Species- and Sex-specific Multiple PCR Amplifications of Partial Cytochrome b Gene and Zfx/Zfy Introns from Invasive and Non-invasive Samples of Korean Ungulates

김백준,이항,이상돈 한국유전학회 2009 Genes & Genomics Vol.31 No.5

There are five representative ungulates (e.g., goral, Nemorhaedus caudatus; goat, Capra hircus; roe deer, Capreolus pygargus; water deer, Hydropotes inermis; musk deer, Moschus moschiferus) in the wild of South Korea. Their fecal morphologies are similar to each other between species or sexes, and therefore it is very difficult to identify the species or sex. To distinguish the species and sex of the elusive animals, we developed species- and sex-specific multiple PCR amplifications using pairs of primer sets. Partial cytochrome b gene and Zfx / Zfy introns were targeted for the PCR amplifications. For species identification using a multiple primer set (BJGL-F1 / GT-F1 / RD-F1 / WD-F1 /MD-F1 and BJGL-R / GT-R / RD-R /WD-R /MD-R), all (n = 21 / 21) of the invasive samples were correctly identified. About 71% (n = 15 / 21) of the non-invasive samples were successfully identified. The multiple primer set could determine each species which showed a unique sized PCR fragment. For sex identification using a multiple primer set (BJSexX-F/Y-F and BJSex-R1), about 90% (n = 19 / 21) of invasive samples were correctly determined. About 62% (n = 13 / 21) of the non-invasive samples were successfully identified. Using the multiple primer set, both X- and Y-chromosome linked bands for males (n = 21) and only X-chromosome linked band for females (n = 11) could be detected. The results would be applicable to the species and sex identification of the Korean and the Far-East Asian wild ungulates. There are five representative ungulates (e.g., goral, Nemorhaedus caudatus; goat, Capra hircus; roe deer, Capreolus pygargus; water deer, Hydropotes inermis; musk deer, Moschus moschiferus) in the wild of South Korea. Their fecal morphologies are similar to each other between species or sexes, and therefore it is very difficult to identify the species or sex. To distinguish the species and sex of the elusive animals, we developed species- and sex-specific multiple PCR amplifications using pairs of primer sets. Partial cytochrome b gene and Zfx / Zfy introns were targeted for the PCR amplifications. For species identification using a multiple primer set (BJGL-F1 / GT-F1 / RD-F1 / WD-F1 /MD-F1 and BJGL-R / GT-R / RD-R /WD-R /MD-R), all (n = 21 / 21) of the invasive samples were correctly identified. About 71% (n = 15 / 21) of the non-invasive samples were successfully identified. The multiple primer set could determine each species which showed a unique sized PCR fragment. For sex identification using a multiple primer set (BJSexX-F/Y-F and BJSex-R1), about 90% (n = 19 / 21) of invasive samples were correctly determined. About 62% (n = 13 / 21) of the non-invasive samples were successfully identified. Using the multiple primer set, both X- and Y-chromosome linked bands for males (n = 21) and only X-chromosome linked band for females (n = 11) could be detected. The results would be applicable to the species and sex identification of the Korean and the Far-East Asian wild ungulates.

Species-specific primers in multiplex PCR for Bactrocera minax identification using an internal transcribed spacer

Regmi Prakriti,Tsai Cheng-Lung,Lin Ming-Ying,Chuang Yi-Yuan,Yeh Wen-Bin 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.4

Bactrocera minax (Enderlein), commonly known as the Chinese citrus fly, is a citrus pest native to China and nearby countries. B. minax can cause substantial losses in citrus orchards. B. minax can be spread to countries free from it by the global trade of citrus and by travelers carrying citrus. Timely, convenient, and accurate identi fication of B. minax is essential in preventing its spread. In the present study, the nuclear ribosomal internal transcribed spacer 2 (ITS2) amplicon was used to design species-specific primer pairs that enable B. minax to be distinguished from 11 other fruit fly species. Four forward and four reverse species-specific primers were designed, and out of all possible sets of species-specific primer pairs obtained after intermixing them, seven sets of species-specific primer pairs were able to accurately identify B. minax. For B. minax identification, specific fragments ranging from 83 to 431 base pairs in length were amplified. The validity of the specific band only in B. minax was determined by visually inspecting the gel profile of the PCR product. B. minax was correctly identified using the designed species-specific primer pairs, with no cross-amplification with the 11 other fruit fly species included in the experiments. In addition to saving DNA sequencing costs, the application of these speciesspecific primer pairs facilitates rapid identification of B. minax, with identification in border scenarios being completable within 2–3 h.

