mutations in the pfmdr1 gene, often associated with chloroquine-resistance. 66% of the samples from Guiné-Bissau showed a correlation with chloroquine-resistance while 73% of the samples from São Tomé and Angola altogether had the 86Tyr mutation. The present study on West African isolates and clones showed, for the first time, the presence of a double point mutation in the pfmdr1 gene one being found, up to now, only in South America isolates of Plasmodium falciparum.

The Impact of Five VDR Polymorphisms on Multiple Sclerosis Risk and Progression: a Case-Control and Genotype-Phenotype Study.

PubMed

Křenek, Pavel; Benešová, Yvonne; Bienertová-Vašků, Julie; Vašků, Anna

2018-04-01

Vitamin D receptor polymorphisms have been the target of many studies focusing on multiple sclerosis. However, previously reported results have been inconclusive. The objective of this study was to investigate the association between five vitamin D receptor polymorphisms (EcoRV, FokI, ApaI, TaqI, and BsmI) and multiple sclerosis susceptibility and its course. The study was carried out as a case-control and genotype-phenotype study, consisted of 296 Czech multiple sclerosis patients and 135 healthy controls. Genotyping was carried out using polymerase chain reaction and restriction analysis. In multiple sclerosis men, allele and/or genotype distributions differed in EcoRV, TaqI, BsmI, and ApaI polymorphisms as compared to controls (EcoRV, p a = 0.02; Taq, p g = 0.02, p a = 0.02; BsmI, p g = 0.02, p a = 0.04; ApaI, p g = 0.008, p a = 0.005). In multiple sclerosis women, differences in the frequency of alleles and genotypes were found to be significant in ApaI (controls vs multiple sclerosis women: p g = 0.01, p a = 0.05). Conclusive results were observed between multiple sclerosis women in the case of EcoRV [differences in Expanded Disability Status Scale (p = 0.05); CT genotype was found to increase the risk of primary progressive multiple sclerosis 5.5 times (CT vs CC+TT p corr = 0.01, sensitivity 0.833, specificity 0.525, power test 0.823)] and FokI [borderline difference in Multiple Sclerosis Severity Score (p = 0.05)]. Our results indicate that the distribution of investigated vitamin D receptor polymorphisms is a risk factor for multiple sclerosis susceptibility and progression in the Czech population. The association between disease risk and polymorphisms was found to be stronger in men. The association of disease progression with polymorphisms was observed only in women.

The rs4285184 polymorphism of the MGAT1 gene as a risk factor for obesity in the Mexican population.

PubMed

Tapia-Rivera, José C; Baltazar-Rodríguez, Luz M; Cárdenas-Rojas, Martha I; Álvarez, Alan; Bustos-Saldaña, Rafael; Delgado-Enciso, Iván; Valdez-Velázquez, Laura L; Guzmán-Esquivel, José; Ramírez-Flores, Mario

2017-02-23

Obesity is a factor that contributes to the morbidity of certain diseases and to worldwide mortality. MGAT1 is a glycosyltransferase involved in the synthesis of protein-bound and lipid-bound oligosaccharides and its polymorphisms are possibly involved in the etiology of obesity. We investigated the association of the rs4285184 polymorphism of the MGAT1 gene with obesity in adults in the State of Colima, Mexico. A case-control study was conducted that included 244 subjects. All of them were grouped according to their percentage of body fat, determined through bioelectrical impedance, and they were genotyped for the rs4285184 polymorphism of the MGAT1 gene through PCR-RFLP. The results were analyzed for their association with the percentage of body fat. The G allele had a frequency of 49.19 and 38.75% for the cases and controls, respectively (P=.020) (OR 1.53; 95% CI 1.068-2.193). The frequency of the A/G+G/G genotype was 75% in the obese patients, which was significantly higher compared with the 57.5% of the control group (P=.004) (OR 2.217; 95% CI 1.287-3.821). The presence of the rs4285184 polymorphism of the MGAT1 gene increased the risk for developing body fat associated with obesity in the Mexican population. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Genetic modification of ALAD and VDR on lead-induced impairment of hearing in children.

PubMed

Pawlas, Natalia; Broberg, Karin; Olewińska, Elżbieta; Kozłowska, Agnieszka; Skerfving, Staffan; Pawlas, Krystyna

2015-05-01

Polymorphisms in the δ-aminolevulinic acid dehydratase (ALAD) and the vitamin D receptor (VDR) genes may modify lead metabolism and neurotoxicity. Two cohorts of children were examined for hearing [pure-tone audiometry (PTA), brain stem auditory evoked potentials (BAEP)], acoustic otoemission (transient emission evoked by a click) and blood-lead concentrations (B-Pb). The children were genotyped for polymorphisms in ALAD and VDR. The median B-Pbs were 55 and 36μg/L in the two cohorts (merged cohort 45μg/L). B-Pb was significantly associated with impaired hearing when tested with PTA (correlation coefficient rS=0.12; P<0.01), BAEP (rS=0.18; P<0.001) and otoemission (rS=-0.24; P<0.001). VDR significantly modified the lead-induced effects on PTA. Carriers of the VDR alleles BsmI B, VDR TaqI t and VDR FokI F showed greater toxic effects on PTA, compared to BsmI bb, VDR TaqI TT and VDR FokI ff carriers. No significant interaction was found for ALAD. Lead impairs hearing functions in the route from the cochlea to the brain stem at low-level exposure, and polymorphisms in VDR significantly modify these effects. Copyright © 2015 Elsevier B.V. All rights reserved.

Association of The IDH1 C.395G>A (R132H) Mutation with Histological Type in Malay Brain Tumors

PubMed

Mohamed Yusoff, Abdul Aziz; Zulfakhar, Fatin Najwa; Sul’ain, Mohd Dasuki; Idris, Zamzuri; Abdullah, Jafri Malin

2016-12-01

Background: Brain tumors, constituting one of the most deadly forms of cancer worldwide, result from the accumulation of multiple genetic and epigenetic alterations in genes and signaling pathways. Isocitrate dehydrogenase enzyme isoform 1 (IDH1) mutations are frequently identified in primary brain tumors and acute myeloid leukemia. Studies on IDH1 gene mutations have been extensively performed in various populations worldwide but not in Malaysia. This work was conducted to study the prevalence of IDH1 c.395G>A (R132H) hotspot mutations in a group of Malaysian patients with brain tumors in order to gain local data for the IDH1 mutation profile in our population. Methods: Mutation analysis of c.395G>A (R132H) of IDH1 was performed in 40 brain tumor specimens by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and then verified by direct sequencing. Associations between the IDH1 c.395G>A (R132H) mutation and clinicopathologic characteristics were also analyzed. Results: The IDH1 c.395G>A (R132H) mutation was detected in 14/40 patients (35%). A significant association was found with histological tumor types, but not with age, gender and race. Conclusions: IDH1 is frequently mutated and associated with histological subtypes in Malay brain tumors. Creative Commons Attribution License

Association of The IDH1 C.395G>A (R132H) Mutation with Histological Type in Malay Brain Tumors

PubMed Central

Yusoff, Abdul Aziz Mohamed; Zulfakhar, Fatin Najwa; Sul’ain, Mohd Dasuki; Idris, Zamzuri; Abdullah, Jafri Malin

