• Clinical and Experimental Pediatrics
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• 제52권12호
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• pp.1358-1363
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• 2009

목 적:호흡기 감염의 증상이 없는 소아들을 대상으로 다중 역전사중합효소연쇄반응법(multiplex reverse transcription-polymerase chain reaction; mRT-PCR)을 이용하여 비인두 상주균의 이환율을 알아보고자 하였다. 방 법:2008년 7월 25일부터 28일까지 33명의 소아들을 대상으로 비강 면봉채취법으로 검체를 채취하였으며, 이들은 검체 채취 당시 심각한 호흡기 감염의 증상이 없었다. 모아진 검체에서 DNA를 추출한 후 multiplex primer set ($Seeplex^{(R)}$ PneumoBacter ACE Detection, Seegene, Seoul, Korea)로 PCR을 진행하였다. 증폭된 반응산물은 2% agarose gel과 전기 영동 자동화 시스템인 screen tape system (Lab901, Scottland, UK)에 각각 전기 영동하여 확인하였다. 결 과:전체 33명의 소아 중 남아는 15명 여아는 18명이었으며, 나이는 3.2세에서 16.3세로 중앙값은 8.2세였다. mRT-PCR 결과 30명(90.9%)의 소아들에서 양성을 보였으며(S. pneumoniae, H. influenzae, C. pneumoniae, B. pertussis), 이들 중 13명(39.4%)에서 2가지 이상의 균이 검출되었다. 균의 종류로는 12명(36.4%)에서는 S. pneumoniae와 H. influenzae, 1명(3.0%)에서는 S. pneumoniae, H. influenzae와 C. pneumoniae이 검출되었다.. 결 론:mRT-PCR은 비인두 상주균의 동정에 있어서 민감도가 높은 방법으로 생각된다. 하지만 비인두 상주균에 대한 PCR 결과가 소아들의 임상 양상과 어느 정도 일치할지에 대해서는 더 많은 연구가 필요할 것으로 생각된다.

• Clinical and Experimental Pediatrics
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• 제58권11호
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• pp.446-450
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• 2015

Purpose: Meningitis is among the most common infections affecting the central nervous system. It can be difficult to determine the exact pathogen responsible for the infection and patients are often treated with empiric antibiotics. This study was conducted to identify the most common clinical characteristics of enteroviral meningitis in children and evaluate the diagnostic efficacy of reverse transcriptase-polymerase chain reaction (RT-PCR) for early detection of an enterovirus. Methods: We analyzed the medical records of children admitted to Korea University Medical Center and diagnosed with meningitis on the basis of cerebrospinal fluid (CSF) analysis and RT-PCR from CSF and other samples from January 2010 to August 2013. Results: A total of 333 patients were enrolled and classified into four groups based on diagnosis: enteroviral meningitis (n=110), bacterial meningitis (n=23), other viral meningitis (n=36), and unknown etiology (n=164). Patients with bacterial meningitis were younger than those in the other groups (P<0.001). Pleocytosis in CSF was similar across all groups. Of patients in the enteroviral meningitis group, 92.7% were diagnosed based on RT-PCR findings. Mean length of hospital stay for patients with enteroviral meningitis was 6.08 days, which was significantly shorter than that for patients with meningitis of bacterial etiology (19.73 days, P<0.001). Conclusion: Diagnosis of enteroviral meningitis before viral culture results are available is possible using RT-PCR. Accurate diagnosis reduces the length of hospital stay and helps to avoid unnecessary empiric antibiotic treatment.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1384-1387
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• 2005

Hantavax(R) is one of the killed Hantavirus vaccines, and is commercially available in South Korea. This vaccine was developed by inactivation of virus isolated from infected suckling mouse brain with formalin. Although Hantavax(R) can induce neutralizing antibodies in vaccinees, the strength of this induction and the duration of the humoral immune response are controversial issues. In this study, we studied the native conformation of the killed vaccine by antigen-capture reverse transcriptase polymerase chain reaction with patient and vaccinee sera containing neutralizing antibodies against Hantavirus. The results showed that Hantavax(R) could bind HTNV patient and vaccinee sera like live virus, suggesting that the integrity of the viral epitope is maintained in Hantavax(R) and induces the protective antibodies, even though the virus was inactivated with formalin.

