• 한국양봉학회지
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• 제33권4호
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• pp.227-237
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• 2018

The majority parasitic bee mites of Thailand in genus Tropilaelaps are infesting colonies of native bees (Apis dorsata) and introduced bees (A. mellifera). The investigation aims to study morphological and genetic variation of Tropilaelaps mites infected different hosts. Adult mites were collected from honey bee brood throughout Thailand. Traditional and geometrical morphometrics were measured on photograph by using TPS program. Additional, COI gene variations were examined by PCR-RFLP and nucleotides sequencing. Tree of mites relationships were constructed by NJ and MP assumptions. Morphometric results indicated T. mercedesae were major species infesting on A. dorsata and A. mellifera. Mophological variation represented at anal and epigynial plate, which the shape of the anal plate apex margin has been key character to identify between T. mercedesae (bell to blunt shape) and T. koenigerum (pear shape). However, the discriminant analysis suggested that geometric results were potential to classify Thai Tropilaelaps populations from different hosts better than traditional morphometric. Otherwise, PCR-RFLP clearly detected the site of Dra I and Xba I digestion of Thai Tropilaelaps morphotypes. The COI sequences of T. koenigerum were founded infesting only A. dorsata in Thailand and four sequences that related to the Thai T. mercedesae morphotypes. The NJ and MP tree were clearly classified Thai Tropilaelaps species which were suggested both from morphological and molecular analysis. This information might be basically of taxonomic status, but this should have implication for controlling these mites in Thailand and other countries.

• The Plant Pathology Journal
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• 제25권4호
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• pp.322-327
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• 2009

A total of 23 isolates of Botrytis cinerea causing the grey mold were collected from infected ginseng in several fields of Korea. The sensitivity to carbendazim and the mixture of carbendazim plus diethofencarb was determined through a mycelial inhibition test on PDA amended with or without fungicides. B. cinerea isolates were classified as 3 phenotypes, which were the first phenotype resistant to both of carbendazim and the mixture ($Car^RMix^R$), the second one resistant to carbendazim and sensitive to the mixture ($Car^RMix^S$), and the last one sensitive to both of them ($Car^RMix^S$). Carbendazim resistance correlated with a single mutation $\beta$-tubulin gene of B. cinerea amplified with primer pair tubkjhL and tubkjhR causing a change of glutamate to alanine at amino acid position 198. Furthermore, the substitution of valine for glutamate led the resistance to carbendazim and the mixture at the same position of amino acid. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis using the restriction endonuclease, Tsp451 and BstUI allowed differentiation of the PCR fragment of $\beta$-tubulin gene of $Car^SMix^S$ isolates from that of $Car^RMix^R$ and $Car^RMix^S$ isolates. This method will aid in a fast detection of resistance of carbendazim and the mixture of carbendazim plus diethofencarb in B. cinerea in ginseng field.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2003년도 추계학술발표대회 논문요약집
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• pp.134-135
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• 2003

The geographic expansion of the toxic dinoflagellates genus Alexandrium has been shown to be world wide ranging. The members of the genus Alexandrium ocnstituted of 20-30 species did not show substantial differences in their morphology, which is mostly referred in the 'tamarensis species complex', except some species. Though rDNA sequences variations are very few and pseudogene types are so diverse that it is difficult to use them as the specific markers. In this study, we outlined Korean and Japanese A, tamarense and A. catenella regional isolates by phylogenetic analysis inferred from no cutting alignments of LSU rDNA D1-D2 and SSU rDNA sequences to group these regional isolates. The results were compared to RFLP patterns of PCR products targeted chloroplast DNA. Lastly screening of highly repeated microsatellite DNA which is frequently used for population analysis in eukaryotes was conducted. A. catenella regional strains identified by the sequencing of rDNA D1-D2 domain were divided into at least 3 groups of type E, CMC and Chinese type, divergence root may not be deep comparing with that of A. tamarense whose pseudogenes are very variable. Results of RFLP pattern and the phylogeny of the unknown gene targeting chloroplast showed that Korean and Japanese A. catenella regional isolates were divided into 3 types: Korean, Japanese and the third CMC types. Population-specific PCR amplification with Japanese A. catenella type-specific PCR primers was useful method for population analysis of A. catenella. Various types of satellite sequences such as 5 nucleotides repeats were obtained from A. tamarense and A. catenella. The 5 nucleotides repeats were primed at the both 3'and 5' ends, and these repeats were prominent as longer repeated motifs. This repeated DNA was intercalated as internal sequences containing various types subrepeats. It is expected that these satellite DNA would be a useful molecular population marker through detail comparison among Alexandrium regional isolates to trace their transferring pathway and to prevent their human-associated their regional extents.