2016-01-01

Background: Brain tumors, constituting one of the most deadly forms of cancer worldwide, result from the accumulation of multiple genetic and epigenetic alterations in genes and signaling pathways. Isocitrate dehydrogenase enzyme isoform 1 (IDH1) mutations are frequently identified in primary brain tumors and acute myeloid leukemia. Studies on IDH1 gene mutations have been extensively performed in various populations worldwide but not in Malaysia. This work was conducted to study the prevalence of IDH1 c.395G>A (R132H) hotspot mutations in a group of Malaysian patients with brain tumors in order to gain local data for the IDH1 mutation profile in our population. Methods: Mutation analysis of c.395G>A (R132H) of IDH1 was performed in 40 brain tumor specimens by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and then verified by direct sequencing. Associations between the IDH1 c.395G>A (R132H) mutation and clinicopathologic characteristics were also analyzed. Results: The IDH1 c.395G>A (R132H) mutation was detected in 14/40 patients (35%). A significant association was found with histological tumor types, but not with age, gender and race. Conclusions: IDH1 is frequently mutated and associated with histological subtypes in Malay brain tumors. PMID:28125199

Influence of polymorphisms in insulin-like growth factor-1 on the risk of osteoporosis in a Chinese postmenopausal female population

PubMed Central

Li, Feng; Xing, Wen-Hua; Yang, Xue-Jun; Jiang, Hai-Yan; Xia, Hong

2015-01-01

We conducted a case-control study in a Chinese postmenopausal population, and explore the potential role of the promoter region variation of the IGF-1 gene in bone mineral density and osteoporosis risk. 485 postmenopausal women with a primary diagnosis of osteoporosis and 485 age-matched controls were selected between 2012 and 2014. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was used for rs35767, rs2288377 and rs5742612 of IGF-1 genotyping. By conditional regression analysis, individuals carrying TT genotype and CT+TT genotype of rs35767 were found to be correlated with an elevated risk of osteoporosis, with adjusted ORs (95% CI) of 1.90 (1.23-2.93) and 1.35 (1.04-1.76), respectively. Our study found that CT+TT genotype of rs35767 was significantly associated with moderate increased risk of osteoporosis in smokers and drinkers, and the ORs (95% CI) were 2.11 (1.06-4.20) and 2.36 (1.29-4.32), respectively. We found that those carrying CT+TT genotype of rs35767 had a significant lower BMD levels at L1-L4 vertebrae, femoral neck, total hip and trochanter compared to those with CC genotype. Our study suggests that TT genotype and CT+TT genotype of IGF-I rs35767 were associated with risk of osteoporosis and BMD levels. PMID:26191288

Apoptosis, production of MMP9, VEGF, TNF-alpha and intracellular growth of M. tuberculosis for different genotypes and different pks5/1 genes.

PubMed

Yorsangsukkamol, Juthaporn; Chaiprasert, Angkana; Palaga, Tanapat; Prammananan, Therdsak; Faksri, Kiatichai; Palittapongarnpim, Prasit; Prayoonwiwat, Narapon

2011-09-01

A previous study of IS6110 RFLP and spoligotyping of M. tuberculosis isolates from 152 Thai patients with tuberculous meningitis revealed a significantly higher percentage (57%) of the Beijing genotype as compared to isolates obtained from pulmonary tuberculosis. We postulated that the M. tuberculosis Beijing genotype is likely to be more virulent than others. Ten M. tuberculosis cerebrospinal fluid (CSF) isolates from five RFLP groups, together with different characteristics of pks15/1, M. tuberculosis H37Rv and M. bovis BCG, were investigated for their virulence in vitro. In this study, THP-1 cells were used as host cells to determine the intracellular growth and the induction of MMP9, VEGF, TNF-alpha and apoptosis. Determinations of the cytokine production and apoptosis were based on available commercial kits using ELISA techniques. No significant difference in intracellular multiplication was found between the M. tuberculosis CSF isolates. Three isolates, consisting of 2 Nonthaburi and 1 heterogeneous isolate, were found to stimulate high TNF-alpha and MMP-9 production during the early infection period.They were isolated from 3 different patients, 2 of whom died with initial stages II and III. This result suggested that there might be an association between TNF-alpha and MMP-9 production that could account for the specific virulent nature of Nonthaburi strains. VEGF production was determined and comparable levels were found in all isolates. No significant apoptosis was detected in M. tuberculosis CSF isolates. No significant differences suggesting that the 2 Beijing strains are more virulent than the others were observed. The predominance of the Beijing strains in cases of tuberculous meningitis (TBM) in Thai patients is not a result of their hypervirulence.

Prognostic significance of CCND1 (cyclin D1) overexpression in primary resected non-small-cell lung cancer.

PubMed Central

Betticher, D. C.; Heighway, J.; Hasleton, P. S.; Altermatt, H. J.; Ryder, W. D.; Cerny, T.; Thatcher, N.

1996-01-01

Amplification of the CCDN1 gene encoding cyclin D1 was examined by Southern blotting and multiplex polymerase chain reaction (PCR) and occurred in 8 of 53 patients (15%) with primary resected non-small-cell lung cancer (NSCLC). These tumours and 17 additional tumours with a normal gene copy number showed overexpression of cyclin D1 (25/53, 47%), as assessed by immunostaining using a monoclonal antibody. In 22/25 cases, cyclin D1 was localised in the cytoplasm, but some (7/25) had simultaneous nuclear staining. This result is in marked contrast to that reported in breast, hepatocellular and colorectal carcinoma studies where immunostaining was invariably nuclear. Examination of a restriction fragment length polymorphic (RFLP) site within the 3'untranslated region of the cDNA following reverse transcriptase (RT)-PCR (29/53 informative cases) showed a strong association between cytoplasmic staining and imbalance in allele-specific message levels. Cyclin D1 overexpression was associated with a poorly differentiated histology (P = 0.04), less lymphocytic infiltration of the tumour (P = 0.02) and a reduction in local relapse rate (P = 0.01). The relative risk of local relapse was 9.1 in tumours without cyclin D1 overexpression (P = 0.01, Cox regression analysis). We conclude that genetic alteration of cyclin D1 is a key abnormality in lung carcinogenesis and may have diagnostic and prognostic importance in the treatment of resectable NSCLC. Images Figure 1 Figure 2 Figure 3 PMID:8562333

Progressive Outer Retinal Necrosis Combined with Vitreous Hemorrhage in a Patient with Acquired Immunodeficiency Syndrome

PubMed Central

You, Yong Sung; Lee, Sung Jin; Lee, Sung Ho; Park, Chang Hyun

2007-01-01

Purpose To describe an unusual case of rapidly progressive outer retinal necrosis (PORN) with vitreous hemorrhage in a 41-year-old woman with acquired immunodeficiency syndrome (AIDS), who had retinitis developed from what was probably varicellar-zoster virus combined with cytomegalovirus (CMV) and herpes simplex type 1,2, as proven by the polymerase chain reaction restriction fragment length polymorphism method (PCR-RFLP). Methods This study is a case report detailing clinical follow-up and an aqueous humor test by PCR-RFLP. Results The deep, white retinal lesions coalesced and progressively expanded in a circumferential manner, with sparing of the perivascular retina. However, retinal and vitreous hemorrhages, unusual findings for PORN, could be noted around the optic nerve. Varicellar-zoster virus (VZV), cytomegalovirus (CMV), and herpes simplex types 1,2 (HSV-1,2) were detected in the aqueous humor by PCR. Conclusions PORN has been described as a variant of necrotizing herpetic retinopathy, occurring particularly in patients with AIDS. Although the etiologic agent has been reported to be VZV, concurrent or combined etiologic agents can include HSV-1, HSV-2, and CMV in AIDS patients. Therefore, combined antiviral therapy with acyclovir and ganciclovir could be more reasonable as an initial therapy. PMID:17460434

Association of PDCD1 polymorphism to systemic lupus erythematosus and rheumatoid arthritis susceptibility.

