RT-PCR 기법을 이용한 mRNA의 정량

예성수 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.2

Although steady state levels of individual RNA transcripts have traditionally been measured by northern blotting, in situ hybridization and nuclease protection assays, these techniques are limited by their sensitivity. For analysis of mRNA expression, RT-PCR is very sensitive technique. It is capable of detecting moderately expressed transcripts from a single cell. Though mainly used for qualitative studies, several modifications of this method have been developed that allow quantitative analyses to be performed. Quantification of transcription via RT-PCR can be approached using either relative RT-PCR or competitive RT-PCR strategy. Quantification by relative RT-PCR involves normalization to a housekeeping gene that is amplified within the same reaction as the target gene. Competitive RT-PCR, as the name implies, utilizes synthetic competitor RNA within the same reaction as the sample RNA. Recently the introduction of the new procedure based on fluorescence-kinetic RT-PCR enables quantification of the PCR product in real-time. The newest form of quantitative RT-PCR is termed real-time RT-PCR, as measurements are taken throughout the reaction due to oligonucleotide cleavage by DNA polymerase that release a fluorescent dye. The purpose of this review is to summarize the RT-PCR-based quantification methods of mRNA expression for useful clinical applications.

Basic Research Workshop : Basic Research Workshop : Polymerase chain reaction (PCR)

( Kyun Hwan Kim ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.2

The polymerase chain reaction (PCR) is a biochemical technology to amplify a few copies of DNA generating thousands to millions of copies of a particular DNA sequence in molecular biology. The PCR was invented in 1983 by Kary Mullis and is now a common and often indispensable technique used for a variety of applications in medical and biological research labs. In 1993, Dr. Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR. Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs, although some techniques allow for amplification of fragments up to 40 kb in size. The application of PCR include: the selective DNA isolation, DNA cloning for sequencing, functional analysis of genes, amplification and quantification DNA, diagnosis of hereditary diseases, identification of genetic fingerprints such as forensic sciences and paternity testing, and the detection and diagnosis of infectious diseases (pathogens). Recently, the real-time polymerase chain reaction (RT-PCR), also called quantitative real time polymerase chain reaction (RT-qPCR) or kinetic polymerase chain reaction is widely used to amplify and simultaneously quantify a targeted DNA molecule. Real Time-PCR enables both detection and quantification. The quantification can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. In this talk, the basic principles, history, and applications of PCR/RT-PCR will presented.

구제역바이러스 신속진단을 위한 pan-serotype reverse transcription loop-mediated isothermal amplification (RT-LAMP) 진단법

임다래 ( Da-rae Lim ),박유리 ( Yu-ri Park ),박선영 ( Sun-young Park ),김혜령 ( Hye-ryung Kim ),박민지 ( Min-ji Park ),구복경 ( Bok-kyung Ku ),나진주 ( Jin-ju Nah ),유소윤 ( So-yoon Ryoo ),위성환 ( Sung-hwan Wee ),전효성 ( Hyo-sung 한국동물위생학회(구 한국가축위생학회) 2018 韓國家畜衛生學會誌 Vol.41 No.1

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at 62°C and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was 10², 10³ and 10³ TCID₅₀/mL for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

Quantitative Evaluation of Viability- and Apoptosis-Related Genes in Ascaris suum Eggs under Different Culture-Temperature Conditions

Yong-Man Yu,You-Hang Cho,Young-Nam Youn,Juan Hua Quan,In-Wook Choi,Young-Ha Lee 대한기생충학열대의학회 2012 The Korean Journal of Parasitology Vol.50 No.3

Ascaris suum eggs are inactivated by composting conditions; however, it is difficult to find functional changes in heat-treated A. suum eggs. Here, unembryonated A. suum eggs were incubated at 20˚C, 50˚C, and 70˚C in vitro, and the gene expression levels related to viability, such as eukaryotic translation initiation factor 4E (IF4E), phosphofructokinase 1 (PFK1), and thioredoxin 1 (TRX1), and to apoptosis, such as apoptosis-inducing factor 1 (AIF1) and cell death protein 6 (CDP6), were evaluated by real-time quantitative RT-PCR. No prominent morphological alterations were noted in the eggs at 20˚C until day 10. In contrast, the eggs developed rapidly, and embryonated eggs and hatched larvae began to die, starting on day 2 at 50˚C and day 1 at 70˚C. At 20˚C, IF4E, PFK1, and TRX1 mRNA expression was significantly increased from days 2-4; however, AIF1 and CDP6 mRNA expression was not changed significantly. IF4E, PFK1, and TRX1 mRNA expression was markedly decreased from day 2 at 50˚C and 70˚C, whereas AIF1 and CDP6 mRNA expression was significantly increased. The expressions of HSP70 and HSP90 were detected for 9-10 days at 20˚C, for 3-5 days at 50˚C, and for 2 days at 70˚C. Taken together, incremental heat increases were associated with the rapid development of A. suum eggs, decreased expression of genes related to viability, and earlier expression of apoptosis-related genes, and finally these changes of viability- and apoptosis-related genes of A. suum eggs were associated with survival of the eggs under temperature stress.

