• Journal of Veterinary Science
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• 제23권4호
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• pp.51.1-51.10
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• 2022

Background: Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives: The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods: LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results: Our results showed that the LMTIA assay could detect the 10⁴ dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions: The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.

• 대한임상검사과학회지
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• 제51권3호
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• pp.309-315
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• 2019

자궁경부암은 전 세계적으로 네번째를 차지하는 여성암이다. 자궁경부암의 대부분 원인은 인유두종 바이러스의 감염이다. 인유두종 바이러스를 검출하기 위해 다양한 분자진단학적 방법들이 고안되었다. 분자진단학적 방법 중의 real-time PCR은 목표 DNA 또는 RNA의 정량과 민감도 향상을 목표로 도입되었다. 특히, real-time PCR 과정은 수행 전에 RNA 추출 및 상보적인 DNA 합성 과정이 필요하다. 따라서 본 연구에서는 민감하고 적합한 상보적인 DNA 합성 키트를 알아보기 위해서 상보적인 DNA 합성에 이용되는 두 개의 상용화된 키트를 평가하였다. 자궁경부암 세포주에서 두개의 상보적인 DNA 합성 키트의 $R^2$과 효율성을 비교한 결과 차이가 없었다. 그러나 Invitrogen 키트보다 Takara 키트가 $10^2$ 및 $10^3$ SiHa 세포주에서 P<0.001를 나타내었고 $10^1$ 및 $10^2$ HeLa 세포주에서도 P<0.001를 나타내었다. 이를 통해 Takara 키트가 Invitrogen키트보다 민감도가 높음을 알 수 있었다. 또한 40개의 탈락세포검체의 8, 4, 2, 1 mL을 이용하여 상보적인 DNA 합성 키트를 비교한 결과 Invitrogen 키트보다 Takara 키트가 8, 4, 1 mL에서 P<0.01 및 0.5 mL에서 P<0.05을 나타내어 임상 검체를 이용하였을 때에도 Takara 키트가 Invitrogen 키트보다 민감도가 높음을 알 수 있었다. 본 연구는 적합한 상보적인 DNA 합성 키트를 확인하기 위해 수행되었으며, 상보적인 DNA 합성 키트가 real-time PCR 결과 다양성에 영향을 미친다는 것을 시사하였다.

• 미생물학회지
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• 제41권3호
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• pp.168-176
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• 2005

갯벌 퇴적물에 존재하는 병원성 해양미생물인 Vibrio vulnificus를 신속하고 정확하게 검출하기 위해 PCR, Southern hybridization 방법과 real-time PCR을 수행하여 검출 민감도를 비교하였다. 갯벌 퇴적물로부터 bead beater를 이용한 물리적 방법으로 DNA 조추출액을 얻고 상용화된 키트 (Geneclean turbo Kit)를 이용하여 부식물질(humic substances)을 제거하였다. 병원성에 관련된 3 종의 유전자(hemolysin, vvhA; phosphomannomutase, pmm; metalloprotease, vvpE)를 대상으로 설계한 프라이머 셋을 동시에 사용하는 multiplex PCR 방법과 Southern hybridization과 병행한 방법(PCR/Southern hybridization)을 수행하였다. Real-time PCR은 hemolysin 유전자(vvhA)에 특이한 프라이머와 TaqMan 탐침을 사용하였다. 전처리하지 않은 갯벌 퇴적물의 경우, PCR/Sourthern hybridization과 real-time PCR 방법의 검출 민감도는 퇴적물 1 g 당 약 $10^2$ 개의 세포 수준이었다. 농후처리액(APW; alkaline peptone water)으로 $35^{\circ}C$에서 $2{\~}3$시간, 8시간 중균 배양할 경우 갯벌 퇴적물 1 g 당 $2{\~}10$개 세포가 존재할 때 PCR/Southern hybridization 방법과 real-time PCR 방법으로 각각 검출할 수 있었다. 전처리 과정을 포함하여 real-time PCR은 $6{\~}7$시간, PCR/Sourthern hybridization은 약 36시간이 소요되었다.

• 한국임상수의학회지
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• 제36권5호
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• pp.245-247
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• 2019

Taylorella equigenitalis (T. equigenitalis), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa) are sexually transmittable bacteria known to cause venereal diseases (VD) in horses. T. equigenitalis causes contagious equine metritis (CEM), which is a considerable concern for equine breeding industry. K. pneumoniae and P. aeruginosa may cause endometritis and infertility in susceptible mares. The purpose of this study was to investigate the prevalence of these bacteria among breeding Thoroughbreds in South Korea. External genital swabs were collected from 178 breeding Thoroughbreds, including 11 stallions and 167 mares. The samples were tested using a commercial multiplex real-time PCR kit. T. equigenitalis, P. aeruginosa, and K. pneumoniae were present in 5.6%, 7.3%, and 5.6% of tested Thoroughbreds, respectively. The results highlight the need for regular testing of South Korean Thoroughbreds, particularly those used for breeding, for these bacteria. The regular pre-breeding test for these bacteria will prevent health complications for the horse and financial losses for the owner as a result of VD.