PubMed

do Canto, Luisa Matos; Farias, Ticiana Della Justina; Medeiros, Mayara Delagnelo; Coêlho, Cíntia Callegari; Sereia, Aline Fernanda Rodrigues; de Carlos Back, Lia Kubelka Fernandes; de Mello, Filipe Martins; Zimmermann, Adriana Fontes; Pereira, Ivânio Alves; de Souza, Ilíada Rainha

This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Association of PDCD1 polymorphism to Systemic Lupus Erythematosus and Rheumatoid Arthritis susceptibility.

PubMed

Canto, Luisa Matos do; Farias, Ticiana Della Justina; Medeiros, Mayara Delagnelo; Coêlho, Cíntia Callegari; Sereia, Aline Fernanda Rodrigues; Back, Lia Kubelka Fernandes de Carlos; Mello, Filipe Martins de; Zimmermann, Adriana Fontes; Pereira, Ivânio Alves; Souza, Ilíada Rainha de

2015-07-17

This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg Equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Designing and conducting in silico analysis for identifying of Echinococcus spp. with discrimination of novel haplotypes: an approach to better understanding of parasite taxonomic.

PubMed

Spotin, Adel; Gholami, Shirzad; Nasab, Abbas Najafi; Fallah, Esmaeil; Oskouei, Mahmoud Mahami; Semnani, Vahid; Shariatzadeh, Seyyed Ali; Shahbazi, Abbas

2015-04-01

The definitive identification of Echinococcus species is currently carried out by sequencing and phylogenetic strategies. However, the application of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns is not broadly used as a result of heterogeneity traits of Echinococcus genome in different regions of the world. Therefore, designing and conducting a standardized pattern should indigenously be considered in under-studied areas. In this investigation, an in silico mapping was designed and developed for eight Echinococcus spp. on the basis of regional sequences in Iran and the world. The numbers of 60 Echinococcus isolates were collected from the liver and lungs of 15 human, 15 sheep, 15 cattle, and 15 camel cases in Semnan province, Central Iran. DNA samples were extracted and examined by polymerase chain reaction of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) and PCR-RFLP via Rsa1 endonuclease enzyme. Moreover, 15 amplicons of cytochrome oxidase 1 (Cox1) were directly sequenced in order to identify the strains/haplotypes. PCR-RFLP and phylogenetic analyses revealed firmly the presence of the G1 and G6 genotypes with heterogeneity (three novel haplotypes) of Cox1 gene although no other expected genotypes were found in the region. Finding shows that the identification of novel haplotypes along with discrimination of Echinococcus spp. through regional patterns can unambiguously illustrate the real taxonomic status of parasite in Central Iran.

Possible interaction between MAOA and DRD2 genes associated with antisocial alcoholism among Han Chinese men in Taiwan.

PubMed

Wang, Tso-Jen; Huang, San-Yuan; Lin, Wei-Wen; Lo, Hsin-Yi; Wu, Pei-Lin; Wang, Yu-Shan; Wu, Yi-Syuan; Ko, Huei-Chen; Shih, Jean-Chen; Lu, Ru-Band

2007-01-30

subjects who were recruited for the study, 73 participants were diagnosed with Antisocial ALC and 158 subjects were diagnosed with antisocial personality disorder without alcoholism (Antisocial Non-ALC). The DRD2 TaqI A and MAOA-uVNTR (variable number of tandem repeat located upstream) polymorphisms were not found to be associated with Antisocial ALC. However, an association between DRD2 TaqI A polymorphisms and Antisocial ALC was shown only after stratification for the MAOA-uVNTR 4-repeat polymorphism. Additionally, after multiple logistic regressions, we found that, under stratification of MAOA-uVNTR 4-repeat polymorphism and in comparison with the DRD2 A1/A1 genotype as a reference group, the DRD2 A1/A2 genotype has a possible protective effect against alcoholism in individuals with antisocial personality disorder (ASPD). We concluded that the possible interactions between MAOA-uVNTR polymorphism and DRD2 TaqI A polymorphism might be related to Antisocial ALC among Han Chinese men in Taiwan.

Association between ADH1C and ALDH2 polymorphisms and alcoholism in a Turkish sample.

PubMed

Ayhan, Yavuz; Gürel, Şeref Can; Karaca, Özgür; Zoto, Teuta; Hayran, Mutlu; Babaoğlu, Melih; Yaşar, Ümit; Bozkurt, Atilla; Dilbaz, Nesrin; Uluğ, Berna Diclenur; Demir, Başaran

2015-04-01

Polymorphisms in the genes encoding alcohol metabolizing enzymes are associated with alcohol dependence. To evaluate the association between the alcohol dehydrogenase 1C (ADH1C) Ile350Val and aldehyde dehydrogenase 2 (ALDH2) Glu504Lys polymorphisms and alcohol dependence in a Turkish sample. 235 individuals (115 alcohol-dependent patients and 120 controls) were genotyped for ADH1C and ALDH2 with PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Association between the polymorphisms and family history, daily and maximum amount of alcohol consumed was investigated. The associations between alcohol dependence, severity of consumption and family history and the polymorphisms were analyzed by chi-square or Fisher's exact test where necessary. Relationship between genotypes and dependence related features was evaluated using analysis of variance (ANOVA). The -350Val allele for ADH1C (ADH1C*2) was increased in alcohol-dependent patients (P = 0.05). In individuals with a positive family history, the genotype distribution differed significantly (P = 0.031) and more patients carried the Val allele compared with controls (P = 0.025). Genotyping of 162 participants did not reveal the -504Lys allele in ALDH2. These findings suggest that ADH1C*2 is associated with alcohol dependence in the Turkish population displaying a dominant inheritance model. ADH1C*2 allele may contribute to the variance in heritability of alcohol dependence. The ALDH2 -504Lys/Lys or Glu/Lys genotypes were not present in alcohol-dependent patients, similar to that seen in European populations and in contrast to the findings in the Asian populations.

Microsatellite-based fine mapping of the Van der Woude syndrome locus to an interval of 4.1 cM between D1S245 and D1S414

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sander, A.; Schmelzle, R.; Murray, J.C.

