• Journal of Veterinary Science
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• 제21권2호
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• pp.22.1-22.9
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• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• Parasites, Hosts and Diseases
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• 제60권4호
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• pp.295-299
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• 2022

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

• Journal of Applied Biological Chemistry
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• 제58권4호
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• pp.383-387
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• 2015

국내 유통 식품 수입 식품 중 토끼와 고양이 고기의 혼입 여부를 알아내고 불법 도축된 고양이 고기를 토끼 고기나 다른 고기로 속여 판매하는 것을 방지하기 위해 토끼와 고양이를 동시에 검출할 수 있는 polymerase chain reaction (PCR) 법을 개발하였다. 토끼와 고양이의 종 특이 프라이머는 미토콘드리아의 cytochrome b 유전자를 대상으로 하였고 개발된 프라이머를 가공식품에 활용하는 것을 고려하여 PCR 산물의 크기는 토끼 101 bp, 고양이 191 bp로 최소화 하였다. 프라이머의 특이성은 총 21종의 동물을 대상으로 검토하였다. 개발된 검출법의 검출 한계는 시료 DNA를 희석하여 PCR과 Bioanalyzer로 확인한 결과 토끼는 0.005 ng, 고양이는 0.0005 ng이었다.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.801-806
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• 2002

A multiplex competitive polymerase chain reaction (PCR) method was developed for the rapid identification and quantification of Leuconostoc mesnteroides and Lactobacillus plantarum populations which are the key microorganisms in kimchi fermentation. The strain-specific primers were designed to selectively amplify the target genes encoding 165 rRNA of L. plantarum and dextransucrase of L. mesenteroides. There was a linear relationship between the band intensity of PCR products and the number of colony forming units of each model organism. The PCR quantification method was compared with a traditional plate-counting method f3r the enumeration of the two lactic acid bacteria in a mixed suspension culture and also applied to a real food system, namely, watery kimchi. The population dynamics of the two model organisms in the mixed culture were reliably predictable by the competitive PCR analysis.

• 대한수의학회지
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• 제57권2호
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• pp.79-88
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• 2017

The study of canine vector-borne diseases in the Philippines started in the 1970s but only gained interest in the past decade. Characterization of such diseases in the Philippines remains incomplete, thus, it is necessary to obtain additional information on the prevalence and diversity of canine tick-borne diseases in the country. In this study, blood samples were obtained at two veterinary clinics in Metro Manila, Philippines from 114 dogs suspected of having canine tick-borne pathogens. Polymerase chain reaction (PCR) was performed on whole blood DNA extracts followed by sequencing, and the following pathogens were detected: Hepatozoon (H.) canis (5.26%), Babesia (B.) vogeli (5.26%), Ehrlichia (E.) canis (4.39%), and Anaplasma platys (3.51%). Additionally, a set of multiplex PCR primers were developed to detect H. canis, Babesia spp. (B. canis and B. vogeli), and E. canis in canine blood. Multiplex and conventional single-reaction PCR results for the 114 dog blood samples were similar, except for one H. canis sample. Multiplex PCR is, therefore, a useful tool in screening infected dogs in veterinary clinics. This study's results, together with those of previous studies in the country, show that canine vector-borne pathogens are an emerging veterinary concern in the Philippines.

• 농업과학연구
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• 제48권4호
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• pp.1051-1065
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• 2021

Recent times have seen sustained increases in genetically modified (GM) crops not only for cultivation but also for the utility of food and feed worldwide. Domestically, commercial planting and the accidental or unintentional release of living modified (LM) crops into the environment are not approved. Many detection methods had been devised in an effort to realize effective management of the safety of agricultural genetic resources. In order to develop event-specific polymerase chain reaction (PCR) markers for LM crops, we analyzed the genetic information of LM crops. Genetic components introduced into crops are of key importance to provide a basis for the development of detection methods for LM crops. To this end, a total of 18 varieties from four major LM crop species (maize, canola, cotton, and soybeans) were subjected to an analysis. The genetic components included introduced genes, promoters, terminators and selection markers. Thus, if proper monitoring techniques and single or multiplex PCR strategies that rely on selection markers can be established, such an accomplishment can be regarded as a feasible solution for the safe management of staple crop resources.

• 대한수의학회지
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• 제43권2호
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• pp.231-237
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• 2003

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.

• Gut and Liver
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• 제12권6호
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• pp.648-654
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• 2018

Background/Aims: Dual priming oligonucleotide-based multiplex polymerase chain reaction (DPO-based PCR) can detect the presence of clarithromycin resistance without culture. The aim of this study was to investigate the cost-effectiveness of DPO-based PCR for Helicobacter pylori eradication. Methods: From 2015 to 2016, medical records of patients who received H. pylori eradication therapy were analyzed. Patients were divided into two groups: tailored group patients who were treated based on DPO-based PCR and empirical group patients. Eradication rate and medical cost, including diagnostic tests, eradication regimens, and $^{13}C$-urea breath tests, were compared between the two groups. Cost for one successful eradication was calculated in each group. The expected cost of eradication for empirical treatment was investigated by varying the treatment duration and eradication rate. Results: A total of 527 patients were analyzed (tailored group 208, empirical group 319). The eradication success rate of the first-line therapy was higher in the tailored group compared to that in the empirical group (91.8% vs 72.1%, p<0.01). The total medical cost for each group was $114.8{\pm}14.1U.S.$ dollars (USD) and $85.8{\pm}24.4USD$, respectively (p<0.01). The total medical costs for each ultimately successful eradication in the tailored group and in the empirical group were 120.0 USD and 92.4 USD, respectively. The economic modeling expected cost of a successful eradication after a 7- or 14-day empirical treatment was 93.8 to 111.4 USD and 126.3 to 149.9 USD, respectively. Conclusions: Based on economic modeling, the cost for a successful eradication using DPO-based PCR would be similar or superior to the expected cost of a successful eradication with a 14-day empirical treatment when the first-line eradication rate is ${\leq}80%$.

• 대한의생명과학회지
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• 제5권1호
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• pp.95-100
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• 1999

일반적으로 임상검사실에서 vancomycin resistant enterococci (VRE)를 검출하는 일은 어렵고, 시간이 많이 들며, 검체처리 비용도 많이 든다. 따라서 본 실험은 임상검체에서 분리된 세균으로부터 VRE를 신속하게 확인하고, 진단하기 위한 방법으로서 다중 중합효소 연쇄반응을 확립하였다. 본 실험에 사용된 primer는 장구균에 특이 한 유전자인 vanA, vanB, vanC-1, vanC-2/3 각각의 염기서열을 기초로 primer를 제작하고, 다중 중합효소 연쇄 반응을 실시하여 임상검체로부터 분리된 VRE 유전자의 type및 분포율을 조사하고자 하였다. 국내에서 분리된 75주의 장구균을 대상으로 다중 중합효소 연쇄반응을 실시한 결과 36주의 분리균주에서 vancomycin에 대해 높은 저항성을 보이는 vanA 유전자를 가진 것으로 나타났다. 그리고 18주에서는 vancomycin에 낮은 저항성을 내성을 보이는 vanC-1 또는 vanC-2/3 유전자를 보유한 것으로 나타났다. 따라서 본 실험에서 확립한 다중 중합효소 연쇄 반응 기법은 신속한 VRE 진단 방법으로 이용할 수 있을 것이다.

• Journal of Plant Biotechnology
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• 제49권3호
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.