Multiplex PCR−Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats

Kim, Won-Keun,No, Jin Sun,Lee, Seung-Ho,Song, Dong Hyun,Lee, Daesang,Kim, Jeong-Ah,Gu, Se Hun,Park, Sunhye,Jeong, Seong Tae,Kim, Heung-Chul,Klein, Terry A.,Wiley, Michael R.,Palacios, Gustavo,Song, Ji U.S. Department of Health and Human Services * Cen 2018 Emerging Infectious Diseases Vol.24 No.2

<P>Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by <I>Rattus norvegicus</I> and <I>R. rattus</I> rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR–based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of <I>R. norvegicus</I> rats in South Korea during 2000–2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.</P>

Antiviral Properties of Probiotic Mixtures against Rotavirus in the Rat

박재은,이도경,김민지,김경태,최경순,서재구,하남주,Park, Jae Eun,Lee, Do Kyung,Kim, Min Ji,Kim, Kyung Tae,Choi, Kyung Soon,Seo, Jae Goo,Ha, Nam Joo The Microbiological Society of Korea 2014 미생물학회지 Vol.50 No.4

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. Rotavirus is a major cause of acute gastroenteritis in young children in developed and developing countries. The use of probiotics for the treatment of gastrointestinal diseases is both safe and easily accessible. In this study, we evaluated the anti-rotaviral activities of probiotic mixtures in a Sprague-Dawley rat. 24 litters with their dams were randomly assigned to four groups; placebo, phosphate buffered saline (PBS), and two probiotic mixture (PRO-1 and PRO-2) groups. All rats were inoculated with rotavirus at dose of 8 log plaque forming units per rat at 5 days old. Animals in the PRO-1 and PRO-2 groups were orally administered probiotic mixtures 1 or 2, respectively, at a dose of 8 log colony forming units daily during 4 days. For control purposes, placebo and PBS groups were orally administered the same amount of placebo (containing maltose and polydextrose) or PBS once daily for 4 days, respectively. Antiviral analysis was performed by real-time quantitative PCR (RT-qPCR) and observing intestinal villi. As a result, weights of small intestines were greater in the PRO-1, PRO-2 groups than in control groups. Villi were short and villous epithelial necrosis was exhibited in control groups, but these morphological changes were not observed in PRO-1, PRO-2 treated rats. RT-qPCR analysis showed that VP7 gene level of rotavirus in fecal samples and small intestinal epithelial cells were lower in the PRO-1 and PRO-2 groups. These findings suggest that probiotic mixtures may be useful probiotics for the treatment of or as alternative therapies for rotaviral gastroenteritis.

결핵균 감염에 의한 대식세포에서의 TNFα 유전자 발현 증가

송호연 순천향의학연구소 2001 Journal of Soonchunhyang Medical Science Vol.7 No.2

Mycobactetium tuberculosis is capable of growing and survival dormant for a long time within macrophages. This might be due to a balance between antimicrobial activity of host cell and pathogencity of M. tuberculosis. This study examined gene regulation in human monocyte derived THP-1 cell infected with mycobacteria. Gene expression was evaluated by differential display RT-PCR and was compared to uninfected cells. Results were confirmed by real time quantitative RT-PCR(TaqMan). Among many up or down-regulated clones, 25 colnes were sequenced and compared with known genes on GenBank. One of over-expressed clones from THP-1 cells infected with mycobacteria was identical to human tumor necrosis factorα(TNFα). In particular, the expression of TNFα was highly over-expressed in THP-1 cells infected with M. tuberculosis H_(37)Ra(avirulent) than H_(37)Rv(virulent). This may in fact be a mycobactetial virulence tactic.

mRNA sequence analysis and quantitative expression of the ADAMTS4 gene in the thoroughbred horse

문재우,안궁,배진한,남규휘,조병욱,박경도,이학교,양영목,김태훈,성환후,한규동,김희수 한국유전학회 2012 Genes & Genomics Vol.34 No.4

Cartilage increases flexibility of motion and helps protect the body from physical shock. Strong physical shock or some biological factor could cause joint disease. ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4)has been related to degradation of aggrecans in cartilage. It has been associated with joint disease, which could influence the ability of horses to exercise. Here, we performed sequence analysis and expression profiling of the ADAMTS4 gene in thoroughbred horses. Quantitative real-time RT-PCR data indicated that higher expression of the ADAMTS4 gene appeared in the cartilage tissues compared to those of pancreas, stomach,lung and colon. The expression pattern was also higher in the muscle tissues after exercise than before exercise. These data could be of great use for further studies in relation to both horse racing and joint disease.

Validation of One-Step Real-Time RT-PCR Assay in Combination with Automated RNA Extraction for Rapid Detection and Quantitation of Hepatitis C Virus RNA for Routine Testing in Clinical Specimens

( Byoung Guk Kim ),( Hye Sung Jeong ),( Sun Young Baek ),( Jin Ho Shin ),( Jae Ok Kim ),( Kyung Il Min ),( Seung Rel Ryu ),( Bok Soon Min ),( Do Keun Kim ),( Yong Seok Jeong ),( Sue Nie Park ) 한국미생물생명공학회 2005 Journal of microbiology and biotechnology Vol.15 No.3

Development of a multiplex qRT-PCR assay for detection of African swine fever virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus

Yating Chen,Kaichuang Shi,Huixin Liu,Yanwen Yin,Jing Zhao,Feng Long,Wenjun Lu,Hongbin Si 대한수의학회 2021 Journal of Veterinary Science Vol.22 No.6

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.