• Title/Summary/Keyword: PCR-restriction fragment length polymorphism (PCR-RFLP)

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Tissue Distribution, SNP Detection and Association Study with Immune Traits of Porcine LBP and CD14 Genes

  • Liu, H.Z.;Li, X.Y.;Liu, B.;Yu, M.;Ma, Y.H.;Chu, M.X.;Li, K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.8
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    • pp.1080-1087
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    • 2008
  • Lipopolysaccharide binding protein (LBP) and CD14 protein play important roles in the defense against infection of Gram-negative bacteria. In the present study, tissue distribution and polymorphism of porcine LBP and CD14 genes were analyzed. Real-time PCR results showed that the porcine LBP gene was especially highly expressed in liver, while CD14 gene was highly expressed in liver and spleen tissues. A 1,732 bp cDNA fragment of porcine LBP gene and a 1,682 bp genomic DNA fragment of CD14 gene were isolated. Polymorphisms were identified in these two fragments and showed that there were 14 potential SNPs in the porcine LBP gene and 3 potential SNPs in the porcine CD14 gene. Three SNPs, 292G/A (Gly/Ser), 1168G/A (Ala/Thr) of the LBP gene and -61G/A of the CD14 gene, were genotyped using restriction fragment length polymorphism (RFLP) method. Association analyses indicated that polymorphism of the 292G/A locus was significantly associated with porcine immune traits hematocrit (HCT), IgG and delayed-type hypersensitivity (DTH) (p<0.01), and the 1168G/A locus was significantly associated with HCT and mean corpuscular volume (MCV) traits (p<0.05). No significant association was found between the -61G/A locus and immune traits of the pig. Our data indicated that the LBP gene was significantly associated with immune traits of pig. Also, we identified some SNPs which may be useful markers for disease-resistant breeding of pigs.

Allelic Characterization of IGF2 and H19 Gene Polymorphisms in Molar Tissues

  • Piyamongkol, Wirawit;Suprasert, Prapaporn
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.9
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    • pp.4405-4408
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    • 2016
  • Background: To investigate the characteristics of allelic distribution of IGF2 and H19 gene polymorphisms in molar tissues compared to normal placentas. Materials and Methods: Forty-nine specimens of molar tissues as well as 100 control normal placental tissues, delivered on the same days, were collected. Polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) on 2% agarose gel electrophoresis was conducted to determine the allelic distribution. The ApaI polymorphism within exon 9 of IGF2 and the RsaI polymorphism within exon 5 of H19 were employed to identify the allelic distribution of the IGF2 and H19 genes, respectively. Then the data for these genes in the molar and normal placenta tissues were compared. Results: The allelic distribution of IGF2 genes found in molar tissue were 21 (42.9%) aa (undigested), 10 (20.4%) ab (heterozygous) and 18 (36.7%) bb (digested), while in normal placenta tissue the values were 22 (22%) aa, 51 (51%) ab, and 27 (27%) bb. The allelic distribution of H19 in molar tissues was 8 (16.2%) aa (undigested), 8 (16.3%) ab (heterozygous) and 33 (67.4%) bb (digested) and in normal placental tissue was 16 (16%) aa, 36 (36%) ab and 48 (48%) bb in normal placenta tissue. These results were significantly different with P values of 0.001 and 0.037 for the allelic distribution of IGF2 and H19, respectively. Conclusions: Molar tissues showed significant differences of allelic distribution of IGF2 and H19 from normal placenta tissues.

Diagnosis of Pigs Producing PSE Meat using DNA Analysis (DNA검사기법을 이용한 PSE 돈육 생산 돼지 진단)