• Parasites, Hosts and Diseases
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• 제55권6호
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• pp.601-606
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• 2017

Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures ($T_ms$) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified $T_ms$ at $85.8^{\circ}C$ and $88.6^{\circ}C$, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using $qPCR-T_ms$ analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

• Asian-Australasian Journal of Animal Sciences
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• 제27권10호
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• pp.1487-1492
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• 2014

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.

• 대한수의학회지
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• 제39권4호
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• pp.778-785
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• 1999

To detect CDV RNA in clinical samples and differentiate prevailing CDV virulent strains affecting susceptible animals from attenuated vaccine strains, we performed RT-PCR, RFLP, and sequencing. CDV specific primers were generated from the middle part of nucleocapsid gene. The expected size of PCR products, 519 bp, was observed in tissues of Jindo dog, poodle dog, badger, fourteen of nineteen blood samples as well as 5 vaccine strains including domestic and imported products. The PCR products obtained from tissues and PBMCs of infected animals were digested to 317- and 202-bp fragments by Bam HI, but the products obtained from four of five vaccine strains and Lederle strain were not digesed by Bam HI. Only one vaccine strain of which the PCR products were digested by Bam HI was confirmed as imported vaccine, modified Synider Hill strain. Based on seqencing data obtained from the 519-bp products, it was confirmed that Bam HI restriction site tends to be conserved in field isolates compared to the commercially available attenuated vaccine strains. Partial nucleotide sequences of CDV NP gene obtained from tissues of Jindo dog, poodle and badger shared 100% homology each other, whereas the nucleotide sequences showed 96.3, 96.5, 93.6 and 93.4% homology with Yanaka (virulent), Han95 (virulent), Lederle (attenuated) and Onderstepoort (attenuated) strain, respectively.

• 대한임상검사과학회지
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• 제45권2호
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• pp.66-72
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• 2013

Fine Needle Aspiration Biopsy Cytology (FNABC), which is known as the most accurate and cost-effective method for diagnosis of the thyroid nodule, may still result in indeterminate cases that are cellular paucity and show minor nuclear atypia. However, most cases are associated with suspicion of papillary thyroid carcinoma (PTC). A B-type Raf kinase (BRAF) mutation was found in about half of PTCs which is currently helping us to differentiate malignancies from benign lesions. Cases studied included 46 histological, confirmed PTC cases. FNABC 102 cell paucity and 74 atypia benign cases were previously diagnosed as suspicious of PTC using cytologic examination. These cases were analyzed for BRAF mutation by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with a new restriction enzyme. In this study, the sensitivity and specificity were calculated and, BRAF mutation was detected by means of a histological method in 23 of 46 cases of PTC and no mutation was found in 22 cases. However, one case was not detected. In using FNABC, BRAF mutation was detected in 6 of 102 cases in cell paucity and in 11 of 74 cases in the atypia. Two cases were not detected in the atypia. The sensitivity and specificity of PCR-RFLP in FNABC were 60% and 97.4% respectively. Assessment of Formalin Fixed Paraffin Embedding (FFPE) block demonstrated similarly a 51.1% positive and 48.9% negative in PTC. Evaluation of BRAF mutation revealed high specificity and low sensitivity in using FNABC method. This study suggests that BRAF mutation analysis should be useful for the clinical diagnosis of PTC in FNABC with cytological findings suspicious for PTC.

• 한국물환경학회지
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• 제24권1호
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• pp.19-29
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• 2008

Hot air sparging is a groundwater remediation technique, in which organic contaminants volatilized into hot air from the saturated to vadose zone. In the laboratory diesel (10,000 mg TPH/kg) was spiked in contaminated saturated aquifer soil. The hot air ($34.9{\pm}2.7^{\circ}C$) was injected in intermittent (Q=1,500 mL/min, 10 minute injection and 10 minute idle) modes. We performed microcosm tests using the groundwater samples to assess TPH reductive remediation activity. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for hot air sparging experiment with sludge soil samples that were closely related to Bacillus (149 bp, Firmicutes), Methlobacterium (149 bp, Euryarchaeotes), Pseudomonas (492 bp, ${\gamma}$-Proteobacteria), etc., in the clone library. In this study we find that TPH-water was reduced to 78.9% of the initial value in this experiment aquifer. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil fate of microorganisms in natural microbial community.

