• 한국동물위생학회지
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• 제39권2호
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• pp.107-116
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• 2016

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

• Fisheries and Aquatic Sciences
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• 제11권3호
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• pp.133-139
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• 2008

Molecular techniques, including restriction fragment length polymorphism(RFLP) and polymerase chain reaction-single strand conformational polymorph isms(PCR-SSCP), were developed to identify salted, dried threadfin(Eleutheronema tetradactylum) and white herring(Ilisha elongata) fish. Using PCR with universal primers, conserved 367-bp fragments of the cytochrome b gene were amplified from fresh fish samples and sequenced. The sequences were then searched for specific restriction sites. The digestion of the PCR products with the endonucleases AvaI, FokI, MboII, and MspI generated RFLP, which was used to identify the commercial products. Similarly, the amplified PCR-SSCP products were developed and the products tested. Overall, similar patterns were found in the majority of the fresh and processed products. Based on the results, both RFLP and PCR-SSCP were useful in determining and validating the authenticity of the fish species used to prepare the commercial salted, dried products. A similar approach can be applied to other species.

• Asian-Australasian Journal of Animal Sciences
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• 제15권9호
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• pp.1237-1243
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• 2002

We have developed a reliable and noninvasive method for swine genotyping of single locus nuclear gene with aged single hair follicles delivered by general mail. The method is based on booster and nested PCR amplification with step-wise increase of primers and dNTPs concentrations followed by restriction endonuclease digestion. To establish this method, the ryanodine receptor (RYR 1) locus which is an economically important trait in swine industry was employed for genotyping experiment. The 3-step PCR amplication method is much less dependent on the quantity and quality of template DNA and produces enough amplification product for the detection on the ethidium bromide-stained gel such as RFLP analysis. A total of 120 pigs were subjected to the RYR 1 genotyping analysis using three-step PCR method which amplified enough quantity of PCR products from the aged single hair follicles for RFLP analysis and genotyping results were identical to the results of the corresponding ethanol-fixed skeletal muscle tissue. This approach will be a great help for porcine breeders and investigators in genotyping of swine. They can receive genotyping results later by simply plucking single hairs of their pigs at farm and sending them in general mail to the diagnostic laboratory which eliminates the inconveniences to collect ear tissue or blood cells from pigs, or the investigator's need for travel to farms in order to collect fresh hair samples.

• KSBB Journal
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• 제8권2호
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• pp.104-109
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• 1993

한 개의 pladsmid에 Gus gene과 Hygromycin resistant gene(Hpt)을 합친 vector를 개발하였다. 이 재조합된 DNA를 벼의 건조한 종자에 imbibition시켜 형질전환했을 때, hygromycin 배지에서 선별한 결과 그 발현율은 single Hpt vector와 차이 가 없었으며, 주로 뿌리의 생장점이나 자엽초에서 Gus expression을 볼 수 있였다. 또한 hygromycin 배지에서 선별되어 성체가 된 개체에서 그 genomic DNA를 뽑아 PCR을 한 결과 1Kb Hpt gene을 확인하였다. 그리고 생체에서 추출한 총 RNA에서 cDNA를 만든 후 reverse transcription PCR을 통하여, 외부 유전자의 발현을 증명하였다.

• Parasites, Hosts and Diseases
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• 제53권1호
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• pp.77-83
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• 2015

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.

• Journal of Genetic Medicine
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• 제9권1호
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• pp.11-16
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• 2012

Purpose: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. Materials and Methods: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. Results: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. Conclusion: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

• 대한의생명과학회지
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• 제13권1호
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• pp.71-73
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• 2007

For the rapid detection of objective pathogenic bacteria from colon biopsy specimens, multiplex PCR (polymerase chain reaction) method was developed. The major objective bacteria in this study were Shiga-like toxin producing E. coli O157:H7, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Shigella spp. Salmonella spp. and Yersinia spp.. To detect simultaneously 7 kinds of pathogenic bacteria in single reaction tube, multiplex PCR system was executed using 6 sets of primers used in single PCR system for the respective bacteria. The results in this research might be applied for the detection of pathogenic bacteria form colon biopsy samples.