16S-23S rRNA Intergenic Spacer Region을 이용한 Vibrio ichthyoenteri Species-specific Primer 개발

문영건,허문수,Moon Young-Gun,Heo Moon-Soo 한국미생물학회 2005 미생물학회지 Vol.41 No.2

Rotifer와 병든 넙치 자어로부터 분리된 2개의 균주는 표현형적인 특성 확인 결과 Vibrio ichthyoenteri로 확인이 되었다. V. ichthyoenteri를 검출하기 위래 고감도 PCR 방법 개발을 하기 위래 V. ichthyoenteri 16S-23S rRNA intergenic spacer region(ISR)을 분석하였고, V. ichthyoenteri중 특이적 primer를 개발하였다. V. ichthyoenteri 의 ISR를 분석한 결과 1개의 다형성 ISR type서열을 포함하고 있었다. ISR서열은 길이는 348bp이며 tRNA gene을 가지고 있지 않았다. 이 서열을 가지고 이미 알려진 다른 Vibrio 종의 ISR 서열과 mutiple alignment를 수행한 결과 여러 영역에서 높은 가변성을 나타내어 가변 부위를 표적으로 하여 V. ichthyoenteri를 검출하기 위한 종 특이적 primer를 제작하였다. 제작된 primer의 특이성을 확인하기 위해 Vibrio 표준균주 19종의 genomic DNA와 분리균주 18 group에 genomic DNA 그리고 V. ichthyoenteri와 가장 유사한 서열을 가지고 있다고 알려진 Vibrio 종의 genomic DNA를 가지고 시험하였다. 그 결과 본 연구에서 제작된 종 특이적 primer를가지고 PCR 반응을 하면 V. ichthyoenteri를 검출 할 수가 있다. Two bacterial isolates obtained from rotifer and diseased olive flounder larvae, Paralichthys olivaceus, were identified as Vibrio ichthyoenteri based on the results of phenotypic characterization. In an attempt to develop rapid PCR method for the detection of V. ichthyoenteri, we examined the 16S-23S rRNA intergenic spacer region(ISR) of V. ichthyoenteri and developed species-specific primer for V. ichthyoenteri. Analysis of the ISR sequences showed that V. ichthyoenteri contains one type of polymorphic ISRs. The size of ISRs was 348 bp length and did not contain tRNA genes. Mutiple alignment of representative sequences from different V. species revealed several domains of high sequence variability, and allowed to design species-specific primer for detection of V. ichthyoenteri. The specificity of the primer was examined using genomic DNA prepared from 19 different V. species, isolated 18group Vibrio species and most similar sequence of other known Vibrio species. The results showed that the PCR reaction using species-specific primer designed in this study can be used to detect V. ichthyoenteri.

16S rDNA 유전자를 이용한 Bordetella 근연종으로부터 Bordetella pertussis의 감별 PCR

정상운,문유미,성화영,강연호,유재연 대한감염학회 2008 Infection and Chemotherapy Vol.40 No.1

Background : Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. Materials and Methods : The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. Results : As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. Conclusion : In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed. 목 적 : 백일해의 원인균인 B. pertussis는 성장속도가 늦기 때문에 PCR 검출방법은 백일해 진단의 초기단계에서 유용하다. 현재 BP primer가 PCR 검출에 광범위하게 사용되나 BP primer는 최근에 B. holmesii와의 교차반응성이 보고되었다. 따라서 PCR 단계에서 B. pertussis와 B. holmesii의 감별이 필요하다. 이러한 이유로 본 논문에서는 16S rDNA sequence를 기반으로 하는 진단용 primer 를 새로 고안하였고, 그 효용성을 측정하였다. 재료 및 방법 : 특이적 PCR primer들은 다양한 Bordetella 속의 16S rDNA 유전자들의 정렬된 서열 집합체로부터 고안되었다. 고안된 primer의 특이도는 임상적으로 중요한 4개 Bordetella 종 (B. pertussis, B. holmesii, B. parapertussis, B. bronchiseptica)을 사용하여 평가하였다. 민감도 또한 평가하였고 그 결과를 BP primer와 비교하였다. 결 과 : 결과적으로, 새롭게 개발된 primer는 B. holmesii를 포함하는 다른 Bordetella 종들로부터 B. pertussis를 성공적으로 감별하였다. 민감도분석에서는 B. pertussis에 대한 16S-F2/16S-R1 primer의 검출한계는 5 pg의 genomic DNA와 105 cells/mL의 세포부유액까지 였다. 또한 임상검체에 대해서 BP primer set와 동일한 결과가 확인되었다. 결 론 : 본 연구에서 오직 B. pertussis에 대해 특이적인 PCR primer를 16S rDNA를 기반으로 하여 개발하였고, 이는 B. holmesii와의 교차반응성을 나타내지 않았다. 또한 본 연구에서 새롭게 개발된 primer의 임상검체에 대한 적용성도 확인되었다.