• BMB Reports
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• 제45권3호
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• pp.165-170
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• 2012

We report here that an ethanol extract of Tetracera scandens, a Vietnamese medicinal plant, has anti-HIV activity and possesses strong inhibitory activity against HIV-1 reverse transcriptase (RTase). Using a MT-4 cell-based assay, we found that the T. scandens extract inhibited effectively HIV virus replication with an $IC_{50}$ value in the range of 2.0-2.5 ${\mu}g$/ml while the cellular toxicity value (CC50) was more than 40-50 ${\mu}g$/ml concentration, thus yielding a minimum specificity index of 20-fold. Moreover, the anti-HIV efficacy of the T. scandens extract was determined to be due, in part, to its potent inhibitory activity against HIV-1 RTase activity in vitro. The inhibitory activity against the RTase was further confirmed by probing viral cDNA production, an intermediate of viral reverse transcription, in virus-infected cells using quantitative DNA-PCR analysis. Thus, these results suggest that T. scandens can be a useful source for the isolation and development of new anti-HIV-1 inhibitor(s).

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.2923-2928
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• 2015

Background: Gallbladder carcinoma (GBC) has been stated as an Indian disease, with the highest number of cases being reported from certain districts of northeast India, which has an ethnically distinct population. Unfortunately there are no scientific reports on the underlying molecular mechanisms associated with the pathogenesis of the disease from this region. Aim: The present study evaluated the role of differential expression of human telomerase reverse transcriptase (hTERT) in the development of gall bladder anomalies. Materials and Methods: Blood and tissue samples were collected from patients undergoing routine surgical resection for clinically proven cases of gallbladder disease {cholelithiasis (CL, n=50), cholecystitis (CS, n=40) and GBC (n=30) along with adjacent histopathologically proved non-neoplastic controls (n=15)} with informed consent. Whole blood was also collected from age and sex matched healthy controls (n=25) for comparative analysis. Differential hTERT mRNA expression was evaluated by semi-quantitative rt-PCR and real-time PCR based analysis using ${\beta}$-actin as an internal control. Evaluation of differential hTERT protein expression was studied by Western blot analysis and immunoflourescence. Statistical analysis for differential expression and co-relation was performed by SPSSv13.0 software. Results: Gallbladder anomalies were mostly prevalent in females. The hTERT mRNA and protein expression increased gradiently from normal

• Asian-Australasian Journal of Animal Sciences
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• 제31권4호
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• pp.467-472
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• 2018

Objective: Molecular cloning and bioinformatics analysis of annexin A2 (ANXA2) gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer (Cervus Nippon hortulorum) and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). Results: The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period). Conclusion: ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

• Journal of Animal Science and Technology
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• 제46권5호
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• pp.841-848
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• 2004

살균된 우유에 오염된 Salmonella의 신속한 검출 방법을 공중 보건의 보호를 위하여 중요하다. RT-PCR 방법은 생균과 사균을 분별하여 검출할 수 있는 분자유전학적 방법이다. 본 연구의 RT-PCR 방법은 Salmonella의 type 1 fImbriae의 단량체 단백질을 암호화하는 fimA 유전자의 mRNA를 주형으로 하여 DNA를 증폭하는 방법으로서 활력이 있는 Salmonella를 검출할수 있기 위하여 개발되었다. RNA 시료를 RQRNase-free DNase로 처리하여 오염된 DNA를 파괴하여 RT-PCR 반응에서 주형으로서 DNA가 합성되는 것을 방지할 수 있었다. 이 RT-PCR 방법은 Salmonella 7균주를 검출하였으나 Escherichia coli, Shigella sonnei, Citrobacter freundii, 및 Klebsiella pneumoniae 는 검출하지 않았다. 우유 내에서 열처리된 $10^7/ml과 10^6/ml$ 세균수의 Salmonella는 검출되 었으나 $10^5{\sim}100/ml$는 검출되지 않았다. RT-PCR를 검출할 수 있는 활력이 있는 Salmonella의 최소 세균수는 100/ml이었다.