1995-01-01

Van der Woude syndrome (VWS) is an autosomal dominant craniofacial disorder characterized by lip pits, clefting of the primary or secondary palate, and hypodontia. The gene has been localized, by RFLP-based linkage studies, to region 1q32-41 between D1S65-REN and D1S65-TGFB2. In this study we report the linkage analysis of 15 VWS families, using 18 microsatellite markers. Multipoint linkage analysis places the gene, with significant odds of 2,344:1, in a 4.1-cM interval flanked by D1S245 and D1S414. Two-point linkage analysis demonstrates close linkage of VWS with D1S205 (lod score [Z] = 24.41 at {theta} = .00) and with D1S491 (Z =more » 21.23 at {theta} = .00). The results revise the previous assignment of the VWS locus and show in an integrated map of the region 1q32-42 that the VWS gene resides more distally than previously suggested. When information about heterozygosity of the closely linked marker D1S491 in the affected members of the VWS family with a microdeletion is taken into account, the VWS critical region can be further narrowed, to the 3.6-cM interval between D1S491 and D1S414. 38 refs., 3 figs., 2 tabs.« less

Direct sequencing of FAH gene in Pakistani tyrosinemia type 1 families reveals a novel mutation.

PubMed

Ijaz, Sadaqat; Zahoor, Muhammad Yasir; Imran, Muhammad; Afzal, Sibtain; Bhinder, Munir A; Ullah, Ihsan; Cheema, Huma Arshad; Ramzan, Khushnooda; Shehzad, Wasim

2016-03-01

Hereditary tyrosinemia type 1 (HT1) is a rare inborn error of tyrosine catabolism with a worldwide prevalence of one out of 100,000 live births. HT1 is clinically characterized by hepatic and renal dysfunction resulting from the deficiency of fumarylacetoacetate hydrolase (FAH) enzyme, caused by recessive mutations in the FAH gene. We present here the first report on identification of FAH mutations in HT1 patients from Pakistan with a novel one. Three Pakistani families, each having one child affected with HT1, were enrolled over a period of 1.5 years. Two of the affected children had died as they were presented late with acute form. All regions of the FAH gene spanning exons and splicing sites were amplified by polymerase chain reaction (PCR) and mutation analysis was carried out by direct sequencing. Results of sequencing were confirmed by restriction fragment length polymorphism (PCR-RFLP) analysis. Three different FAH mutations, one in each family, were found to co-segregate with the disease phenotype. Two of these FAH mutations have been known (c.192G>T and c.1062+5G>A [IVS12+5G>A]), while c.67T>C (p.Ser23Pro) was a novel mutation. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. Most of the HT1 patients die before they present to hospitals in Pakistan, as is indicated by enrollment of only three families in 1.5 years. Most of those with late clinical presentation do not survive due to delayed diagnosis followed by untimely treatment. This tragic condition advocates the establishment of expanded newborn screening program for HT1 within Pakistan.

Influence of backwashing on the microbial community in a biofilm developed on biological activated carbon used in a drinking water treatment plant.

PubMed

Kasuga, I; Shimazaki, D; Kunikane, S

2007-01-01

The influence of backwashing on the biofilm community developed on biological activated carbon (BAC) used in a drinking water treatment plant was investigated by means of bacterial cell enumeration and terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting analysis of bacterial and eukaryotic ribosomal RNA genes (rDNA). After backwashing, the attached bacterial abundance in the top layer of the BAC bed decreased to 64% of that before backwashing. The community level changes caused by backwashing were examined through the T-RFLP profiles. In the bacterial 16S rDNA analysis, the relative abundances of some terminal-restriction fragments (T-RFs) including the Planctomycetes-derived fragment increased; however, the relative abundances of some T-RFs including the Betaproteobacteria-derived fragments decreased. In the eukaryotic 18S rDNA analysis, the relative abundances of some T-RFs including the protozoan Cercozoa-derived fragments increased; however, the relative abundances of some T-RFs including the metazoan Chaetonotus- and Paratripyla-derived fragments decreased. The T-RFLP analysis suggests that backwashing can cause changes in the relative compositions of microorganisms in a BAC biofilm in the top layer of the bed.

Bifidobacterium pseudolongum are efficient indicators of animal fecal contamination in raw milk cheese industry

PubMed Central

2011-01-01

Background The contamination of raw milk cheeses (St-Marcellin and Brie) from two plants in France was studied at several steps of production (raw milk, after addition of rennet - St-Marcellin - or after second maturation - Brie -, after removal from the mold and during ripening) using bifidobacteria as indicators of fecal contamination. Results Bifidobacterium semi-quantitative counts were compared using PCR-RFLP and real-time PCR. B. pseudolongum were detected in 77% (PCR-RFLP; 1.75 to 2.29 log cfu ml-1) and 68% (real-time PCR; 2.19 to 2.73 log cfu ml-1) of St-Marcellin samples and in 87% (PCR-RFLP; 1.17 to 2.40 log cfu ml-1) of Brie cheeses samples. Mean counts of B. pseudolongum remained stable along both processes. Two other populations of bifidobacteria were detected during the ripening stage of St-Marcellin, respectively in 61% and 18% of the samples (PCR-RFLP). The presence of these populations explains the increase in total bifidobacteria observed during ripening. Further characterization of these populations is currently under process. Forty-eight percents (St-Marcellin) and 70% (Brie) of the samples were B. pseudolongum positive/E. coli negative while only 10% (St-Marcellin) and 3% (Brie) were B. pseudolongum negative/E. coli positive. Conclusions The increase of total bifidobacteria during ripening in Marcellin's process does not allow their use as fecal indicator. The presence of B. pseudolongum along the processes defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. B. pseudolongum was more sensitive as an indicator than E. coli along the two different cheese processes. B. pseudolongum should be used as fecal indicator rather than E. coli to assess the quality of raw milk and raw milk cheeses. PMID:21816092

Higher risk of progressing breast cancer in Kurdish population associated to CDH1 -160 C/A polymorphism

PubMed Central

Zarei, Farzaneh; Menbari, Mohammad Nazir; Ghaderi, Bayazid; Abdi, Mohammad; Vahabzadeh, Zakaria

2017-01-01

There is an increasing interest about studying possible effects of genetic polymorphisms and risk of cancer progression. E-cadherin (CDH1) involves in many important cellular processes including cell-cell interactions, cell development and genetic changes of this molecule has been associated with greater tumor metastasis. The present study was aimed to evaluate the possible role of CDH1 -160 C/A polymorphism as a potential risk factor for breast cancer in Kurdish population. This case-control study consisted of 100 breast cancer patients and 200 healthy controls. Clinicopathological findings of all individuals were reported and immunohistochemistry staining was carried out on tissue samples. The CDH1 -160 C/A genotype was determined by polymerase chain reaction- restriction fragment length polymorphism method (PCR-RFLP). CDH1 -160 C/A polymorphism was differently distributed between patient and control groups. The A allele of CDH1 -160 C/A polymorphism significantly increased in patients compared to controls. In addition we found that the A allele of this polymorphism might be a potential risk factor for progression of breast cancer in our studied population. Patients with A allele of CDH1 -160 C/A was in higher risk to progress invasive ductal carcinoma. The A allele was also correlated with high grade and stage IV and also with metastatic tumors in studied subjects. The CDH1 -160 C/A polymorphism is correlated with clinicopathologial findings of breast cancer patients. The A allele of CDH1 -160 C/A may be a risk factor for progression of breast cancer in Kurdish patients. PMID:29285016

Higher risk of progressing breast cancer in Kurdish population associated to CDH1 -160 C/A polymorphism.