• Asian Pacific Journal of Cancer Prevention
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• 제17권7호
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• pp.3065-3069
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• 2016

Single nucleotide polymorphism (SNP) detection has been used extensively for genetic association studies of diseases including cancer. For mass, yet accurate and more economic SNP detection we have optimized tetra primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) to detect three SNPs in the cytochrome P450 2E1 (CYP2E1) gene locus; i.e. rs3813865, rs2070672 and rs3813867. The optimization system strategies used were (1) designing inner and outer primers; (2) determining of their optimum primer concentration ratios; and (3) determining of the optimum PCR annealing temperature. The tetra primer ARMS PCR result could be directly observed using agarose gel electrophoresis. The method succesfully determined three SNPs in CYP2E1 locus, the results being consistent with validation using DNA sequencing and restriction fragment length polymorphisms (RFLP).

• Clinical and Experimental Reproductive Medicine
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• 제32권4호
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• pp.293-300
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• 2005

연구목적: 단일 유전자 이상에 대한 착상전 유전진단을 성공적으로 시행하기 위해서는 효과적이고 신뢰도가 높은 PCR 방법의 확립이 중요하다. 본 연구에서는 alkaline lysis와 duplex nested PCR 방법을 단일 림프구와 할구의 유전자 분석에 적용하여 그 효용성을 확인하고자 하였다. 재료 및 방법: 단일 유전자의 이상이 확인된 Duchenne muscular dystrophy (DMD), ornithine transcarbamylase (OTC) 결핍증과 epidermolysis bullosa (EB) 가계의 대상자들에서 채취한 단일 림프구와 공여 받은 배아의 할구를 이용하여 각각 PCR, restriction fragment length polymorphism (RFLP)와 direct DNA sequencing 분석을 시행하였다. 이러한 분석에서 유전자 증폭률 (amplification rate)과 두개의 allele 중에서 하나의 allele이 증폭되지 않는 allele drop-out (ADO) 빈도에 대해 살펴보았다. 결 과: 단일 림프구와 할구를 이용한 PCR 방법의 유전자 증폭률은 DMD에서 91.1%와 81.8%, OTC 결핍증에서 96.0%와 78.1%, EB에서 91.3%와 90.0%를 각각 나타냈으며, ADO 빈도는 OTC 결핍증에서 13.3%, EB에서 16.8%로 관찰되었다. 결 론: 본 연구에서 적용한 alkaline lysis와 duplex nested PCR 방법은 단일 유전자에 대한 착상전 유전진단에 성공적으로 적용할 수 있는 방법으로 생각되며, ADO 빈도를 최소화할 수 있는 효율적인 방법의 개발에 대한 지속적인 연구가 필요하다.

• Clinical and Experimental Reproductive Medicine
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• 제23권1호
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• pp.109-114
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• 1996

General PCR technique alone has a limitation for preimplantation genetic diagnosis(PGD) using single blastomere. Recntly developed primer extension preamplification(PEP) technology amplifies the whole genome and thus, simultaneous multiple locus analysis became possible. In this study, we report the efficacy of PEP-PCR for PGD in three muscular dystrophy carriers undergoing IVF-ET. A total of 37 blastomeres were obtained from 40 embryos at six to eight cell stage in three IVF cycles in two DMD and one BMD carriers. Whole genome from single blastomeres were amplified using I5-base oligonucleotide random primers. PCR amplified products of exon 45 in the dystrophin gene and alphoid X/Y loci for gender determination were analysed by 2% metaphor gel electrophoresis. A total of 37 PEP-PCR replicates from 37 single blastomeres from 40 embryos and 37 blanks were performed. We obtained the reliable results for exon 45 and alphoid X/Y. Transfer of female embryos and unaffected male embryo was attempted in three couples. Unfortunately, pregnancy was not achieved in these cases. PEP-PCR is a reliable and efficient PGD method in multiple locus analysis using single blastomere.