만성 타액선염에서 Cathelicidin의 발현

정지용,정승원,우정수,황순재,이흥만 대한이비인후과학회 2005 대한이비인후과학회지 두경부외과학 Vol.48 No.2

Background and Objectives:Salivary secretions and the secreted IgA in the secretions play a critical role in maintaining oral health via innate host defense mechanism. Cathelicidins are a family of peptides thought to provide an innate defensive barrier against a variety of potential microbial pathogens. LL-37, an antimicrobial peptide, is the only Cathelicidin protein so far identified in humans. The purpose of this study was to examine the expression of Cathelicidin in human salivary glands and to investigate upregulation of Cathelicidin in inflammatory conditions. Materials and Method:Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed on 20 salivary gland tissues, of which 10 were normal and 10 were chronic sialadenitis. Results:Cathelicidin mRNA transcripts were detected in the normal salivary glands and chronic sialadenitis. The level of Cathelicidin mRNA in chronic sialadenitis was significantly increased compared with that in the normal salivary gland. Cathelicidin protein was expressed in the glandular epithelium of the normal salivary gland and chronic sialadenitis. Conclusion:The results indicate that Cathelicidin might play an important role in the innate host defense of human salivary glands.

Detection of Norovirus in Contaminated Ham by Reverse Transcriptase-PCR and Nested PCR

Seok Ryel Kim,Duwoon Kim,Ki-Sung Kwon,In-Gyun Hwang,Myung-Joo Oh 한국식품과학회 2008 Food Science and Biotechnology Vol.17 No.3

In order to enhance the efficacy of norovirus detection by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR, this study developed a norovirus mRNA concentration method using poly oligo dT-conjugated magnetic beads. An efficient norovirus detection protocol was performed on commercial ham using 2 viral elution buffers (glycine buffer and Tris beef extract buffer) and 2 concentration solutions [polyethylene glycol (PEG) and zirconium hydroxide]. The different approaches were verified by RT-PCR and nested PCR. This method was performed on ham in less than 8 hr by artificial inoculation of serial dilutions of the virus ranging from 1,000 to 1 RT-PCR unit/mL. The viral extraction and concentration method had 10-fold higher sensitivity using the combination of Tris beef extract buffer and PEG as compared to glycine buffer and zirconium hydroxide. This method proved that RT-PCR and nested PCR have the sensitive ability to detect norovirus in commercial ham, in that norovirus was successfully detected in artificially contaminated samples at a detection level as low as 1-10 RT-PCR unit/mL. Overall, such a detection limit suggests this protocol is both quick and efficient in terms of its potential use for detecting norovirus in meat products.

Analysis of clinical information and reverse transcriptase-polymerase chain reaction for early diagnosis of enteroviral meningitis

Jin, Dahee,Heo, Tae Hoon,Byeon, Jung Hye,Kim, Gun-Ha,Kim, Mi Kyung,Eun, So-Hee,Eun, Baik-Lin The Korean Pediatric Society 2015 Clinical and Experimental Pediatrics (CEP) Vol.58 No.11

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.

Reference gene selection for RT-qPCR analysis in two invasive whiteflies after the acquisition of vectored or non-vectored viruses

Zhen-Hong Lv,Hui-Peng Pan,Wei Zhang,Tian-Bo Ding,Dong Chu 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.1

Custom reference gene selection is essential for reverse transcriptase-quantitative polymerase chain reaction(RT-qPCR) in different species of insects and various experiment conditions. In this study, 14 candidate referencegenes (HSP40, HSP20, HSP70, HSP90, v-ATPase, RPL29, EF-1, SDHA, Actin, PPIA, GAPDH, MyosinL,NADH, and γ-tubulin) were analyzed using five different programs including ΔCt method, BestKeeper, geNorm,NormFinder, and ReFinder to validate their use as reference genes in two invasive whiteflies, Bemisia tabaci Band Q, after acquiring the vectored virus, Tomato yellow leaf curl virus (TYLCV), or ingesting the non-vectoredvirus, Tomato spotted wilt virus (TSWV), respectively. The results showed that HSP40, v-ATPase, and EF-1 werethe most stable genes in B. tabaci B (B. tabaci B feeding on the healthy, TYLCV- and TSWV-infected tomatoplant), PPIA, SDHA, and RPL29 were the most stable genes in B. tabaci Q (B. tabaci Q feeding on the healthy,TYLCV- and TSWV-infected tomato plant). In addition, EF-1, RPL29, and HSP20 were the most stable referencegenes in B. tabaci B and Q. These findings provide the basis for future RT-qPCR-based studies on whitefly-virus interactions. Meanwhile,this report may set a precedent for reference gene selection in insects after the ingestion of non-vectored viruses.