PubMed

Zarei, Farzaneh; Menbari, Mohammad Nazir; Ghaderi, Bayazid; Abdi, Mohammad; Vahabzadeh, Zakaria

2017-01-01

There is an increasing interest about studying possible effects of genetic polymorphisms and risk of cancer progression. E-cadherin (CDH1) involves in many important cellular processes including cell-cell interactions, cell development and genetic changes of this molecule has been associated with greater tumor metastasis. The present study was aimed to evaluate the possible role of CDH1 -160 C/A polymorphism as a potential risk factor for breast cancer in Kurdish population. This case-control study consisted of 100 breast cancer patients and 200 healthy controls. Clinicopathological findings of all individuals were reported and immunohistochemistry staining was carried out on tissue samples. The CDH1 -160 C/A genotype was determined by polymerase chain reaction- restriction fragment length polymorphism method (PCR-RFLP). CDH1 -160 C/A polymorphism was differently distributed between patient and control groups. The A allele of CDH1 -160 C/A polymorphism significantly increased in patients compared to controls. In addition we found that the A allele of this polymorphism might be a potential risk factor for progression of breast cancer in our studied population. Patients with A allele of CDH1 -160 C/A was in higher risk to progress invasive ductal carcinoma. The A allele was also correlated with high grade and stage IV and also with metastatic tumors in studied subjects. The CDH1 -160 C/A polymorphism is correlated with clinicopathologial findings of breast cancer patients. The A allele of CDH1 -160 C/A may be a risk factor for progression of breast cancer in Kurdish patients.

Partial isodisomy for maternal chromosome 7 and short stature in an individual with a mutation at the COL1A2 locus.

PubMed

Spotila, L D; Sereda, L; Prockop, D J

1992-12-01

Uniparental disomy for chromosome 7 has been described previously in two individuals with cystic fibrosis. Here, we describe a third case that was discovered because the proband was homozygous for a mutation in the COL1A2 gene for type I procollagen, although his mother was heterozygous and his father did not have the mutation. Phenotypically, the proband was similar to the two previously reported cases with uniparental disomy for chromosome 7, in that he was short in stature and growth retarded. Paternity was assessed with five polymorphic markers. Chromosome 7 inheritance in the proband was analyzed using 12 polymorphic markers distributed along the entire chromosome. Similar analysis of the proband's two brothers established the phase of the alleles at the various loci, assuming minimal recombination. The proband inherited only maternal alleles at five loci and was homozygous at all loci examined, except one. He was heterozygous for an RFLP at the IGBP-1 locus at 7p13-p12. The results suggest that the isodisomy was not complete because of a recombination event involving the proximal short arms of two maternal chromosomes. In addition, the phenotype of proportional dwarfism in the proband suggests imprinting of one or more growth-related genes on chromosome 7.

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe.

PubMed

Moreira, A R; Paolicchi, F; Morsella, C; Zumarraga, M; Cataldi, A; Fabiana, B; Alicia, A; Piet, O; van Soolingen, D; Isabel, R M

1999-12-01

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated 'A', 'B', 'C' and 'E') were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern 'A', was found in 46 isolates (75%). The second, pattern 'E', included 8 isolates (13%), while the third, pattern 'B', included 6 isolates (10%). Pattern 'C' was found for only one isolate. All of the deer isolates were classified as pattern 'A', while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern 'A', was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns 'B', 'C' and 'E', were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.

The role of xenobotic metabolism MGST1 gene polymorphism in colorectal cancer patients.

PubMed

Akil, Fardah; Akil, H A M; Lutfie, A M; Wibowo, Wahyu S; Miskad, Upik; Yusuf, Irawan

2012-10-01

to asses the role of Microsomal Glutathione S-Transferase1 (MGST1) gene as one of enzym metabolism that plays in enviromental factor. using case-control study, subjects with age less than 50 years were collected from teaching hospital Makassar between 2008-2010. Frozen or routinely processed tumour samples biopsy and peripheral blood were obtained from 35 CRC patients undergoing surgery and endoscopic examination with 61 subject as control. CRC cases were diagnosis by clinical examination and confirm by histopathology without familial aggregation of CRC. DNA resequencing was conducted for the 3 kb genomic DNA region MGST1 using PCR-restriction fragment length polymorphism (PCR-RFLP). from 96 subject, two varian single nucleotide polymorphisms (SNPs) 16454T>G and 16416G>A MGST1 were identified. Significant CRC association (p= 0.047) was detected in GG genotipe SNP 16454T>G MGST1 with 3.5 fold risk (95% confidence interval (CI) 0.962-13.191). the results suggest that MGST1 gene polymorphisms as one of environment gene may contribute to CRC risk in younger age (<50 years old).

Programmed death-1 gene polymorphism (PD-1.5 C/T) is associated with gastric cancer.

PubMed

Savabkar, Sanaz; Azimzadeh, Pedram; Chaleshi, Vahid; Nazemalhosseini Mojarad, Ehsan; Aghdaei, Hamid Asadzadeh

2013-01-01

This study aimed to determine the association between PD-1.5C/T (rs2227981, +7785) and the risk of gastric cancer (GC) in an Iranian population. Gastric cancer is the fourth most common cancer in the world. The programmed death 1 (PD-1) is a member of the CD28 super family. PD-1 is a negative regulator of T-cell effector mechanisms which decrease immune responses against cancer. we conducted case- control study to investigate the association of PD-1.5 C/T polymorphism in 122 GC patients and 166 control individuals. DNA was extracted from blood specimens. Genotypes were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The frequency of CC, CT and TT genotypes was 53.6%, 42.2% and 4.2% in control group and 41%, 54.1% and 4.9% in gastric cancer patients respectively. CC genotype was more frequent in control individuals than in patients but we found no statically significant association. The frequencies of PD-1.5CT genotypes were significantly higher in GC patient compared with control individuals (OR= 1.77, 95% CI= 1.077-2.931; P=0.026). Allele distribution was similar in patients and healthy individuals (p = 0.061).Frequency of C and T alleles was 74.7%, 25.3% in control individuals and 68.03% and 31.97% in gastric cancer patients respectively. These results suggest that PD-1.5 C/T polymorphism may affect the GC risk and prognosis in an Iranian population.

The CYP7A1 gene rs3808607 variant is associated with susceptibility of tuberculosis in Moroccan population

PubMed Central

Qrafli, Mounia; Amar, Youssef; Bourkadi, Jamaleddine; Ben Amor, Jouda; Iraki, Ghali; Bakri, Youssef; Amzazi, Saaîd; Lahlou, Ouafae; Seghrouchni, Fouad; El Aouad, Rajae; Sadki, Khalid

2014-01-01

Introduction Despite the medical progress in treatment. Tuberculosis (TB) continues to be a serious global health problem. A genome-wide linkage study identified a major susceptibility locus on chromosomal region 8q12-q13 in Moroccan TB patients. The CYP7A1 gene is located in this region and codes for cholesterol 7a-hydroxylase, an enzyme involved in cholesterol catabolism. Methods We selected three SNPs (rs3808607, rs8192875 and rs8192879) and studied their genotype and allele frequencies distribution in patients with pulmonary (PTB) or pleural TB (pTB), and compared them to Healthy Controls (HC). Genotyping of rs8192875 and rs8192879 SNPs was carried out using the Taq Man SNP genotyping Assay while rs3808607 was investigated by PCR-RFLP. Results We reported here for the first time a statistically significant increase in the AA homozygote genotype frequency of rs3808607 in PTB patients compared to HC (p = 0.02, OR = 1.93, 95% CI: 1.93 (1.07;3.49). The increased risk of developing TB was maintained when we combined the groups of patients (PTB-pTB) (p = 0.01, OR= 1.91, 95% CI = (1.07 - 3.42). In contrast, no genetic association was observed between the rs8192875 or rs8192879 polymorphisms and TB. Conclusion Our investigations suggest that rs3808607 may play a role in susceptibility to TB in a Moroccan population. PMID:25360185

Partial isodisomy for maternal chromosome 7 and short stature in an individual with a mutation at the COL1A2 locus.