  • Chung Eui-Ryong;Chung Ku-Young
    • Food Science of Animal Resources
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    • v.24 no.4
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    • pp.349-354
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    • 2004
  • Stress-susceptible pigs have been known as the porcine stress syndrome (PSS), swine PSS, also known as malignant hyperthermia (MH), is characterized as sudden death and production of poor meat quality such as PSE (pale, soft and exudative) meat after slaughtering. PSS and PSE meat cause major economic losses in the pig industry. A point mutation in the gene coding for the ryanodine receptor (RYR1) in porcine skeletal muscle, also known calcium (Ca$^{2+}$) release channel, has been associated with swine PSS and halothane sensitivity. We used the PCR-RFLP(restriction fragment length polymorphism) and PCR-SSCP (single strand conformation polymorphism) methods to detect the PSS gene mutation (C1843T) in the RYR1 gene and to estimate genotype frequencies of PSS gene in Korean pig breed populations. In PCR-RFLP and SSCP analyses, three genotypes of homozygous normal (N/M), heterozygous carrier (N/n) and homozygous recessive mutant (n/n) were detected using agarose or polyacrylamide gel electrophoresis, respectively. The proportions of normal, carrier and PSS pigs were 57.1, 35.7 and 7.1% for Landrace, 82.5, 15.8 and 1.7% far L. Yorkshire, 95.2, 4.8 and 0.0% for Duroc and 72.0, 22.7 and 5.3% for Crossbreed. Consequently, DNA-based diagnosis for the identification of stress-susceptible pigs of PSS and pigs producing PSE meat is a powerful technique. Especially, PCR-SSCP method may be useful as a rapid, sensitive and inexpensive test for the large-scale screening of PSS genotypes and pigs with PSE meat in the pork industry.y.

Effects of Heart Fatty Acid-binding Protein Genotype on Intramuscular Fat Content in Duroc Pigs Selected for Meat Production and Meat Quality Traits

  • Uemoto, Yoshinobu;Suzuki, Keiichi;Kobayashi, Eiji;Sato, Syushi;Shibata, Tomoya;Kadowaki, Hiroshi;Nishida, Akira
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.5
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    • pp.622-626
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    • 2007
  • Using multi-trait animal model BLUP, selection was conducted over seven generations for growth rate (DG), real-time ultrasound loin-eye muscle area (LEA), backfat thickness (BF), and intramuscular fat content (IMF) to develop a new line of purebred Duroc pigs with enhanced meat production and meat quality. This study was intended to investigate the relationship between restriction fragment length polymorphism (RFLP) of a heart fatty acid-binding protein (H-FABP) gene and intramuscular fat content (IMF) of this Duroc purebred population. The present experiment examined the RFLP of 499 slaughtered pigs. The DNA was separated from the blood or ear tissue of the pigs, which were slaughtered at 105 kg of body weight. Intramuscular fat content of the longissimus muscle was measured using chemical analysis. A significant difference was detected in the breeding value of IMF among the H-FABP PCR RFLP genotypes. The AA genotype has a significantly larger positive effect on the IMF breeding value than do the Aa and aa genotypes for the MspI RFLP. In addition, the DD genotype has a significantly greater positive effect on IMF breeding value than the Dd and dd genotypes for the HaeIII RFLP. For the HinfI RFLP, the hh genotype has a significantly larger positive effect on IMF breeding value than the HH genotype. Multiple regression analysis was performed using the IMF breeding values as the dependent variable and the three H-FABP genotypes as independent variables. Results revealed that the contribution of the genotypes to variation in IMF breeding values was approximately 40%. These results demonstrated that H-FABP RFLPs affect IMF in this Duroc population.

MUTYH Association with Esophageal Adenocarcinoma in a Han Chinese Population

  • Kong, Feng;Han, Xue-Ying;Luan, Yun;Qi, Tong-Gang;Sun, Chao;Wang, Jue;Hou, Hua-Ying;Jiang, Yu-Hua;Zhao, Jing-Jie;Cheng, Guang-Hui
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.11
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    • pp.6411-6413
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    • 2013
  • Adenocarcinoma of esophagus (AE) is a complex disease, affected by a variety of genetic and environmental factors. Much evidence has shown that the MutY glycosylase homologue (MUTYH) plays a key role in the pathogenesis of many cancers. However, there have been no reports on influence on AE in the Han Chinese population. The objective of this study was to investigate this issue. A gene-based association study was conducted using three single nucleotide polymorphisms(SNPs) reported in previous studies. The three SNPs (rs3219463, rs3219472, rs3219489) were genotyped in 207 unrelated AE patients and 249 healthy controls in a case-control study using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The results revealed that the genotype distribution of rs3219472 differed between the case and control groups (OR=1.66,95%CI=1.11-2.48, P=0.012), indicating that an association may exist between MUTYH and AE. These findings support a signifcant role for MUTYH in AE pathogenesis in the Han Chinese population.