다중 PCR 분석법을 이용한 참조기, 부세, 흑조기 및 긴가이석태의 신속한 종판별법 개발

노은수(Eun Soo Noh),이미난(Mi-Nan Lee),김은미(Eun-Mi Kim),박중연(Jung Youn Park),노재구(Jae Koo Noh),안철민(Cheul Min An),강정하(Jung-Ha Kang) 한국생명과학회 2017 생명과학회지 Vol.27 No.7

참조기는 민어과에 속하는 우리나라의 중요한 산업 어종 중 하나이다. 최근 과도한 남획과 해양 환경의 변화로 참조기의 어획량이 줄어들자 일부 유통과정에서 유사어종인 부세, 흑조기 및 긴가이석태를 참조기로 둔갑시키는 사례가 빈번하게 발생하고 있다. 이에 본 연구에서는 종 특이 primer를 사용하여 참조기, 부세, 흑조기 및 긴가이 석태를 신속하게 분석할 수 있는 방법을 마련하였다. 약 1,400 bp의 미토콘드리아 COI 유전자 분석을 통하여 종간 특이성을 나타내는 단일염기다형성 유전자를 탐색하였으며, PCR 증폭산물의 크기를 고려하여 4개의 종 특이적 정방향 primer를 제작하였다. 단일 PCR을 이용한 종간 교차반응을 통하여 최적의 PCR 조건을 확립하였으며, 이후 제작된 4개의 정방향 primer를 혼합하여 4종에 대한 다중 PCR 반응을 진행하였다. 증폭된 산물은 전기영동을 통해 크기에 따라 1,540 bp, 1,013 bp, 470 bp 그리고 182 bp로 분리되었으며, 각각 참조기, 흑조기, 부세, 긴가이석태로 명확하게 판별이 가능하였다. PCR 민감도 측정에서도 모든 종에서 0.1 ng/μl의 농도까지 검출 가능함을 확인하였다. 따라서, 본 연구에서 개발된 참조기와 유사어종에 대한 종특이 다중 PCR 분석법은 정확도와 민감도가 우수하여, 불법 유통가능성이 있는 제품에 대한 신속하고 정확한 판별로 식품안전관리에 효과적으로 활용될 것이라 사료된다. In order to rapidly identify four drums species, Larimichthys polyactis, L. crocea, Atrobucca nibe, and Pseudotolithus elongates, a highly efficient and quick method has been developed using multiplex polymerase chain reaction (PCR) with species-specific primers. Around 1.4 kbp of the mitochondrial COI gene sequences from the four drums species were aligned, and species-specific forward primers were designed, based on the single nucleotide polymorphism (SNP). The optimal conditions for PCR amplification were selected through cross-reactivity, using a gradient PCR method. The PCR results demonstrated species-specific amplification for each species at annealing temperatures between 50 and 62℃. Multiplex species-specific PCR (MSS-PCR) amplification reactions with four pairs of primers were performed for sixteen specimens of each species. MSS-PCR lead to a species-specific amplification of a 1,540 bp fragment in L. polyactis, 1,013 bp in A. nibe, 474 bp in L. crocea, and 182 bp in P. elongates, respectively. The four different sizes of each PCR product can be quickly and easily detected by single gel electrophoresis. The sensitivity of the MSS-PCR of the DNA was up to 0.01 ng/μl as a starting concentration for the four different species tested. These results suggest that MSS-PCR, with species- specific primers based on SNP, can be a powerful tool in the rapid identification of the four drums species, L. polyactis, L. crocea, A. nibe, and P. elongates.

Species- and Sex-specific Multiple PCR Amplifications of Partial Cytochrome b Gene and Zfx/Zfy Introns from Invasive and Non-invasive Samples of Korean Ungulates

Baek Jun Kim,Hang Lee,Sang Don Lee 한국유전학회 2009 Genes & Genomics Vol.31 No.5

There are five representative ungulates (e.g., goral, Nemorhaedus caudatus; goat, Capra hircus; roe deer, Capreolus pygargus; water deer, Hydropotes inermis; musk deer, Moschus moschiferus) in the wild of South Korea. Their fecal morphologies are similar to each other between species or sexes, and therefore it is very difficult to identify the species or sex. To distinguish the species and sex of the elusive animals, we developed species- and sex-specific multiple PCR amplifications using pairs of primer sets. Partial cytochrome b gene and Zfx/Zfy introns were targeted for the PCR amplifications. For species identification using a multiple primer set (BJGL-F1/GT-F1/RD-F1/WD-F1/MD-F1 and BJGL-R/GT-R/RD-R/WD-R/MD-R), all (n=21/21) of the invasive samples were correctly identified. About 71% (n=15/21) of the non-invasive samples were successfully identified. The multiple primer set could determine each species which showed a unique sized PCR fragment. For sex identification using a multiple primer set (BJSexX-F/Y-F and BJSex-R1), about 90% (n=19/21) of invasive samples were correctly determined. About 62% (n=13/21) of the non-invasive samples were successfully identified. Using the multiple primer set, both X- and Y-chromosome linked bands for males (n=21) and only X-chromosome linked band for females (n=11) could be detected. The results would be applicable to the species and sex identification of the Korean and the Far-East Asian wild ungulates.

