• Title, Summary, Keyword: PCR-RFLP method

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Identification of Cryptosporidium in Environmental Sample using Nested PCR-RFLP and DNA Sequencing (Nested PCR-RFLP 및 DNA Sequencing을 이용한 환경시료에서의 크립토스포리디움 동정)

  • Park, Sangjung;Jeong, Hyanghee
    • Journal of Korean Society on Water Environment
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    • v.24 no.6
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    • pp.817-822
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    • 2008
  • In order to identify various Cryptosporidium species in environment, nested PCR-RFLP and DNA sequencing method were used. The sensitivity of nested PCR-RFLP based on 18s rRNA gene was shown to 1 oocyst. Therefore, we applied nested PCR-RFLP method to environmental samples. As a result, only 4 samples out of 8 samples confirmed as Cryptosporidium parvum by standard method of Cryptosporidium were identified as Cryptosporidium parvum by nested PCR-RFLP and DNA sequencing method. The rest of 4 samples among 8 samples were identified as Cryptosporidium muris, Cryptosporidium bailey. Therefore, in addition to standard method of Cryptosporidium, supplementary verification through nested PCR-RFLP and DNA sequencing should be needed to give more accurate information about risk of Cryptosporidium.

Development of a Monitoring System for Water-borne Bacteria by a Molecular Technique, PCR-RFLP-sequence Analysis

  • Lee, Ji-Young;Jeong, Eun-Young;Lee, Kyu-sang;Seul-Ju;Kim, Jong-Bae;Kang, Joon-Wun;Lee, Hye-Young
    • Biomedical Science Letters
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    • v.9 no.3
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    • pp.139-144
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    • 2003
  • Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

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Genetic Diagnosis of Beckwith Wiedemann Syndrome using Methylation Specific PCR-RFLP Method (Methylation Specific PCR-RFLP 방법을 이용한 Beckwith Wiedemann Syndrome의 진단)

  • Kim, Gu-Hwan;Lee, Jin-Joo;Choi, Seung-Hoon;Lee, Joo-Yeon;Lee, Beom-Hee;Yoo, Han-Wook
    • Journal of Genetic Medicine
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    • v.7 no.2
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    • pp.133-137
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    • 2010
  • Purpose: Beckwith-Wiedemann syndrome (BWS) is an overgrowth malformation syndrome caused by a methylation abnormality at chromosome 11p15, consisting of two imprinting centers, BWSIC1 (IGF2, H19) and BWSIC2 (LIT1, KvDMR). This study evaluated the applicability of a methylation-specific (MS) PCR RFLP method for the genetic diagnosis of BWS. Materials and Methods: A total of 12 patients were recruited based on clinical findings. Karyotyping was performed using peripheral blood leukocytes, and genomic DNA was treated with bisulfate and amplified using methylation-specific primers. RFLP was conducted with restriction enzymes in differentially methylated regions of LIT1, H19, and IGF2. Results: The 12 BWS patients had normal karyotypes. Abnormal methylation patterns in the BWSIC2 (LIT1) region were identified in seven patients (58.3%) using the MS-PCR RFLP method. Conclusions: The MS-PCR RFLP method is a simple, economical genetic test. It detected genetic abnormalities in 50-60% of BWS patients, suggesting that it can be used as a screening test. A more precise method is required, however, to enhance the detection rate of genetic abnormalities, especially in BWSIC1 region.

Discrimination of Hanwoo from Holstein/black Angus meat by PCR-RFLP of MC1R gene (MC1R 유전자의 PCR-RFLP를 이용한 한우육과 젖소육/black Angus 수입육의 구분)

  • Kim, Tae-Jung;Lee, Jae-Il
    • Korean Journal of Veterinary Research
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    • v.45 no.3
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    • pp.335-339
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    • 2005
  • The melanocortin 1 receptor (MC1R) plays an important role in regulation of melanin pigment synthesis within mammalian melanocytes. Mutations within the gene encoding MC1R have been shown to explain coat color variations within several mammalian species including cattle. To develope a rapid and accurate method for the identification of Hanwoo, we performed a modified PCR-RFLP analysis of MC1R gene using single nucleotide polymorphism (SNP) within MC1R as a target. A size of 538 bp (537 bp for Hanwoo) was amplified by PCR, digested with Hpa II, and electrophoresed on a 1.5% agarose gel. A PCR product from Hanwoo showed a single band of 537 bp, whereas two fragments of 328 bp and 210 bp were detected in both Holstein and Black angus. The current result suggests that the PCR-RFLP using our primers and enzyme digestion system would be very accurate, easy and reproducible method to discriminate between Hanwoo and Holstein/Black angus meat.

