• Journal of Veterinary Science
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• 제22권6호
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• pp.87.1-87.12
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• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• 생명과학회지
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• 제25권2호
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• pp.136-150
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• 2015

Pseudomonas syringae pv. tabaci 11528은 담배를 숙주로 하여 wildfire disease를 일으키는 식물 병원성 세균이다. P. syringae pv. tabaci psyR deletion mutant를 이용하여 swarming motility, tabtoxin 생산능, siderophore 생산능, AHL 생산능 등의 phenotypic test를 수행하였다. psyR deletion mutant는 wild-type 균주보다 swarming motility가 증가하였고, tabtoxin 생산 또한 증가하였다. 하지만 siderophore와 AHL 생산능은 감소하였고 virulence 또한 지연되었다. 이러한 결과로 PsyR이 QS regulator로 작용한다는 사실과 더불어 병원성 유전자의 조절에도 관여한다는 것을 확인하였다. PsyR이 각각의 병원성 유전자의 발현을 조절하는 regulator들에게 미치는 영향을 전사단계에서 확인하기 위해 fur, gacA, psyI, prhI, prhA, hrpR, hrpA 유전자들을 정량적 real-time PCR (qRT-PCR) 방법으로 확인하였다. 또한 PsyR에 의한 병원성 유전자 조절이 DNA상에 직접적으로 결합하여 일어나는 것인지 아니면 다른 경로를 통해 간접적으로 일어나는 것인지를 확인할 필요가 있어 정제한 PsyR 단백질과 병원성 관련 유전자들의 upstream region 서열을 이용하여 electrophoretic mobility shift assay (EMSA)를 수행한 결과 본 연구에서 선정한 병원성 관련 유전자들이 PsyR에 의해 직접적으로 조절되지는 않는다는 사실을 밝혔다.

• 대한임상검사과학회지
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• 제51권1호
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• pp.64-70
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• 2019

Dirofilaria immitis (D. immitis)는 개의 심폐사상충증을 일으키는 선형사상충이다. 이 기생충에 감염된 개는 감염 후기 단계에서 하나 이상의 증상과 혈관 주위의 염증을 동반한 심화된 심장 질환을 보인다. 감염 초기단계에 특이적이고 효율적으로 D. immitis를 검출하기 위해서, 선행연구에서 밝혀낸 D. immitis의 cytochrome c oxidase subunit I (COI)와 개의 glyceraldehyde-3-phosphate dehydrogenase (GAPDH)를 검출하는 특이적인 프라이머와 프로브를 이용하여 이중 TaqMan qPCR 방법을 개발했다. 양성 대조군인 플라스미드 유전자는 TA-cloning vector와 D. immitis의 COI나 개의 GAPDH로 구성되었다. 단일과 이중 TaqMan qPCR 방법은 특이적인 프라이머와 프로브, 그리고 게놈 유전자나 플라스미드 유전자로 수행했다. 프라이머의 농도를 최적한 후, 본 연구에서 개발한 이중 반응은 D. immitis의 COI와 개의 GAPDH를 동일 시료에서 동시에 검출했다. 검출 한계는 단일과 이중 방법 모두 25 copies였고, 두 방법 모두 좋은 선형성과 높은 민감도, 그리고 우수한 PCR 효율을 보여주었다. 병원체를 검출하기 위한 이중 방법은 단일 방법에 비해 비용과 노동력, 시간이 적게 든다. 따라서 이중 TaqMan qPCR 방법의 개발은 많은 수의 시료로부터 동시에 효율적으로 D. immitis 검출과 정량이 가능하게 할 것이다.

• 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.

• Journal of Microbiology and Biotechnology
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• 제22권11호
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• pp.1575-1579
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• 2012

Paenibacillus polymyxa is known to be a plant-growth-promoting rhizobacterium. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection and quantitation of P. polymyxa using a primer pair based on the sequence of a membrane-fusion protein for the amplification of a 268 bp DNA fragment. This study reports that the qPCR-based method is applicable for the rapid and sensitive detection of P. polymyxa and can be used as an alternative method for agricultural soil monitoring.

