• Genomics & Informatics
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• 제11권4호
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• pp.277-281
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• 2013

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

• 한국어병학회지
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• 제24권2호
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• pp.65-73
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• 2011

패류 내에 오염되어 있는 바이러스의 검출에 있어서 정성 정량적 분석이 기능하며 신속하고 간편한 방법의 개발을 이루고자 하였다. 5g의 굴 중장선 조직을 Glycine buffer 및 PEG 8000 용액을 사용하여 제조한 농축 시료 (T5g-D)와 50mg의 굴 중장선 조직으로부터 직접적으로 제조한 시료(sT5Omg-D)를 대상으로 2-step PCR을 실시하여 검출감도를 비교하였다. 동일한 1 ${\mu}l$의 DNA template T5g-D와 sT50mg-D를 사용하였을 때, 35cycles의 1-step PCR에서 양성의 결과를 얻을 수 있었다. 인위적으로 sT50mg-D 혼합 시료를 사용하여 2-step PCR (35cycles)을 실시하였을 때 T5g-D를 사용한 것과 비교하여 뚜렷한 차이를 나타내지 않았다. 또한 megalocytivirus에 오염된 양성시료 0.5~50mg을 사용하여 조제한 각각 $50{\mu}l$의 template DNA 1 ${\mu}l$를 사용하여 qPCR을 하였을 때 6.14E+00~1.2E+02/${\mu}l$의 농도를 함유하고 있는 것으로 나타났다. 조제한 template DNA에 6.14E+00 copies/${\mu}l$ 이하의 농도가 있을 때는 qPCR에서 positive의 결과를 얻을 수 없었다. 결론적으로 패류 내 mega1ocytivirus의 오염 유무 판단을 위한 2-step PCR 및 qPCR에서 50mg의 중장선을 사용하는 것은 i) 굴의 조직으로부터 오염된 megalocytivirus의 정성 및 정량을 위한 최소 검출 한계에 벼하여 충분한 양이었으며 ii) 개체별 분석이 가능하다는 장점이 있었으며 iii) 이를 토대로 국내에서 서식하고 있는 굴에서 megalocytivirus의 오염을 확인할 수 있었다.

• Communications for Statistical Applications and Methods
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• 제16권6호
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• pp.1037-1046
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• 2009

Copy number variations(CNVs) are known as one of the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing real-time polymerase chain reaction(PCR), invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed. PCR followed by a quantitative oligonucleotide ligation assay(qOLA) was developed for quantifying CNVs. The aim of this study was to compare the two methods for detecting and quantifying the CNVs of duplicated gene: the published pyrosequencing assay(pyro_CNV) and the newly developed qOLA_CNV. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares(RMSs) of bias and standard deviation of qOLA_CNV were 2.09 and 0.45, respectively. These values are less than half of those of pyro CNV.

• 미생물학회지
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• 제50권4호
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• pp.296-301
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• 2014

로타바이러스는 선진국과 개발도상국의 영 유아에게 급성위장염을 일으키는 주요원인이다. 위장질환의 치료를 위한 유산균의 사용은 안전하며 간단하게 이용할 수 있다. 본 연구는 Sprague-Dawley 랫드에서 유산균혼합물의 로타바이러스에 대한 항 바이러스 효능을 조사하였다. 24마리의 새끼와 그들의 어미를 무작위로 네 그룹으로 나누었다; placebo, phosphate buffered saline (PBS)와 유산균 혼합물-1, 유산균 혼합물-2 그룹. 5일령인 모든 랫드에게 8 log plaque forming units의 농도로 로타바이러스를 접종하고 유산균혼합물-1, 유산균 혼합물-2 그룹은 4일 동안 하루에 한번 각각 8 log colony forming units의 농도로 유산균 혼합물을 경구 투여하였다. 대조군인 placebo와 PBS 그룹은 4일 동안 하루에 한번 각각 동일한 양의 placebo (말토오스, 폴리덱스트로스 포함)와 PBS를 경구 투여하였다. 항 바이러스 효능분석을 위해 Real-time quantitative PCR (RT-qPCR)과 소장융모관찰을 수행하였으며, 그 결과 유산균 혼합물-1, 유산균 혼합물-2 그룹의 소장무게는 대조군 보다 무거웠다. 대조군의 융모는 길이가 짧아지고 융모상피세포의 괴사가 일어났지만 유산균 혼합물-1과 유산균 혼합물-2 그룹에서는 이러한 형태학적 변화를 관찰할 수 없었다. RT-qPCR 분석에서는 유산균 혼합물-1과 유산균 혼합물-2 그룹의 분변샘플과 소장상피세포에서 로타바이러스의 VP7 유전자 레벨이 낮았다. 이러한 연구결과는 유산균혼합물이 로타바이러스 위장염에 대한 대체요법이나 치료에 유용하게 사용될 수 있음을 시사한다.

