• 생명과학회지
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• 제26권9호
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• pp.1007-1014
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• 2016

본 연구에서는 국산 참서대과 어류 5종(개서대, 참서대, 칠서대, 박대, 용서대) 및 수입산 참서대과(Cynoglossidae) 어류 5종(기니개서대, 긴개서대, 큰비늘개서대, 큰입개서대, 세네갈 개서대)을 대상으로 분자생물학적 방법을 이용한 신속하고 정확한 종판별법을 검토하였다. 참서대과 어류 10종에 대한 최적의 종 특이 프라이머를 선정하기 위하여 약 1,500 bp의 COI 유전자를 분석하였으며, 종간 특이적인 단일염기다형성 유전자가 3’ 말단에 위치하도록 프라이머를 제작하였다. 제작된 10종에 대한 종특이 정방향 프라이머는 동일한 PCR조건과 전기영동을 통해 육안으로 식별이 가능할 정도의 PCR 산물의 상대적인 크기를 고려하였다. 다중 PCR 분석을 위해 종특이 정방향 프라이머는 모두 혼합되어 사용되었으며, 그 결과 세네갈개서대(208 bp), 용서대(322 bp), 큰입개서대(493 bp), 큰 비늘개서대(754 bp), 박대(874 bp), 칠서대(952 bp), 참서대(1,084 bp), 긴개서대(1,198 bp), 개서대(1,307 bp), 기니개서대(1,483 bp)에 해당하는 종 특이적인 증폭을 확인하였다. 또한 이들의 PCR 민감도를 측정한 결과 0.1~1.0 ng/μ l의 농도까지 검출이 가능한 것을 확인 하였다. 본 연구에서 확인된 참서대과 어류 10종에 대한 종특이 프라이머는 특이도 및 민감도가 우수하며 향후 수산물의 수출입 및 유통 관리에 사용이 이루어진다면 정확한 종명 표기가 가능하여 국민 먹거리 안전을 위한 효과적인 방법이 될 것이다.

• 미생물학회지
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• 제48권4호
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• pp.309-313
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• 2012

본 연구는 multiplex PCR 방법을 적용하여 질 내부에 서식하는 유산균을 동시에 그리고 신속하게 검출하는 방법을 개발하는 것을 목적으로 수행되었다. 세균의 감염에 의한 질염 증상이 없는 건강한 20대 여성 166명으로부터 질 분비물을 채취하였다. 건강한 여성의 질 내부에 서식하는 것으로 확인된 Lactobacillus 속에 속하는 6종의 유산균을 종 특이 multiplex PCR primer를 제작하기 위해 선정하였다. Multiplex primer I은 L. iners, L. crispatus, L. gasseri를 선택적으로 검출하기 위해 특성화하였고 multiplex primer II는 L. acidophilus, L. jensenii, L. fermentum를 선택적으로 검출하기 위해 특성화하였다. Multiplex PCR 기술을 이용하여 분석한 결과 L. crispatus (77%)가 가장 높은 빈도로 검출되었고 L. acidophilus (57%)과 L. jensenii (57%)는 상대적으로 높은 빈도로 검출되었다. L. iners (59%)는 L. acidophilus (57%)와 L. jensenii (57%) 보다 높은 빈도로 검출되었지만 항생제 치료 과정 후 또는 세균성 질염의 경우에도 발견되는 균종으로 건강한 여성에게서 주로 서식하는 종이라고 결론 내리기는 어렵다. 결론적으로, 특정 유산균의 특이적인 primer를 이용한 multiplex PCR 기술은 질의 건강상태의 변화 또는 질의 질병으로부터 회복 과정을 예측할 수 있는 도구로서 유용할 것으로 생각된다.