PubMed Central

Spotila, L D; Sereda, L; Prockop, D J

1992-01-01

Uniparental disomy for chromosome 7 has been described previously in two individuals with cystic fibrosis. Here, we describe a third case that was discovered because the proband was homozygous for a mutation in the COL1A2 gene for type I procollagen, although his mother was heterozygous and his father did not have the mutation. Phenotypically, the proband was similar to the two previously reported cases with uniparental disomy for chromosome 7, in that he was short in stature and growth retarded. Paternity was assessed with five polymorphic markers. Chromosome 7 inheritance in the proband was analyzed using 12 polymorphic markers distributed along the entire chromosome. Similar analysis of the proband's two brothers established the phase of the alleles at the various loci, assuming minimal recombination. The proband inherited only maternal alleles at five loci and was homozygous at all loci examined, except one. He was heterozygous for an RFLP at the IGBP-1 locus at 7p13-p12. The results suggest that the isodisomy was not complete because of a recombination event involving the proximal short arms of two maternal chromosomes. In addition, the phenotype of proportional dwarfism in the proband suggests imprinting of one or more growth-related genes on chromosome 7. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:1463018

[Analysis for the association between genetic polymorphisms of XRCC1, XPD, XRCC3, CCND1 and the latency of the occupational chronic benzene poisoning].

PubMed

Xu, Jian-ning; Huang, Hui-long; Wang, Quan-kai; Wang, Ya-wen; Yang, Min; Zheng, Yu-xin

2007-03-01

To explore the association between genetic polymorphisms of XRCC1, XPD, XRCC3 and CCND1 and latency of occupational chronic benzene poisoning. 80 patients diagnosed with occupational chronic benzene poisoning were investigated. PCR-RFLP was applied to detect the single nucleotide polymorphisms of C26304T, G27466A, G28152A, G36189A of XRCC1, C22541A, C23591T, A35931C of XPD, C18067T of XRCC3 and G870A of CCND1. Their relationship with the latency of chronic benzene poisoning was analyzed by Kaplan-Meier method. The association of XRCC1 G27466A subgroup with the latency of chronic benzene poisoning was observed, as well as that of CCDN1G870A subgroup. The benzene-exposed workers with XRCC1 27466G/G homozygous wild genotype developed chronic benzene poisoning 6.9 years later than those had homozygous (27466A/A) or heterozygous (27466G/A) mutant alleles. On the other hand, the latency developing chronic benzene poisoning was longer in workers with homozygous (CCND1 870A/A) or heterozygous (CCND1 870G/A) mutant alleles than in those carrying 870G/G homozygous wild genotype (14.9 vs. 8.7 years). The polymorphisms of XRCC1 and CCND1 potentially modify the latency of the chronic benzene poisoning among workers exposed to benzene.

PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant Mycobacterium tuberculosis▿

PubMed Central

Caws, Maxine; Tho, Dau Quang; Duy, Phan Minh; Lan, Nguyen Thi Ngoc; Hoa, Dai Viet; Torok, Mili Estee; Chau, Tran Thi Hong; Van Vinh Chau, Nguyen; Chinh, Nguyen Tran; Farrar, Jeremy

2007-01-01

PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates. PMID:17428939

Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.

PubMed

Fischer, Cristine Dossin Bastos; Ikuta, Nilo; Canal, Cláudio Wageck; Makiejczuk, Aline; Allgayer, Mariangela da Costa; Cardoso, Cristine Hoffmeister; Lehmann, Fernanda Kieling; Fonseca, André Salvador Kazantzi; Lunge, Vagner Ricardo

2013-12-01

Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution - 10(0.7) TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains. Copyright © 2013 Elsevier B.V. All rights reserved.

Vitamin D Receptor Gene Polymorphisms Associated with Childhood Autism

PubMed Central

Cieślińska, Anna; Kostyra, Elżbieta; Chwała, Barbara; Moszyńska-Dumara, Małgorzata; Fiedorowicz, Ewa; Teodorowicz, Małgorzata

2017-01-01

Background: Autism spectrum disorder (ASD) is a group of heterogeneous, behaviorally defined disorders whereby currently no biological markers are common to all affected individuals. A deregulated immune response may be contributing to the etiology of ASD. The active metabolite of vitamin D3 has an immunoregulatory role mediated by binding to the vitamin D receptor (VDR) in monocyte, macrophages, and lymphocytes. The effects of vitamin D and interaction with the VDR may be influenced by polymorphism in the VDR gene. Methods: Genetic association of four different VDR polymorphisms (Apa-I, Bsm-I, Taq-I, Fok-I) associated with susceptibility to the development of autism in children was investigated. Results: We uniquely found an association between the presence of the T allele at position Taq-I and presence of the a allele at position Apa-I of the VDR gene with decreased ASD incidence. There was also an association between female gender and the presence of the T allele. We found no statistical significant correlation between VDR single nucleotide polymorphisms (SNPs) and vitamin D3 concentration in serum of ASD children. Conclusion: Genetic polymorphism in two SNP in VDR may be correlated with development of ASD symptoms by influencing functionality of vitamin D3 metabolism, while vitamin D3 levels were not significantly different between ASD and non-ASD children. PMID:28891930

Relationship between ambient air pollution and DNA damage in Polish mothers and newborns.

PubMed Central

Whyatt, R M; Santella, R M; Jedrychowski, W; Garte, S J; Bell, D A; Ottman, R; Gladek-Yarborough, A; Cosma, G; Young, T L; Cooper, T B; Randall, M C; Manchester, D K; Perera, F P

1998-01-01

Industrialized regions in Poland are characterized by high ambient pollution, including polycyclic aromatic hydrocarbons (PAHs) from coal burning for industry and home heating. In experimental bioassays, certain PAHs are transplacental carcinogens and developmental toxicants. Biologic markers can facilitate evaluation of effects of environmental PAHs on the developing infant. We measured the amount of PAHs bound to DNA (PAH-DNA adducts) in maternal and umbilical white blood cells. The cohort consisted of 70 mothers and newborns from Krakow, Poland, an industrialized city with elevated air pollution. Modulation of adduct levels by genotypes previously linked to risk of lung cancer, specifically glutathione S-transferase MI (GSTM1) and cytochrome P4501A1 (CYP1A1) Msp restriction fragment length polymorphism (RFLP), was also investigated. There was a dose-related increase in maternal and newborn adduct levels with ambient pollution at the women's place of residence among subjects who were not employed away from home (p < or = 0.05). Maternal smoking (active and passive) significantly increased maternal (p < or = 0.01) but not newborn adduct levels. Neither CYP1A1 Msp nor GSTM1 polymorphisms was associated with maternal adducts. However, adducts were significantly higher in newborns heterozygous or homozygous for the CYP1A1 Msp RFLP compared to newborns without the RFLP (p = 0.04). Results indicate that PAH-induced DNA damage in mothers and newborns is increased by ambient air pollution. In the fetus, this damage appears to be enhanced by the CYP1A1 Mspl polymorphism. Images Figure 1 PMID:9646044

Community structure and succession regulation of fungal consortia in the lignocellulose-degrading process on natural biomass.