Genetic identification of anisakid nematodes isolated from largehead hairtail (Trichiurus japonicus) in Korea

  • Kim, Jeong-Ho;Nam, Woo-Hwa;Jeon, Chan-Hyeok
    • Fisheries and Aquatic Sciences
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    • v.19 no.5
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    • pp.26.1-26.8
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    • 2016
  • Background: The nematode species belonging to genus Anisakis occur at their third larval stage in numerous marine teleost fish species worldwide and known to cause accidental human infection through the ingestion of raw or undercooked fish or squids. They may also draw the attention of consumers because of the visual impact of both alive and dead worms. Therefore, the information on their geographical distribution and clear species identification is important for epidemiological survey and further prevention of human infection. Results: For identification of anisakid nematodes species isolated from largehead hairtail (Trichiurus japonicus), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of internal transcribed spacers of ribosomal DNA were conducted. Mitochondrial cytochrome c oxidase subunit 2 gene was also sequenced, and phylogenetic analysis was conducted. From the largehead hairtail (n = 9), 1259 nematodes were isolated in total. Most of the nematodes were found encapsulated throughout the viscera (56.2 %, 708/1259) or moving freely in the body cavity (41.5 %, 523/1259), and only 0.3 % (4/1259) was found in the muscles. By PCR-RFLP, three different nematode species were identified. Anisakis pegreffii was the most dominantly found (98.7 %, 1243/1259) from the largehead hairtail, occupying 98.7 % (699/708) of the nematodes in the mesenteries and 98.1 % (513/523) in the body cavity. Hybrid genotype (Anisakis simplex ${\times}$ A. pegreffii) occupied 0.5 %, and Hysterothylacium sp. occupied 0.2 % of the nematodes isolated in this study. Conclusions: The largehead hairtail may not significantly contribute accidental human infection of anisakid nematode third stage larvae because most of the nematodes were found from the viscera or body cavity, which are not consumed raw. But, a high prevalence of anisakid nematode larvae in the largehead hairtail is still in concern because they may raise food safety problems to consumers. Immediate evisceration or freezing of fish after catch will be necessary before consumption.

Stem Rot on Adzuki Bean (Vigna angularis) Caused by Rhizoctonia solani AG 4 HGI in China

  • Sun, Suli;Xia, Changjian;Zhang, Jiqing;Duan, Canxing;Wang, Xiaoming;Wu, Xiaofei;Lee, Suk-Ha;Zhu, Zhendong
    • The Plant Pathology Journal
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    • v.31 no.1
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    • pp.67-71
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    • 2015
  • During late August and early September 2011, stem rot symptoms were observed on adzuki bean plants (Vigna angularis) growing in fields located in Beijing and Hebei Province, China, respectively. In this study, four isolates were obtained from infected stems of adzuki bean plants. Based on their morphology, and sequence and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of the ribosomal DNA internal transcribed spacers (rDNA-ITS) region, the four isolates were identified as Rhizoctonia solani in anastomosis group (AG) 4 HGI. Pathogenicity tests showed that all isolates were strongly pathogenic to adzuki bean and resulted in serious wilt symptoms which was similar to observations in the fields. Additionally, the isolates infected several other crops and induced related rot on the roots and basal stems. To our knowledge, this is the first report of Rhizoctonia solani AG 4 HGI causing stem rot on adzuki bean.