국내 식물검역대상 Phytophthora pinifolia의 PCR 검출을 위한 종 특이적 마커 개발

김나래 ( Na Rae Kim ),최유리 ( You Ri Choi ),서문원 ( Mun Won Seo ),송정영 ( Jeong Young Song ),김홍기 ( Hong Gi Kim ) 한국균학회 2016 韓國菌學會誌 Vol.44 No.2

To establish a rapid and accurate detection of Phytophthora pinifolia, which is a quarantine pathogenic fungus in Korea, a species-specific primer was developed based on the ras-related protein (Ypt1) gene. Species-specific primer based on the DNA sequences of Ypt1 gene amplified 193 bp polymerase chain reaction (PCR) product for P. pinifolia. The primer pair yielded the predicted PCR product size exactly in testing with target pathogen DNAs, but not from the other 10 species of Phytophthora and 14 species of other phytopathogenic fungi. The primer pair also showed only the species-specific amplification curve on realtime PCR on target pathogen DNA. The detection sensitivity of real time PCR using species-specific primer pair was 10 to 100 times higher than conventional PCR, with 1 to 10 pg/μL.

종 특이 프라이머를 이용한 동물성 식품원료의 진위 판별법 개발

김규헌,이호연,김용상,김미라,정유경,이재황,장혜숙,박용춘,김상엽,최장덕,장영미 한국식품위생안전성학회 2014 한국식품위생안전성학회지 Vol.29 No.4

In this study, the detection method was developed using molecular biological technique to distinguishauthenticity of animal raw materials. The genes for distinction of species about animals targeted at Cytochromec oxidase subunit I (COI), Cytochrome b (Cytb), and 16S ribosomal RNA (16S rRNA) genes in mitochondrial DNA. The species-specific primers were designed by that Polymerase Chain Reaction (PCR) product size was around 200bp for applying to processed products. The target 24 raw materials were 2 species of domestic animals, 6 species ofpoultry, 2 species of freshwater fishes, 13 species of marine fishes and 1 species of crustaceans. The results of PCRfor Rabbit, Fox, Pheasant, Domestic Pigeon, Rufous Turtle Dove, Quail, Tree Sparrow, Barn Swallow, Catfish, MandarinFish, Flying Fish, Mallotus villosus, Pacific Herring, Sand Lance, Japanese Anchovy, Small Yellow Croaker,Halibut, Jacopever, Skate Ray, Ray, File Fish, Sea Bass, Sea Urchin, and Lobster raw materials were confirmed 113bp ~ 218 bp, respectively. Also, non-specific PCR products were not detected in compare species by species-specificprimers. The method using primers developed in this study may be applied to distinguish an authenticity of food materialsincluded animal raw materials for various processed products.

Development of a Species-specific PCR Assay for Three Xanthomonas Species, Causing Bulb and Flower Diseases, Based on Their Genome Sequences

백창기,이승열,이부자,예미지,김상목,강인규,차재순,정희영 한국식물병리학회 2015 Plant Pathology Journal Vol.31 No.3

In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP), X. hyacinthi (XH) and X. campestris pv. zantedeschiae (XCZ), based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of 1 pg/μl per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

담수산 복족류 (Gastropoda: Architaenioglossa) 3종 간 신속한 Triplex PCR 종 판별 방법

김용휘,윤봉한,한호섭,방인철 한국패류학회 2023 The Korean Journal of Malacology Vol.39 No.1

This study designed species-specific primers for quick and convenient molecular biological species identification of three species belonging to Architaenioglossa widely distributed in Korea and developed a single triplex PCR primer set by verifying specificity and reproducibility. Species-specific primers were designed to amplify species-specific bands (Pomacea canaliculata, 223 bp; Sinotaia quadrata, 261 bp; Cipangopaludina chinensis malleata, 431 bp) in consideration of positions representing genetic variation within species and between species on the cytochrome c oxidase subunit 1 (co1) gene base sequence of mitochondrial DNA. In addition, conventional PCR verification experiments were performed according to the amplification cycle and concentration range of genomic DNA (gDNA) to verify the amplification efficiency when configuring the triplex PCR primer set. As a result, considering the PCR reaction cycle and sample amount, the minimum PCR reaction cycle and gDNA concentration were found to be each 25 cycles and 10 ng/μL. Therefore, it is judged that the triplex PCR primer set between the three species belonging to Architaenioglossa developed in this study can contribute to accurate species identification in the industrial field with a quick and simple analysis method at the conventional PCR level.