• 대한수의학회지
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• 제54권1호
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• pp.55-57
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• 2014

Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) has extended latent periods of infection. Due to this property, difficulties in the detection of fecal shedder have been raised. A newly designed method for DNA extraction from fecal specimens, mGITC/SC was evaluated in terms of diagnostic efficiency. The detection limit of IS900 real-time PCR was about 50 MAP (1.5 cfu) in 250 mg of feces (6 cfu per g). Also, this DNA extraction method was faster and cheaper than that using commercial kit or other methods. Consequently, the mGITC/SC is an economical DNA extraction method that could be a useful tool for detecting MAP from fecal specimens.

• 대한의생명과학회지
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• 제16권3호
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• pp.193-196
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• 2010

Multiplex PCR-based short tandem repeat (STR) analysis is considered as a good tool for monitoring bone marrow engraftment after sex-mismatched allogeneic transplantation and provides a sensitive and accurate assessment of the contribution of both donor and/or recipient cells in post-transplantation specimens. Forensic STR analysis and quantitative real time PCR are used to determine the proportion of donor versus recipient each contained within the total DNA. The STR markers were co-amplified in a single reaction by using commercial $PowerPlex^{(R)}$ 16 system and $AmpFISTR^{(R)}$ $Identifiler^{(R)}$ / $Yfiler^{(R)}$ PCR amplification kits. Separation of the PCR products and fluorescence detection were performed by ABI $PRIS^{(R)}$ 3100 Genetic Analyzer with capillary electrophoresis. The $GeneMapper^{TM}$ ID software were used for size calling and analysis of STR profiles. Extracted DNA was quantified by the $Quantifiler^{TM}$ Human DNA / Y Human Male DNA Quantification Kit The intent of this study was to analyze the ratio of donor versus recipient cells in the post-transplant peripheral blood, spleen, lung and kidney specimens. Specimens were taken from the traffic accident male victim who had been engrafted from bone marrow female donor. Blood and spleen specimens displayed female donor DNA profile. Kidney specimen showed male recipient DNA profile. Interestingly, lung tissue showed mixed profiles. The findings of this study indicate that the forensic STR analysis using fluorescence labeling PCR combined with capillary electrophoresis is quick and reliable enough to assess the ratio of donor versus recipient cells and to monitor the mixed chimeric patterns.

• 한국동물위생학회지
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• 제43권2호
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• pp.107-112
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• 2020

Foot-and-mouth disease (FMD) and avian influenza (AI) are highly pathogenic viral disease which affects the livestock industry worldwide. Outbreak of these viruses causes great impact in the livestock industry; thus, disease infected animals were immediately disposed. Burial is the commonly used disposal method for deceased animals. However, there is potential for secondary environmental contamination, as well as the risk that infectious agents persisting in the environment due to the limited environmental controls in livestock burial sites during the decomposition of the carcasses. Therefore, this study aimed to investigate the detection of FMD and AI viruses from animal carcass disposal sites using real-time reverse transcription PCR. Soil samples of more than three years post-burial from livestock carcass disposal sites were collected and processed RNA isolation using a commercial extraction kit. The isolated RNA of the samples was used for the detection of FMDV and AIV using qRT-PCR. Based on the qPCR assay result, no viral particle was detected in the soil samples collected from the animal disposal sites. This indicates that 3 years of burial and their carcass disposal method is efficient for the control or at least reduction of spread infections in the surrounding environment.

• 한국토양비료학회지
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• 제44권6호
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• pp.1181-1184
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• 2011

Total aerobic bacteria, Esherichia coli O157:H7, and Salmonella spp. were examined in commercial liquid pig manures. Commercial liquid pig manures (n=33) were collected from muck joint resource recovery plant at April, June, August, October 2009, Korea. Total aerobic bacteria were incubated at $37^{\circ}C$ for 24-48 hrs, and quantified as a colony-forming unit (CFU) $mL^{-1}$. Analysis of Esherichia coli O157:H7 and Salmonella spp. were followed by Korean Food Standards Codex method. Colony of Salmonella spp. was confirmed by API kit and real time polymerase chain reaction (PCR). Total aerobic bacteria isolated from fermented commercial liquid pig manures ranged from 2.8 to $24.3{\times}10^4\;CFU\;mL^{-1}$. Esherichia coli O157:H7 was not detected, and Salmonella spp. showed the low detection frequency at only 1 sample. This study suggests that continuous monitoring in commercial liquid pig manures is required to improve the agricultural food through management of agricultural land contaminated with liquid pig manures.