Occurrence of canine brucellosis in large kennels and characterization of Brucella canis isolates by PCR-RFLP (집단 개사육농장에서의 Canine Brucellosis 발생 및 PCR-RFLP를 이용한 분리주의 특성조사)

  • Kim, Jong-Wan;Lee, Young-Ju;Tak, Ryun-Bin
    • Korean Journal of Veterinary Research
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    • v.43 no.1
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    • pp.67-75
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    • 2003
  • A total of 260 dogs were randomly selected from two different treed kennels that brucellosis has occurred (group 1, 126 dogs), and random selected breed kennel (group 2, 134 dogs), and monitored for Brucella canis (B. canis) by 2-mercaptoethanol rapid slide agglutination test (2ME-RSAT) and bacterial culture method. For the differentiation, PCR-RFLP using omp-31, wbkA and per genes used for 52 of B canis strains (strain I) isolated in this study and 3 of B. canis strains (strain II) isolated in 1994 in Korea. 2ME-RSAT revealed that 63/126 dogs (50.0%) and 12/134 dogs (9.0%) were positive in group I and group II, respectively. Bacterial culture revealed that 47/126 dogs (37.3%) and 5/134 dogs (3.7%) were positive in group I and group II, respectively. As the results of PCR-RFLP, $\underline{omp}-31$ was amplified from all Brucella spp, except B. abortus. All B. canis isolates showed unique PCR-RFLP pattern following digestion with Bmel8I. However, all Brucella spp. showed the same PCR-RFLP pattern following digestion with SalI. PCR-RFLP analysis of wbkA revealed that all Brucella spp. showed the same pattern following digestion with HindIII. PCR-RFLP analysis of per revealed that B. abortus 544 and B. melitensis 63/9 showed the same pattern, but different from B. suis and B. canis following digestion with HindIII.

Identification of Meat Species Using Species-Specific PCR-RFLP Fingerprint of Mitochondrial 12S rRNA Gene (미토콘드리아 12S rRNA 유전자의 종 특이적 PCR-RFLP Fingerprint를 이용한 식육 원료의 판별)

  • Park, Jong-Keun;Shin, Ki-Hyun;Shin, Sung-Chul;Chung, Ku-Young;Chung, Eui-Ryong
    • Food Science of Animal Resources
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    • v.27 no.2
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    • pp.209-215
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    • 2007
  • In order to develop a sensitive and reliable method for the species-specific molecular markers, PCR-RFLP assay of the mitochondrial DNA(mt DNA) 12S rRNA gene was exploited for the identification of the origin of animal meat species including cattle, pig, sheep, goat, horse, deer, chicken, duck and turkey. A specific primer pairs were designed, based on the nucleotide sequences of mt 12S rRNA gene, for the amplification of the highly conserved region in the gene of the animal species using PCR-RFLP technique. mt DNA was isolated from meat samples followed by DNA amplification using PCR with the specific primers. PCR amplification produced an approximately 455 bp fragment in each of these animal meats. To distinguish pleat species, the PCR amplicons were digested with restriction endonucleases Tsp5091 and MboI, respectively, which generates distinct RFLP profiles. The DNA profiles digested with Tsp5091 allowed the clear discrimination in the mammalian meat species and the DNA profiles digested with MboI in poultry meat species. Therefore, the PCR-RFLP profiles of mt 12S rRNA gene could be very useful to identify the origin of the raw materials in the raw meats as well as the processed meat products.