Experience of percutaneous tracheostomy in critically ill COVID-19 patients

김은진,유은형,정치영,김경찬 대한중환자의학회 2020 Acute and Critical Care Vol.35 No.4

Background: Coronavirus disease 2019 (COVID-19) is a highly contagious disease that causes respiratory failure. Tracheostomy is an essential procedure in critically ill COVID-19 patients; however, it is an aerosol-generating technique and thus carries the risk of infection transmission. We report our experience with percutaneous tracheostomy and its safety in a real medical setting. Methods: During the COVID-19 outbreak, 13 critically ill patients were admitted to the intensive care unit (ICU) at Daegu Catholic University Medical Center between February 24 and April 30, 2020. Seven of these patients underwent percutaneous tracheostomy using Ciaglia Blue Rhino. The medical environment, percutaneous tracheostomy method, and COVID-19 reverse transcriptase-polymerase chain reaction (RT-PCR) results were retrospectively reviewed. After treatment, the COVID-19 infection status of healthcare personnel was investigated by RT-PCR. Results: The ICU contained negative pressure cohort areas and isolation rooms, and healthcare personnel wore a powered air-purifying respirator system. We performed seven cases of percutaneous tracheostomy in the same way as in patients without COVID-19. Five patients (71.4%) tested positive for COVID-19 by RT-PCR at the time of tracheostomy. The median cycle threshold value for the RNA-dependent RNA polymerase was 30.60 (interquartile range [IQR], 25.50–36.56) in the upper respiratory tract and 35.04 (IQR, 28.40–36.74) in the lower respiratory tract. All healthcare personnel tested negative for COVID-19 by RT-PCR. Conclusions: Percutaneous tracheostomy was performed with conventional methods in the negative pressure cohort area. It was safe to perform percutaneous tracheostomy in an environment of COVID-19 infection.

C형 간염바이러스 검출을 위한 역전사효소-중합효소연쇄반응 방법들의 비교

이미경,박애자 중앙대학교 의과대학 의과학연구소 1998 中央醫大誌 Vol.23 No.2

HCV-RNA의 검출은 역전사효소를 사용하여 HCV-RNA를 HCV cDNA로 전환한 후 중합효소 연쇄반응으로 검출하는 역전사효소-중합효소 연쇄반응을 이용하며, C형 간염의 진단과 치료 판정에 매우 유용하지만, 검사 과정에 시간과 노력이 많이 들고 위양성과 위음성의 위험이 있어, 실제 임상 검사실에서 일상적으로 시행하기에는 여러 가지 문제점이 있어 왔다. 이에 저자들은 대부분의 시약을 본 검사실에서 만들어 사용하던 본원의 방법과 역전사 반응과 1차 중합효소 연쇄반응이 한 tube에서 시행되도록 고안된 2가지의 HCV RNA detection kit를 비교하고 그 유용성에 대해서 검토하여 보고자, 중앙대학교 부속병원에서 HCV RNA가 검출되었던 양성 검체 30개와 C형 간염 항체는 양성이었으나 HCV RNA가 검출되지 않았던 음성 검체 10개를 대상으로 본 연구를 시행하였다. HCV RNA 양성 검체 30개 중 HCV RNA를 검출해내지 못한 경우는 본원의 방법에서 2, 검체, kit A와 kit B의 방법에서 각각 3 검체로 비슷한 결과를 나타내었고, HCV RNA가 검출되지 않았던 음성 검체 10개 중 1검체에서 kit B에서만 HCV RNA가 검출되었다. 한편, 본원의 검사법이 매우 번거롭고 시간과 노력이 많이 드는데 비해, 2 가지의 HCV detection kit는 모두 RNA 추출과정도 매우 간단하고 cDNA 합성과 1차 PCR이 한 tube에서 동시에 시행된다는 장점을 갖고 있으며, 검사 비용도 비교적 경제적이었다. 그러나 본원의 방법을 포합한 3 가지 방법 모두 상당한 불일치를 보여주고 있어, 동일한 검체의 반복 검사시 문제시되는 재현성의 향상을 시판되는 kit에서도 여전히 남아 있는 것으로 판단되었다. 결론적으로 기존의 방법에 비해 시판되는 kit는 검사 시간과 노동력 절감의 효과는 있으나, 재현성의 향상은 개선되지 않은 것으로 판단되었다. Diagnosis for the hepatitis C virus(HCV) infection currently is based on the detection of anti-HCV antibodies or HCV RNA. The HCV RNA in the patient's sera can detected in the early acute infection stage before antibody appearance or aminotransferase elevation and from the chronic patients who failed to produce antibodies. This technology, however, has not been standadized well and is laborious and expensive. Two HCV detection kits(A and B) were compared with HCV reverse transcriptase-polymerase chain reaction(RT-PCR) method of Choun-Ang University Hospital to establish the most sensitive and relaible method for HCV RNA detection. We tested 30 HCV RNA positive and 10 HCV RNA negative sera. Agreement with three HCV RT-PCR methods was 82.5%. In using HCV detection kits, the reverse transcription and first round PCR step are carried out in a single tube. The lesser manipulations reduced risks of contamination and labour. But the two kits appeared not to be improved in sensitivity and reproducibility of the HCV RT-PCR test.