• 한국물환경학회지
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• 제34권1호
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• pp.46-56
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• 2018

A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

• 대한소아치과학회지
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• 제45권1호
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• pp.32-40
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• 2018

본 연구는 치태의 성숙도에 따라 치태를 서로 다른 색상으로 염색하는 GC Tri Plaque ID $Gel^{TM}$(GC corporation, Tokyo, Japan)을 이용하여 치아 우식 위험도를 평가하고자 하였다. 치아 우식의 발생 및 진행과 연관된 균인 Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp.의 수를 Quantitative real-time polymerase chain reaction (qRT-PCR)로 측정하여 치아 우식 위험도를 보았다. 본 실험은 강릉원주대학교 치과병원 임상시험 심사위원회의 심의를 받고 진행하였다. 강릉원주대학교 치과병원 소아치과에 내원한 전신질환이 없는 건강한 9 - 12세의 초등학생 15명의 치면을 착색제로 염색하였다. 치태의 성숙도에 따라 서로 다른 세가지 색상으로 염색되었으며 색상별로 3개의 실험군인 I군(pink/red), II군(blue/purple), III군(light blue)으로 나누었다. 3개의 실험군에서 각각 DNA를 추출한 후, qRT-PCR을 이용하여 S. mutans, S. sobrinus, Lactobacillus spp.의 수를 측정하였다. 3개의 실험군 사이에 S. mutans, S. sobrinus와 Lactobacillus spp. 균 수의 유의한 차이가 관찰되었으며 3종류의 균 모두 III군에서 가장 많이 관찰되었다(p < 0.05). GC Tri Plaque ID $Gel^{TM}$는 기존 착색제와는 달리 치태의 성숙도에 따라 서로 다른 세가지 색상으로 염색되며, 치태 염색 색상의 차이는 치아 우식 관련 균 수의 차이를 보여주었다. GC Tri Plaque ID $Gel^{TM}$이 치아 우식 위험도를 평가하는 하나의 지표로서 사용될 수 있는 가능성을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.630-636
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• 2013

Recently, there has been a growing body of evidence showing that cellular microRNAs (miRNAs) are involved in virus-host interactions. Numerous studies have focused on analyses of the expression profiles of cellular miRNAs, but the expression patterns of Dicer, which is responsible for the generation of miRNAs, have only rarely been explored in Gallus gallus. We developed a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the relative quantification of the mRNAs of Dicer and ${\beta}$-actin in G. gallus. To apply this method, the expression of Dicer in avian cells after infection with avian leukosis virus subgroup J (ALV-J) was detected using our established duplex real-time RT-PCR. The duplex real-time RT-PCR assay is sufficiently sensitive, specific, accurate, reproducible, and cost-effective for the detection of Dicer in G. gallus. Furthermore, this study, for the first time, demonstrated that ALV-J can induce differential expression of Dicer mRNA in the ALV-J-infected cells.

• 한국미생물·생명공학회지
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• 제48권4호
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• pp.447-454
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• 2020

Veillonella spp. have been reported to be the most prevalent nitrate-reducing bacterial species in the oral cavity. The purpose of this study was to examine the relationship between the abundance of Veillonella spp. and nitrite production after nitrate ingestion. Bacterial samples were obtained from the tongue surfaces of 50 university students. The predominant Veillonella spp., V. atypica, V. dispar, and V. rogosae were identified and enumerated using real-time polymerase chain reaction (qPCR). Salivary nitrate and nitrite were measured before and 30, 60, and 90 min after ingestion of 100 ml of beetroot juice. Increased nitrite concentrations were observed in all participants, with a mean increase of 0.61 (0.42-1.10) mM expressed as the median (interquartile range). Veillonella atypica was detected in 40 subjects (80%), V. dispar in 48 (96%), and V. rogosae in 48 (96%), at quantities ranging from 1.3 × 102 to 2.8 × 107 CFU/ml per subject. The strengths of the correlations of the log colony forming unit (CFU) values of V. atypica, V. dispar, V. rogosae, and the log CFU value of the three species together with the increase in nitrite levels were 0.091, 0.114, -0.228, and 0.060, respectively, none of which were significant (p > 0.05). Our results indicate that the abundance of Veillonella spp. is not related to salivary nitrite production after nitrate ingestion.

• Molecules and Cells
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• 제20권3호
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• pp.348-353
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• 2005

Using in silico approaches and RACE we cloned a full length trinucleotide (CAG) repeat-containing cDNA (cag-3). The cDNA is 2478 bp long and the deduced polypeptide consists of 140 amino acids of which 73 are glutamines. The genomic sequence spans approximately 79 kb on mouse chromosome 7 and the gene is composed of four exons. Standard and real-time PCR analyses of several mouse tissues showed that the gene is exclusively expressed in the brain and is not detected in embryonic stages. Within the brain, it is expressed throughout the forebrain region with predominant expression in the hypothalamus and olfactory bulb and very low levels in the mid- and hindbrain.