• 한국동물위생학회지
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• 제34권4호
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• pp.417-428
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• 2011

Species-specific PCR assay was developed for detection of cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey using mitochondrial 12S rRNA and 16S rRNA as target genes. Also, an internal positive control was used to detect possible false negatives by using 18S rRNA gene. We designed species-specific primers with amplicon length of 190, 219, 350, 467, 241, 119, 171, 229, 111 and 268 bp for cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey respectively. The specificity of the primers was tested against the other 10 non-target animal species and a cross-reaction was not observed. We developed two multiplex PCR assays for the simultaneous identification of Korea's major livestock species (cattle, pig, chicken and duck) and poultry species (chicken, duck, goose and turkey) from analogous samples, retaining the same specificity. The limit of detection of the multiplex PCR assay (cattle, pig, chicken and duck) ranged between 1 pg and 0.1 pg of template DNA extracts from raw meat. Applying multiplex PCR assays to DNA extracts from experimental pork/beef and pork/chicken tested raw and heat-treated ($120^{\circ}C$ for 30 min) mixtures respectively, detection limit was 0.1% level beef in pork, pork in beef and chicken in pork and 1.0% level pork in chicken. In conclusion, this assay using gel-based capillary electrophoresis would be very useful in highly sensitive and rapid identification of animal species or ingredients in minced meat and other meat products.

• 한국식품위생안전성학회지
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• 제16권4호
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• pp.251-257
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• 2001

Listeria monocytogenes와 L. ivanovii는 인간과 동물에 대한 식품 유래성 병원성 세균이다. Listeria 속에는 이들 병원균 외에 비병원성세균인 L. innocua, L, welshimeri, L. seeligeri, L.grayi 등 이 포함되어 있기에, 식품검사에서 배양법을 사용하는 종래의 Listeria의 검출방법은 시간과 많은 실험을 필요로 한다. 이런 이유 등으로 Literia의 종동정과 검출을 위한 신속한 검출법으로 Lis-mix multiplex PCR검출법 (Bubert et al., 1999)이 개발되었다. 본 연구에서는 식품검사에 활용하기 위한 실용적인 Lis-mix multiplex PCR 방법을 개발하고, 아울러 새로운 Siw-mixIII PCR 검출법을 개발하였다. 이 방법을 사용하여 식품에서 분리된 총 69개의 Listeria 균주를 성공적으로 종동정할 수 있었으며, Siw-mixIII multiplex PCR방법을 사용하여 L. ivanovii, L.welshimeri, L.seeligeri를 한번의 PCR로 검출 및 종동정을 할 수 있었다.

• 대한수의학회지
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• 제43권1호
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• pp.103-112
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• 2003

A multiplex PCR assay, which allows simultaneous detection of vancomycin resistant genotypes and Enterococcus species-specific genes, was developed. Vancomycin resistant enterococci (VRE) from chickens and humans could be detected for vanA, vanB, vanC-1, vanC-2, $ddl_{E.faecium}$ and $ddl_{E.faecalis}$ by multiplex PCR. Eight isolates of VRE from humans (n=11) had $ddl_{E.faecium}$ and vanA, and 3 isolates of the VRE had $ddl_{E.faecium}$ and vanB. One isolate of VRE from chickens (n=6) had $ddl_{E.faecium}$ and vanA, and 5 isolates of the VRE had only vanA. E. faecium, E. faecalis, E. gallinarum and E. casseliflavus were also confirmed for the species-specific gene by multiplex PCR. This multiplex PCR could detect E. faecium, E. faecalis, E. gallinarum, E. casseliflavus, vanA, vanB, vanC-1 and vanC-2, simultaneously. The PCR assay established in the present study can be an alternative to time-consuming biochemical tests and antibiotic susceptibility tests of Enterococcus spp.