• Journal of Animal Science and Technology
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• 제44권6호
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• pp.653-660
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• 2002

소의 흰 반점 관련 후보유전자로 c-KIT receptor 유전자를 선정하여, c-KIT receptor 유전자내의 변이를 탐색하고 변이가 흰반점 표현형과 연관성이 있는지를 분석하였다. 한우, Angus, Brown Swiss, Charolais, Hereford, Hols- tein, Limousin 및 Simmental 등 8개 품종의 DNA 시료를 사용하여 c-KIT receptor 유전자의 intron 6번 영역에서 다형성을 조사하고 분석하였다. c-KIT receptor 유전자의 intron 6번 영역에서는 4개의 염기치환이 발견되어, MspⅠ, BsrBⅠ 및 NdeⅠ 제한효소를 이용하여 PCR- RFLP 분석을 실시하였다. Intron 6번을 포함하는 영역의 PCR 산물 크기는 2,440 bp 이었다. MspⅠ다형성은 PCR-RFLP 분석 결과 3개의 대립유전자가 존재하였으며, 한우품종에서는 3개의 대립유전자 모두가 발견되었고, CC 형태의 유전자형을 제외한 5개의 유전자형 (AA, AB, AC, BC 및 BB)을 확인하였다. Angus, Brown Swiss, Hereford, Holstein 및 Simmental 품종에서는 A 대립유전자만을 갖는 것으로 조사되었고, 한우는 44%만 AA 유전자형을 나타내었다. BsrBⅠ 다형성은 2개의 대립유전자로서 3개의 유전자형이 나타나는 것을 확인하였으며, Charo- lais 및 Hereford 품종이 다른 소 품종에 비하여 A 대립유전자의 빈도가 높게 나타났다. NdeⅠ다형성을 분석한 결과 Brown Swiss 품종에서는 NdeⅠ에 의해 절단되는 형태인 A 대립유전자만 관찰되었으며, Holstein 품종은 92%, Simmental 품종은 72%가 절단되는 형태를 나타내어, 모색이 흰색을 띠는 소 품종에서 절단되는 형태가 많았다. 소 c-KIT receptor 유전자의 intron 6번 영역에서 확인된 4개의 염기치환은 품종에 따라 다른 빈도를 보였으나, 이들 염기치환과 흰 반점과의 연관성에 대한 증거는 발견하지 못하였다. 그러므로 소의 흰 반점과 c-KIT receptor 유전자 내의 변이와의 관련성은 다른 영역에 대한 추가적인 분석과, 이미 보고된 다른 모색관련 유전자의 다형성과의 연관성 분석 등과 같은 연구가 필요한 것으로 판단된다.

• 생명과학회지
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• 제26권7호
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• pp.819-825
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• 2016

이 논문은 제주도에서 채집한 구멍갈파래(Ulva pertusa)를 Restriction Fragment Length Polymorphism (RFLP)를 이용하여 세균군집을 조사하였다. RFLP 분석을 위해 Marine agar배지와 R2A배지를 사용하여 145개의 균주가 분리되었으며, 제한효소 HaeⅢ와 RsaⅠ을 이용하여 서로 다른 RFLP 패턴을 구분하였다. RFLP 패턴 결과로부터 균주를 선별하여 16S rRNA 유전자 염기서열 분석 결과, 알려진 균주의 염기서열과 91% 이상의 유사도를 보였다. 주요 계통군은 Proteobacteria (Alpha-proteobacteria, Beta-proteobacteria, Gamma-proteobacteria) (63%), Bacteroidetes (22%), Firmicutes (11%), Actinobacteria (4%)로 4개의 문이 관찰되었고, 7개의 강, 13개의 목, 18개의 과, 27개의 속으로 관찰되었다. 계통학적 분석 결과, 상동성이 97% 미만으로 나타난 10균주가 새로운 속이나 종으로 분류될 가능성이 높게 나타났으며, 신종 후보 균주에 대한 형태학적, 생리학적, 생화학적 등 분류·동정을 위한 추가적인 실험을 수행해야 할 것이다.