Analysis of the spike glycoprotein gene and nonstructural protein gene of transmissible gastroenteritis virus using PCR and RFLP analysis (PCR과 RFLP분석을 이용한 transmissible gastroenteritis virus의 spike glycoprotein gene과 nonstructural protein gene의 분석)

  • Kwon, Hyuk-moo
    • Korean Journal of Veterinary Research
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    • v.36 no.3
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    • pp.627-633
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    • 1996
  • To analyze the genomic diversity of transmissible gastroenteritis virus (TGEV), the N-terminal half of the spike (S) glycoprotein gene and nonstructural protein gene (open reading frames 3 and 3-1) were amplified by reverse transcriptase reaction and polymerase chain reaction (RT-PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. In this study, TGEV Miller (M6) and Purdue (P115) strains were used as reference strains, and two vaccine strains (MSV and STC3) and four Korea isolates (P44, VRI-WP, VRI-41, and VRI-48) were analyzed. All TGEV strains were amplified with three TGEV primer pairs. Although there was some exception in RFLP analysis, this method differentiated TGEV strains into following groups : Miller group (M6 and MSV), Purdue group (PUS, STC3, P44, VRI-WP, VRI-41, and VRI-48). Using Sau3AI and SspI, VRI-48 was differentiated from the Miller and Purdue type viruses. The RT/PCR in conjuction with RFLP analysis was a rapid and valuable tool for differentiating several strains of TGEV. This study revealed the occurences of distinct difference in genome of TGEV strains.

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Identification of Mycobacterium species by rpoB Gene PCR-RFLP (rpoB 유전자의 PCR-RFLP를 이용한 Mycobacterium 균종 동정의 유용성)

  • Yu, Kyong-Nae;Park, Chung-Ho
    • Korean Journal of Clinical Laboratory Science
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    • v.38 no.3
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    • pp.158-165
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    • 2006
  • Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

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RFLP Analysis of cry1 and cry2 Genes of Bacillus thuringiensis Isolates from India

  • Patel, Ketan D.;Ingle, Sanjay S.
    • Journal of Microbiology and Biotechnology
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    • v.22 no.6
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    • pp.729-735
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    • 2012
  • The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

Analysis of Molecular Epidemiological Properties of Staphylococcus aureus Isolates from Domestic Animals and Human Patients by PCR (Polymerase Chain Reaction을 활용한 국내 동물과 사람환자에서 분리한 Staphylococcus aureus 분리주의 분자역학적 특성분석)

  • Woo Yong-Ku;Kim Shin
    • Korean Journal of Microbiology
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    • v.41 no.1
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    • pp.24-37
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    • 2005
  • This study was conducted to analyze the molecular epidemiological properties and to select the most efficient and reliable PCR method on 116 of Staphylococcus aureus (S. aureus) isolates from Korean cattle, black goat, pig, dog, chicken, mouse and also human clinical cases from hospital. The distribution patterns of SSG [species specific genes; coagulase (coa), protein A (spa), nuclease (nuc) and aroA (RsaI) gene] were analyzed by PCR method. Among the SSGs, the nuc-gene was found in all strains $(100\%)$ tested and followed by coa-gene $(87.9\%)$, spa-gene $(91.4\%)$ and aroA-gene $(26.7\%)$, in order. The genetic subtyping by RFLP method was performed on the coa [AluI] and aroA-gene [RsaI] PCR products. The mecA-gene PCR and PCR-RFLP techniques were chosen to detect and verify of MRSA strains. Only the human strains $(12.1\%)$ were detected the positive mecA-gene products (533 bp), which were divided into two specific bands [201 & 332 bp] by HhaI enzyme digestion. On coa-gene and spa-gene typing, coa-gene was typed with ten kinds of genotype and coa-3 type were determined as the most predominant genotype, while spa-gene was divided into eleven kinds of genotype and also spa-7 type were selected the most prevalent genotype based on their genetic variations. On the aroA and coa-gene subtyping by PCR-RFLP, aroA-gene products were discriminated with only seven types of genotype, while coa-gene products were further divided into an eleven genotype, respectively. In comparison of SID values of five PCR based typing methods, the coa-PCR-RFLP (SID0.894) was evaluated the most efficient and reliable tools and followed by coa-PCR (SID0.883) and aroA-PCR-RFLP (SID0.462), in order. In conclusion, we could determined that the coa-PCR-RFLP method was the most suitable genetic analysis tool for S. aureus and MRSA strains from domestic animals and humans.