• 한국어병학회지
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• 제5권2호
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• pp.143-152
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• 1992

Metallothionein은 세포내의 중금속의 농도을 조절하는 주요한 단백질로서 bacteria에서 척추동물에 이르기까지 모든 생명체에서 나타나는 공통된 단백질이다. 비록 metallothionein의 정확한 기능은 알려져 있지 않으나 독성을 나타내는 중금속에 대하여 세포내 방어기작에 관여할 뿐만 아니라 여러다른 유전자의 총괄적 조절기작 및 matalloprotein의 발현에 관여할 것으로 보고있다. 본 연구에서는 Channel Catfish의 metallothionein cDNA 유전자를 poly(A)를 갖는 mRNA로 부터 Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR)에 의하여 cloning하였다. 증폭된 PCR products는 pBluescript SK+의 EcoRV site 및 pUC19의 Smal site에 dT tailing을 하여 cloning하였으며, PCR products는 multicloning site에 있는 EcoRI 및 HindIII 로 절단하여 확인하거나 신속한 PCR screening에 의하여 확인하였다. 여러 PCR clone 중 하나인 pMT150에 대한 DNA 염기서열을 조사한 결과 다른 어류의 metallothionein cDNA 유전자와 높은 유사성을 보였다.

• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.121-127
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• 2009

Sequencing analysis of a 5-kb DNA fragment from Streptomyces venezuelae ATCC 15439 revealed the presence of one 3.1-kb open reading frame(ORF), designated as afsR-sv. The deduced product of afsR-sv(1,056 aa) was found to have high homology with the global regulatory protein AfsR. Homology-based analysis showed that aftR-sv represents a transcriptional activator belonging to the Streptomyces antibiotic regulatory protein(SARP) family that includes an N-terminal SARP domain containing a bacterial transcriptional activation domain(BTAD), an NB-ARC domain, and a C-terminal tetratricopeptide repeat domain. Gene expression analysis by reverse transcriptase PCR(RT-PCR) demonstrated the activation of transcription of genes belonging to pikromycin production, when aftR-sv was overexpressed in S. venezuelae. Heterologous expression of the aftR-sv in different Streptomyces strains resulted in increased production of the respective antibiotics, suggesting that afsR-sv is a positive regulator of antibiotics biosynthesis.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.685-689
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• 2000

Due to the uncultivable nature of Mycobacterium leprae in vitro, the fast, easy, and accurate measurement of the antimicrobial drug susceptibility of this microbe has been difficult. Conventional methods for such testing are subjective, cumbersome, and expensive in some cases. Here, the utility of a reverse transcriptase-PCR (RT-PCR)-based assay for testing was examined and compared with a Buddmeyer-type radiorespirometric assay. The susceptibility of M. leprae to rifampin was determined by probing the presence of M.leprae-specific 18 kDa gene mRNA in M. leprae-infected IC-21 macrophage cells after drug treatment. The results showed that, as the refampin concentration was increased, the 360-bp cDNA products generated by the RT-PCR-based assay decreased in a dose-dependent manner as in the drug susceptibility observed in the Buddmeyer-type assay. The drug susceptibility testing of M. leprae by the RT-PCR based assay was found to be not only faster but also nearly $10^4$-fold more sensitive than the Buddmeyer-type assay. Moreover, it was also found that, unlike the RT-PCR based assay, the same testing by a DNA-PCR resulted in no differences in the 360-bp signal, regardless of the rifampin concentrations used. Accordingly, these results demonstrated that the drug susceptibility of M. leprae can be determined effectively by an RT-PCR-based assay, thereby providing a new, fast, and sensitive testing method.