• 한국해양학회지:바다
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• 제24권3호
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• pp.422-428
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• 2019

어류폐사 와편모조류인 Cochlodinium polykrikoides는 large-subunit(LSU) ribosomal RNA gene을 기반으로 전 세계적으로 4가지 유전타입이 알려져 있으며, 남해안에는 2가지 유전타입이 출현한다고 보고되었다. 본 연구에서는 C. polykrikoides의 동아시아 타입과 필리핀 타입의 남해안 출현양상을 quantitative real-time PCR(qPCR)을 이용하여 3년간(2014~2016년) 조사하였다. 동아시아 타입의 경우 2014~2016년에 40~100% 비율로 남해안 전 정점(통영~완도)에서 검출이 된 반면, 필리핀타입은 대마난류 유입이 강했던 2016년에만 통영~고흥 일부 해역에서 1~2% 비율로 극미량 검출되었다. 위 결과는 동아시아타입이 남해안의 우점 C. polykrikoides 개체군임을 보여주고 있으며, 일부 유영세포는 대마난류를 따라 외해역으로 부터 유입될 수 있음을 시사한다.

• 한국동물위생학회지
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• 제45권1호
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• pp.19-30
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• 2022

In this study, a simple loop-mediated isothermal amplification (LAMP) combined with visual detection method (vLAMP) assay was developed for the rapid and specific detection of African swine fever virus (ASFV), overcoming the shortcomings of previously described LAMP assays that require additional detection steps or pose a cross-contamination risk. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubation for 40 min at 62℃. The assay specifically amplified ASFV DNA and no other viral nucleic acids. The limit of detection of the assay was <50 DNA copies/reaction, which was ten times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). For clinical evaluation, the ASFV detection rate of vLAMP was higher than cPCR and comparable to OIE-recommended qPCR, showing 100% concordance, with a κ value (95% confidence interval) of 1 (1.00~1.00). Considering the advantages of high sensitivity and specificity, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vLAMP assay will be a valuable tool for detecting ASFV from clinical samples, even in resource-limited laboratories.

• Journal of Veterinary Science
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• 제25권2호
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• pp.28.1-28.11
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• 2024

Background: Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. Objectives: This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. Methods: We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. Results: The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. Conclusions: The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.39-46
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• 2016

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (${\beta}-actin$), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

• Bulletin of the Korean Chemical Society
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• 제35권5호
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• pp.1562-1564
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• 2014

• International Journal of Oral Biology
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• 제42권3호
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• pp.143-147
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• 2017

In a previous study, Peptoniphilus mikwangii was isolated from the human oral cavity as a new species. The purpose of this study was to develop P. mikwangii-specific PCR primers. The PCR primers were designed, based on the nucleotide sequence of 16S ribosomal RNA (16S rDNA). The specificity of the primers was tested using genomic DNAs of 3 strains of P. mikwangii and 27 strains (27 species) of non-P. mikwangii bacteria. The sensitivity of primers sensitivity was determined using PCR, with serial dilutions of the purified genomic DNAs (4 ng to 4 fg) of P. mikwangii KCOM $1628^T$. The data showed that P. mikwangii-specific qPCR primers (B134-F11/B134-R1 & B134-F5/B134-R5) could detect only P. mikwangii strains, and 400 fg or 40 fg of P. mikwangii genome DNA. These results suggest that PCR primers are useful in detecting P. mikwangii from the oral cavity.