PubMed

Tian, Baoyu; Wang, Chunxiang; Lv, Ruirui; Zhou, Junxiong; Li, Xin; Zheng, Yi; Jin, Xiangyu; Wang, Mengli; Ye, Yongxia; Huang, Xinyi; Liu, Ping

2014-01-01

The study aims to investigate fungal community structures and dynamic changes in forest soil lignocellulose-degrading process. rRNA gene clone libraries for the samples collected in different stages of lignocellulose degradation process were constructed and analyzed. A total of 26 representative RFLP types were obtained from original soil clone library, including Mucoromycotina (29.5%), unclassified Zygomycetes (33.5%), Ascomycota (32.4%), and Basidiomycota (4.6%). When soil accumulated with natural lignocellulose, 16 RFLP types were identified from 8-day clone library, including Basidiomycota (62.5%), Ascomycota (36.1%), and Fungi incertae sedis (1.4%). After enrichment for 15 days, identified 11 RFLP types were placed in 3 fungal groups: Basidiomycota (86.9%), Ascomycota (11.5%), and Fungi incertae sedis (1.6%). The results showed richer, more diversity and abundance fungal groups in original forest soil. With the degradation of lignocellulose, fungal groups Mucoromycotina and Ascomycota decreased gradually, and wood-rotting fungi Basidiomycota increased and replaced the opportunist fungi to become predominant group. Most of the fungal clones identified in sample were related to the reported lignocellulose-decomposing strains. Understanding of the microbial community structure and dynamic change during natural lignocellulose-degrading process will provide us with an idea and a basis to construct available commercial lignocellulosic enzymes or microbial complex.

IL-1RN and IL-1β Polymorphism and ARV-Associated Hepatotoxicity

PubMed Central

Samani, Dharmesh; Nema, Vijay; Gangakhedkar, R. R.

2018-01-01

The severity of hepatic injury depends upon cytokines. Previous studies associated IL-1RN allele 2 with IL-1β production. Hence, we examined the association of IL-1 RN and IL-1β polymorphisms with ARV-associated hepatotoxicity. Genotyping of IL-1RN (VNTR), IL-1β (-511C/T) polymorphisms was done in 162 HIV-infected patients, 34 with ARV hepatotoxicity, 128 without hepatotoxicity, and 152 healthy controls using PCR and PCR-RFLP method. The haplotypes 1T and 2C enhanced the risk for severe hepatotoxicity (OR = 1.41, P = 0.25; OR = 1.67, P = 0.31). IL-1β-511TT genotype significantly represented among tobacco using HIV-infected individuals compared to nonusers (OR = 3.74, P = 0.05). IL-1β-511TT genotype among alcohol users increased the risk for hepatotoxicity (OR = 1.80, P = 0.90). IL-1β-511CT and -511TT genotypes overrepresented in alcohol using HIV-infected individuals (OR = 2.29, P = 0.27; OR = 2.64, P = 0.19). IL-RN 2/2 and 1/3 genotypes represented higher in nevirapine using hepatotoxicity patients (OR = 1.42, P = 0.64, OR = 8.79, P = 0.09). IL-1β-511CT and -511 TT genotypes among nevirapine users enhanced the risk for severe hepatotoxicity (OR = 4.29, P = 0.20; OR = 1.95, P = 0.56). IL-1β-511CT and -511TT genotypes were overrepresented in combined nevirapine and alcohol using HIV-infected individuals as compared to nevirapine users and alcohol nonusers (OR = 2.56, P = 0.26; OR = 2.84, P = 0.24). IL-1β-511TT genotype with tobacco, alcohol, and nevirapine usage revealed a trend of risk for the development of ARV-associated hepatotoxicity and its severity.

Neer Award 2017: A rapid method for detecting Propionibacterium acnes in surgical biopsy specimens from the shoulder.

PubMed

Holmes, Scott; Pena Diaz, Ana M; Athwal, George S; Faber, Kenneth J; O'Gorman, David B

2017-02-01

Propionibacterium (P) acnes infection of the shoulder after arthroplasty is a common and serious complication. Current detection methods for P acnes involve anaerobic cultures that require prolonged incubation periods (typically 7-14 days). We have developed a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) approach that sensitively and specifically identifies P acnes in tissue specimens within a 24-hour period. Primers were designed to amplify a unique region of the 16S rRNA gene in P acnes that contained a unique HaeIII restriction enzyme site. PCR and RFLP analyses were optimized to detect P acnes DNA in in vitro cultures and in arthroscopic surgical biopsy specimens from patients with P acnes infections. A 564 base-pair PCR amplicon was derived from all of the known P acnes strains. HaeIII digests of the amplicon yielded a restriction fragment pattern that was unique to P acnes. P acnes-specific amplicons were detected in as few as 10 bacterial cells and in clinical biopsy specimens of infected shoulder tissues. This PCR-RFLP assay combines the sensitivity of PCR with the specificity of RFLP mapping to identify P acnes in surgical isolates. The assay is robust and rapid, and a P acnes-positive tissue specimen can be confirmed within 24 hours of sampling, facilitating treatment decision making, targeted antibiotic therapy, and monitoring to minimize implant failure and revision surgery. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Identification of HLA-DP polymorphism with DP alpha and DP beta probes and monoclonal antibodies: correlation with primed lymphocyte typing.

PubMed Central

Bodmer, J; Bodmer, W; Heyes, J; So, A; Tonks, S; Trowsdale, J; Young, J

1987-01-01

Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP beta probes and a DP alpha probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of DPw1, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defined and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies. Images PMID:2885841

Vitamin D receptor gene polymorphism in Egyptian pediatric acute lymphoblastic leukemia correlation with BMD.

PubMed

Tantawy, Maha; Amer, Mahmoud; Raafat, Tarek; Hamdy, Nayera

2016-09-01

We studied the frequencies of the 3' and 5'-end vitamin D receptor (VDR) gene polymorphisms and their correlation with bone mineral density (BMD) in Egyptian pediatric acute lymphoblastic leukemia (ALL) patients receiving calcium and vitamin D supplements. The purpose of this study is to find out the relation between VDR polymorphism and the response to vitamin D intake in pediatric ALL cases who receive corticosteroid therapy which predispose to osteoporosis. This study might shed the light on some genetic variants that are effect the response of individuals to vitamin D therapy. Forty newly diagnosed pediatrics ALL cases were studied. Three SNPs at the 3'-end of the VDR gene (BsmI rs1544410, ApaI rs739837and TaqI rs731236) and two SNPs at the 5'-end (Cdx-2 rs11568820 and GATA rs4516035) were analyzed by Allelic discrimination assay. Of those twenty-six cases with initial BMD data available were further analyzed with regards to the effect of various VDR genotypes/haplotypes on BMD. The genotype frequencies at 3'-end of VDR gene were, TaqI TT 23%, Tt 54% and tt 23%, BsmI bb 19.2%, Bb 65.4% and BB 15.4% and ApaI AA 12%, Aa 27% and aa 61%. The frequencies at the 5'-end were Cdx-2 GG 34.5%, GA 54% and AA 11.5% and GATA AA 8%, AG 50% and GG 42%. Eight and four possible haplotypes were observed at the 3' and 5'-ends of the VDR gene respectively. The Tt genotype was significantly correlated with high BMD as compared to other TaqI genotypes (P = 0.0420). There was a trend towards higher BMD with the genotype Bb as compared to other BsmI genotypes. No statistical significance was found between the other VDR genotypes or haplotypes studied and BMD. This is the first report on VDR gene polymorphisms in Egyptian pediatric ALL patients. The Tt genotype was associated with increased BMD. Our study showed marked genetic heterogeneity in VDR gene in Egyptian pediatric ALL patients.