Molecular Identification of Thrips in Two Medicinal Crops, Cnidium officinale Makino and Ligusticum chuanxiong Hort (일천궁과 토천궁에서 발생하는 총채벌레류의 분자동정)

  • Jung, Chung Ryul;Jeong, Dae Hui;Park, Hong Woo;Kim, Hyun Jun;Jeon, Kwon Seok;Yoon, Jung Beom
    • Korean Journal of Medicinal Crop Science
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    • v.27 no.1
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    • pp.17-23
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    • 2019
  • Background: Cnidium officinale Makino and Ligusticum chuanxiong Hort. are important medicinal crops in Korea. However, there is insufficient information on the identification of thrips, which attack these plants. Until now, one species of thrips has been recorded as a main pest. Methods and Results: To identify the thrips emerging in C. officinale Makino and L. chuanxiong Hort., these plants were independently cultivated in two local areas. Thirty individuals of each plant species were selected randomly and surveyed for the presence of thrips. After confirming the existence of thrips, 100 thrips individuals were collected from each crop using the beating method. To identify thrips species, we performed PCR-restriction fragment length polymorphism (RFLP)-based analysis using ITS2 primer sets. Six thrips species were identified: western flower thrips (Frankliniella occidentalis), flower thrips (F. intonsa), onion thrips (Thrips tabaci), chrysanthemum thrips (T. nigropilosus), chilli thrips (Scirtothrips dorsalis), and grass thrips (Anaphothrips obscurus). The proportion of these species differed between the host plant species. Conclusions: Six thrips species were major pests of two medicinal crops. Integrated pest management is required to control these thrips species, and will enhance the yield and quality of C. officinale and L. chuanxiong.

Lack of Association between the MiR146a Polymorphism and Susceptibility to Thai Childhood Acute Lymphoblastic Leukemia

  • Chansing, Kochpinchon;Pakakasama, Samart;Hongeng, Suradej;Thongmee, Acharawan;Pongstaporn, Wanida
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.5
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    • pp.2435-2438
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    • 2016
  • Background: MiRNAs, small non coding RNAs, play a role in the regulation of hematopoiesis, with effects on cell growth, differentiation, and apoptosis. In addition, MiRNAs are thought to play an important role in tumorigenesis. The miR146a G>C polymorphism can lead to alteration of miR146 expression, which appears to be associated with development and progression of several cancers. This study aimed to investigate the association of the miRNA146a (rs2910164) G>C polymorphism and susceptibility to childhood acute lymphoblastic leukemia (ALL) and clinical outcomes. Materials and Methods: Totals of 100 childhood ALL patients and 200 healthy children were studied for miR146a polymorphisms using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). Results: The frequency of the miR146a G allele in controls was 0.40 compared with 0.38 in ALL patients. There was no association between miRNA146a (rs2910164) G>C polymorphism and susceptibility to childhood ALL (OR=1.484, 95%CI=0.712-3.093, p=0.290). Moreover, the frequencies of miR146a (rs2910164) G>C polymorphism were not associated with demographic data and clinical outcomes in ALL cases. Conclusions: The miRNA146a polymorphism was not significantly associated with susceptibility to Thai childhood ALL or any clinico-pathological variables.

CYP1A1 Genetic Polymorphisms and Risk for Esophageal Cancer: a Case-control Study in Central China

  • Yun, Yu-Xia;Wang, Yan-Ping;Wang, Peng;Cui, Li-Hong;Wang, Kai-Juan;Zhang, Jian-Ying;Dai, Li-Ping
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.11
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    • pp.6507-6512
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    • 2013
  • The purpose of this study was to evaluate the associations of CYP1A1 genetic polymorphisms with the risk of developing esophageal cancer (EC). A case-control study was carried out in a Chinese population in which 157 hospital based EC cases and 157 population based healthy controls with 1:1 match by age and sex were included. PCR based restriction fragment length polymorphisms (PCR-RFLP) were used to detect genotypes in case and control groups. For the CYP1A1 Ile/Val polymorphism, comparing with wild genotype Ile/Ile, both the heterozygote genotype Ile/Val and the combined variant genotype Ile/Val+Val/Val increased the risk of esophageal cancer (OR: 2.05, 95%CI: 1.19-3.54, OR: 1.86, 95%CI: 1.11-3.12). No significant association was found between the CYP1A1 MspI polymorphism and EC. According to analysis of combined genotypes, the TC/AG combined genotype which contained both variant alleles of these two polymorphisms increased the risk of developing EC (OR: 2.12, 95%CI: 1.16-3.85). Our results suggested that genetic polymorphisms of CYP1A1 may increase the susceptibility to EC.