Analysis of clinical information and reverse transcriptase-polymerase chain reaction for early diagnosis of enteroviral meningitis

진다희,허태훈,변정혜,김건하,김미경,은소희,은백린 대한소아청소년과학회 2015 Clinical and Experimental Pediatrics (CEP) Vol.58 No.11

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 transcriptasepolymerase 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.

대유행 인플루엔자(H1N1 2009)에서 신속항원검사 양성률

김영근,김효열,어영,전진경 대한감염학회 2010 감염과 화학요법 Vol.42 No.2

There are few datas on the diagnostic accuracy of rapid antigen test for pandemic influenza A (H1N1). We evaluated the performance of rapid antigen test for the diagnosis of pandemic influenza A (H1N1). During the period from 21 August 2009 to 20 September 2009, 451 patients with influenza-like symptom underwent the rapid antigen test (SD Influenza Antigen, Standard Diagnostics, Yongin, Korea) and real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) at the same time. Rapid antigen test results were positive for 65 of 90 patients with the positive results for pandemic influenza A (H1N1) on rRT-PCR assay. The sensitivity of the rapid antigen test was 72.2% (95% CI, 61.8-81.1) and the specificity was 100% (95% CI, 99.0-100). The positive predictive value was 100% (95% CI, 94.5-100) and negative predictive value was 93.5% (95% CI, 90.6-95.8).

Zinc Status Assessment by Analysis of Mononuclear Cell Metallothionein mRNA Using Competitive-Reverse Transcriptase-Polymerase Chain Reaction

Lee, Soo-Lim,Yoon, Jin-Sook,Kwon, Chong-Suk,Beattie, John H.,Kwun, In-Sook The Korean Society of Food Science and Nutrition 2004 Preventive Nutrition and Food Science Vol.9 No.3

Marginal Zn deficiency is prevalent through the world and yet human zinc status has not been properly assessed due to the lack of a reliable diagnostic indicator. One potential possibility for zinc status assessment using Zn-binding protein, metallothionein (MT)-mRNA, has been proposed. The purpose of the present study was aimed to show whether measurement of mononuclear cell (MNC) MT mRNA, using a competitive-reverse transcriptase-polymerase chain reaction (competitive-RT-PCR) assay, could indicate zinc status in human subjects. In this study, MNC MT-mRNA expression was measured using a competitive-RT-PCR to compare before and after 14 days of zinc supplementation (50 mg Zn/das zinc gluconate). RT-PCR oligonucleotide primers which were designed to amplify both a 278 bp segment of the human MT-2A cDNA and a 198 bp mutant competitor cDNA template from MNCs, were prepared. MT-2A mRNA was normalized by reference to the housekeeping gene, $\beta$-actin, mRNA for which was also measured by competitive-RT-PCR. There was considerable inter-individual variation in MT-mRNA concentration and yet, the mean MT-2A mRNA level increased 4.7-fold after Zn supplementation, as compared to before Zn supplementation. This MT-2A mRNA level was shown as the same pattern and, even more sensitive assay, compared to the conventional plasma and red blood cells (RBCs) Zn assessment in which plasma and RBCs zinc levels increased 2.3- and 1.2-fold, respectively (p<0.05). We suggest that MT competitive-RT-PCR can be a useful assessment tool for evaluating human zinc status.

면역능력이 저하된 소아에서 Multiplex RT-PCR에 의한 비인두 상주균 검출의 의의

김경희,신지혜,김선영 대한혈액학회 2009 Blood Research Vol.44 No.4

Background: The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in immunocompromized children by using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. Methods: We obtained clinical samples by nasopharyngeal swabs from 42 patients with underlying immune deficiency from May 20, 2008 to May 22, 2008. The children were free from signs of respiratory tract infections at the time of sampling. Isolated cDNA was extracted and after this the DNA was examined using a multiplex primer set for pneumonial bacteria detection (SeeplexⓇ PneumoBacter ACE Detection, Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with ethidium bromide and a screentape system (Lab901, Scottland, UK) and then they were compared. The nasopharyngeal swab culture was done simultaneously and this was compared with the results of mRT-PCR. Results: A total of 42 patients (males: 24, females: 18) aged between 1.2 and 16.3 years (median: 9.2 years) were included in this study. The mRT-PCR detected bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 28 patients (66.6%). Of these 28 patients, 4 patients (14.3%) showed more than 2 bacteria: 2 patients were positive for two bacteria (S. pneumoniae and H. influenzae, H.influenzae and B. pertussis) and 2 patients were positive for three bacteria (S. pneumoniae, B. pertussis and C. pneumoniae, C. Pneumoniae, H. influenzae and B. pertussis). S. pneumoniae was cultured in one patient (2.4%). Conclusions: The mRT-PCR is a sensitive tool for the detection of the asymptomatic nasopharyngeal carriages. The clinical significance of the bacteria detected in immunocompromized patients by mRT-PCR will need further evaluation.