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

PubMed

Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

2006-05-25

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

Restriction fragment length polymorphism of the major histocompatibility complex of the dog.

PubMed

Sarmiento, U M; Storb, R F

1988-01-01

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3 + Dw4 + D1, Dw8 + D10, D7 + D16 + D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.

Optimized molecular resolution of cross-contamination alerts in clinical mycobacteriology laboratories.

PubMed

Martín, Ana; Herranz, Marta; Lirola, Miguel Martínez; Fernández, Rosa Fernández; Bouza, Emilio; García de Viedma, Darío

2008-02-14

The phenomenon of misdiagnosing tuberculosis (TB) by laboratory cross-contamination when culturing Mycobacterium tuberculosis (MTB) has been widely reported and it has an obvious clinical, therapeutic and social impact. The final confirmation of a cross-contamination event requires the molecular identification of the same MTB strain cultured from both the potential source of the contamination and from the false-positive candidate. The molecular tool usually applied in this context is IS6110-RFLP which takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions. Our purpose in this study is to evaluate a novel PCR-based method, MIRU-VNTR as an alternative to assure a rapid and optimized analysis of cross-contamination alerts. MIRU-VNTR was prospectively compared with IS6110-RFLP for clarifying 19 alerts of false positivity from other laboratories. MIRU-VNTR highly correlated with IS6110-RFLP, reduced the response time by 27 days and clarified six alerts unresolved by RFLP. Additionally, MIRU-VNTR revealed complex situations such as contamination events involving polyclonal isolates and a false-positive case due to the simultaneous cross-contamination from two independent sources. Unlike standard RFLP-based genotyping, MIRU-VNTR i) could help reduce the impact of a false positive diagnosis of TB, ii) increased the number of events that could be solved and iii) revealed the complexity of some cross-contamination events that could not be dissected by IS6110-RFLP.

Lyme disease with facial nerve palsy: rapid diagnosis using a nested polymerase chain reaction-restriction fragment length polymorphism analysis.

PubMed

Hashimoto, Y; Takahashi, H; Kishiyama, K; Sato, Y; Nakao, M; Miyamoto, K; Iizuka, H

1998-02-01

A 64-year-old woman with Lyme disease and manifesting facial nerve palsy had been bitten by a tick on the left frontal scalp 4 weeks previously. Erythema migrans appeared on the left forehead, accompanied by left facial paralysis. Nested polymerase chain reaction-restriction fragment length polymorphism analysis (nested PCR-RFLP) was performed on DNA extracted from a skin biopsy of the erythema on the left forehead. Borrelia flagellin gene DNA was detected and its RFLP pattern indicated that the organism was B. garinii, Five weeks later, B. garinii was isolated by conventional culture from the erythematous skin lesion, but not from the cerebrospinal fluid. After treatment with ceftriaxone intravenously for 10 days and oral administration of minocycline for 7 days, both the erythema and facial nerve palsy improved significantly. Nested PCR and culture taken after the lesion subsided, using skin samples obtained from a site adjacent to the original biopsy, were both negative. We suggest that nested PCR-RFLP analysis might be useful for the rapid diagnosis of Lyme disease and for evaluating therapy.

Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population

PubMed Central

Silva, Sarah Pagliarini- e-; Santos, Bruna Cunha; de Figueiredo Pereira, Elizangela Mendes; Ferreira, Mari Ellen; Baraldi, Elaine Cristina; Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

2013-01-01

OBJECTIVE: The JAK2 46/1 haplotype has recently been described as a major contributing factor to the development of myeloproliferative neoplasm, whether positive or negative for the JAK2 V617F mutation. The G allele, identified by a single-nucleotide polymorphism known as JAK2 rs10974944, is part of the JAK2 46/1 haplotype. The aim of this study was to verify the association between the presence of the G allele and the development of BCR-ABL-negative chronic myeloproliferative neoplasms in our population. METHODS: Blood and oral mucosa swab samples were obtained from 56 patients of two local Brazilian hospitals who had previously been diagnosed with BCR-ABL-negative chronic myeloproliferative neoplasms. Blood samples from 90 local blood donors were used as controls. The presence of the G allele was assessed using a PCR-RFLP assay after extracting DNA from the samples. RESULTS: The presence of the G allele was strongly associated with the presence of BCR-ABL-negative chronic myeloproliferative neoplasms (p = 0.0001; OR = 2.674; 95% CI = 1.630−4.385) in the studied population. CONCLUSION: In agreement with previous reports, the JAK2 46/1 haplotype, represented in this study by the presence of the G allele, is an important predisposing factor in the oncogenetic development of these neoplasms in our population. PMID:23420150

Association of vitamin D binding protein polymorphism with long-term kidney allograft survival in Hispanic kidney transplant recipients.

PubMed

Vu, Don; Sakharkar, Prashant; Tellez-Corrales, Eglis; Shah, Tariq; Hutchinson, Ian; Min, David I

2013-02-01

Polymorphism of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (VDBP), have been widely explored due to the complex role played by vitamin D in renal transplant outcomes. In this study, we investigated whether polymorphisms of genes encoding VDR and VDBP were associated with allograft survival or acute rejection (AR) among a Hispanic kidney transplant population. A total of 502 Hispanic renal allograft recipients at the St. Vincent Medical Center between 2001 and 2010 were genotyped for four different single nucleotide polymorphisms of VDR: FokI C>T (rs2228570), BsmI G>A (rs1544410), ApaI T>G (rs7975232), and TaqI T>C (rs731236). We also performed genotyping for one common polymorphism in the VDBP gene (rs4588). Survival was significantly improved for patients who were homozygous GG for the rs4588 G>T allele in the VDBP gene (GG vs. GT + TT, OR = 0.63, p = 0.02) while GT genotype was associated with a higher risk of graft loss (GT vs. GG + TT, OR = 1.67, p = 0.01). We found no association for polymorphic markers in VDR with allograft survival and AR. The frequency of the haplotype GTCG (in the order of VDR FokI C>T, BsmI G>A, ApaI T>G, and TaqI T>C), was significantly different in the patients with graft rejection compared to the control (p = 0.007) while ACCA haplotype was found to be associated with graft loss (p = 0.02). Hence, the VDBP G>T polymorphism (rs4588) and two haplotypes (GTCG and ACCA) of VDR appear to be associated with renal allograft outcomes among Hispanic allograft recipients.