• 대한수의학회지
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• 제41권4호
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• pp.535-542
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• 2001

A multiplex PCR technique was developed for detecting specifically each Mycobacterium bovis, M. tuberculosis, M. avium and M. avium subsp, paratuberculosis, respectively, using clinical samples of field cattle. To apply this novel technique to clinical specimens, blood sample was obtained from live cows comprising 11 intradermal tuberculin test (ITT)-positive and 17 ITT-negative and tested by multiplex PCR. Positive results were obtained from 15 cows by the multiplex PCR, showing that 4 (23.5%) of the 17 ITT-negative cows were multiplex PCR positive. The multiplex PCR results also showed that among the 15 positive cows, 7 (46.7%) were infected with M. bovis, 1 (6.7%) with M. tuberculosis and 7 (46.7%) with M. avium. The sensitivity and specificity of multiplex PCR in comparison with those of ITT were 100% and 76.5%. The correlation between the multiplex PCR and ITT assays with blood samples was considered excellent, 85.7% agreement and ${\kappa}=0.72$. The results obtained, using reference mycobacterial strains and typed clinical samples, show that the multiplex PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay. Therefore, multiplex PCR assay is a useful tool for early diagnosis of tuberculosis in live cattle and to identify the species or complex of mycobacterium from clinical samples.

• 한국약용작물학회지
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• 제21권3호
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• pp.165-173
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• 2013

The fruits of Schisandra chinensis have been used as an edible ingredient and traditional medicine in Korea. Due to morphological similarities of dried mature fruits, the correct identification of S. chinensis from other closely related Schisandrae species is very difficult. Therefore, molecular biological tools based on genetic analysis are required to identify authentic Schisandrae Fructus. Random amplifed polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were used to develop an easy, reliable and reproducible method for the authentication of these four species. In this paper, we developed several RAPD-derived species specific SCAR markers and established a multiplex-PCR condition suitable to discriminate each species. These genetic markers will be useful to distinguish and authenticate Schisandrae Fructus and four medicinal plants, S. chinensis, S. sphenanthera, S. repanda and K. japonica, in species level.

• 한국동물위생학회지
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• 제37권4호
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• pp.247-252
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• 2014

The economic impact of swine mycoplasma infection is high. An accurate diagnosis is often difficult and time consuming. We report the development and validation of an effective multiplex polymerase chain reaction (PCR) assay that detects Mycoplasma (M.) hyopneumoniae and M. hyorhinis. The multi detection of M. hyopneumoniae and M. hyorhinis primer set were employed to detect mycoplasma species and typing of the species was performed on the basis of sequence analysis of the PCR product. The target nucleic acid fragments were specifically amplified by M. hyopneumoniae and M. hyorhinis PCR with 16S ribosomal DNA primers. Single and mixed Mycoplasma species DNA templates were used to evaluate the specificity of the multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of M. hyopneumoniae and M. hyorhinis.

• 식물병연구
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• 제24권4호
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• pp.348-352
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• 2018

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed for the detection of Clover yellow vein virus (ClYVV), Peanut mottle virus (PeMoV), and Tomato spotted wilt virus (TSWV), which were recently reported to infect soybean and azuki bean in Korea. Species-specific primer sets were designed for the detection of each virus, and their specificity and sensitivity were tested using mixed primer sets. From among the designed primer sets, two combinations were selected and further evaluated to estimate the detection limits of uniplex, duplex, and multiplex RT-PCR. The multiplex RT-PCR assay could be a useful tool for the field survey of plant viruses and the rapid detection of ClYVV, PeMoV, and TSWV in leguminous plants.

• 한국약용작물학회지
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• 제19권3호
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• pp.162-169
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• 2011

The rhizomes and herbal medicines originating from Acorus gramineus, A. calamus, A. tatarinowii, and A. gramineus var. pusilus, show significant similarity, and the correct identification of species is very difficult. Random Amplified Polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were used to develop a reliable method for identification of these four species. Several distinct SCAR markers were developed from species-specific RAPD amplicons for each species. Furthermore, a useful molecular marker was established for multiplex-PCR, in order to the four species could be distinguished concurrently. These markers allow efficient and rapid identification of closely-related Acorus species and will be useful for standardization of herbal medicines.