• Title/Summary/Keyword: Virus detection

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Development of an Automatic PCR System Combined with Magnetic Bead-based Viral RNA Concentration and Extraction

  • MinJi Choi;Won Chang Cho;Seung Wook Chung;Daehong Kim;Il-Hoon Cho
    • Biomedical Science Letters
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    • v.29 no.4
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    • pp.363-370
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    • 2023
  • Human respiratory viral infections such as COVID-19 are highly contagious, so continuous management of airborne viruses is essential. In particular, indoor air monitoring is necessary because the risk of infection increases in poorly ventilated indoors. However, the current method of detecting airborne viruses requires a lot of time from sample collection to confirmation of results. In this study, we proposed a system that can monitor airborne viruses in real time to solve the deficiency of the present method. Air samples were collected in liquid form through a bio sampler, in which case the virus is present in low concentrations. To detect viruses from low-concentration samples, viral RNA was concentrated and extracted using silica-magnetic beads. RNA binds to silica under certain conditions, and by repeating this binding reaction, bulk samples collected from the air can be concentrated. After concentration and extraction, viral RNA is specifically detected through real-time qPCR (quantitative polymerase chain reaction). In addition, based on liquid handling technology, we have developed an automatic machine that automatically performs the entire testing process and can be easily used even by non-experts. To evaluate the system, we performed air sample collection and automated testing using bacteriophage MS2 as a model virus. As a result, the air-collected samples concentrated by 45 times then initial volume, and the detection sensitivity of PCR also confirmed a corresponding improvement.

Responses of Resistant Genes to Barley Yellow Mosaic Virus (BaYMV) Strains in Korea (국내 분포 보리호위축바이러스(Barley Yellow Mosaic Virus) strain에 대한 저항성 유전자 반응)

  • Park, Jong-Chul;Noh, Tae-Hwan;Park, Chul-Soo;Kang, Chun-Sik;Kang, Mi-Hyung;Lee, Eun-Sook;Lee, Jun-Hee;Lee, Jung-Joon;Kim, Tae-Soo
    • Research in Plant Disease
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    • v.15 no.2
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    • pp.72-76
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    • 2009
  • It was investigated the responses of BaYMV resistant genes to Korean BaYMV(Barley yellow mosaic virus) strains. BaYMV was distributed dominantly with about 51% detection ratio among the three investigated virus such as BaYMV, BaMMV(Barley mild mosaic virus) and SBWMV(Soil-borne wheat mosaic virus) in ELISA test. Double infection with BaYMV and BaMMV was detected also higher as 38.8%, however, BaMMV sole infection ratio was lower with only 1.4%. The 11 BaYMV resistant genes were tested their responses to four Korean BaYMV strains, BaYMV-N, H, I and M. Generally, rym 3 genes showed resistant to Korean BaYMV strains and rym 4m and 5a also was better. Three genes, rym 1+5(Mokusekko-3), rym 3(Ea 52, Baitori) and rym 5a(Solan) showed resistant responses to BaYMV-N type. In -H strain test, seven genes that rym 2(Mihori Hadaka 3), rym 3(Ea 52, Haganemugi, Baitori), rym 4m(Diana, Franka), rym 5a(Solan), rym 7(Hor 3365), rym 9(Bulgarian 347), rym 12(Jochiwon Covered 2) were considered as resistant. The three genes that rym 1+5, rym 3 and rym 5a was effective to -I strain, and rym 3, rym 4m and rym 5a showed resistant to -M strain.

Real-Time RT-PCR for Validation of Reovirus Type 3 Safety During the Manufacture of Mammalian Cell Culture-Derived Biopharmaceuticals (세포배양 유래 생물의약품 생산 공정에서 Reovirus Type 3 안전성 검증을 위한 Real-Time RT-PCR)

  • Lee, Dong-Hyuck;Jeong, Hyo-Sun;Kim, Tae-Eun;Oh, Seon-Hwan;Lee, Jung-Suk;Kim, In-Seop
    • Korean Journal of Microbiology
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    • v.44 no.3
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    • pp.228-236
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    • 2008
  • Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to Reovirus type 3 (Reo-3), and there are several reports of Reo-3 contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the Reo-3 safety, a real-time RT-PCR method was developed for quantitative detection of Reo-3 in cell lines, raw materials, manufacturing processes, and final products as well as Reo-3 clearance validation. Specific primers for amplification of Reo-3 RNA was selected, and Reo-3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $3.2{\times}10^0\;TCID_{50}/ml$. The real-time RT-PCR method was proven to be reproducible and very specific to Reo-3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with Reo-3. Reo-3 RNA could be quantified in CHO cell as well as culture supernatant. When the real-time RT-PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of Reo-3.

Distribution of Hard Ticks based on Environments and Detection of Severe Fever with Thrombocytopenia Syndrome Virus in Sangju city, Korea, 2019 (2019년 경북 상주 지역 환경별 참진드기 분포 조사 및 중증열성혈소판감소증후군 바이러스 검출)

  • Lee, JaeSeok;Moon, KyungHwan;Kim, YeongHo;Park, Ye eun;Jeon, Ji Hyang;Kim, Chae Won;Park, Sean;Woo, Ji Hyeon;Jeong, Yeo Jin;Eom, Jong Won;Lee, Wook-Gyo;Kim, Young Ho
    • Korean journal of applied entomology
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    • v.59 no.3
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    • pp.233-241
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    • 2020
  • Several hard tick species in Ixodidae are medically important pests that transmit infectious disease including severe fever with thrombocytopenia syndrome (SFTS). In Korea, the number of SFTS patients continues to steadily increase since its first report in 2013, and Gyeongsangbuk-do (province) is the second most frequent area of SFTS patient occurrence. In order to investigate the distribution of ticks in Sangju city, we collected ticks using the flagging method from 26 sites comprising urban green area, vulnerable area of management, and rural area, and examined SFTS virus from the collected ticks in 2019. Based on Collection Index (CI = tick number / 1 h / 2 people), CI 143 ticks, including Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis, were collected; the most abundant species among the three tick species was H. longicornis (CI 138, 96.5%). Ninety two percent (CI 131) of ticks were collected in vulnerable area of management, where people cannot easily enter and are not managed by city hall, whereas only 8.4% (CI 12) were collected in the urban green area and rural area. Regarding SFTS virus detection, virus was not investigated from 26 pools containing CI 143 ticks. The results of this study are expected to be used as a recommendation for ensuring the safety of local residents from tick-borne diseases.

Sample size of pooled sera for detection of chicken infectious bronchitis virus infection (닭 전염성 기관지염을 검출하기 위한 합병혈청의 표본크기)

  • Pak, Son-Il
    • Journal of Veterinary Clinics
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    • v.24 no.4
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    • pp.603-607
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    • 2007
  • The sample sizes required to detect at least one chicken infectious bronchitis virus(IBV) infection at flock-level were determined using pooled samples for 48 submissions with different samples in each. A total of serum samples of 9,980 layers from Kangwon, Chungpook and Chungnam province were collected and tested hemagglutination inhibition(HI) antibody titers against IBV both individually and with pooling size of 10. Of the 48 submissions, 72.9% were required less than 5 pools to detect at least one infected pool at 95% confidence level, and the corresponding rate was 77.1% at 90% confidence level. Overall, the number of pools was decreased as the percent of positive pools increased. At two different cut-of HI titer${\geq}9\;and{\geq}10$ for individual samples the seroprevalence was 50.1% and 33.4%, respectively while 59.9% were seropositive for pooled samples at HI $titer{\geq}8$. The correlation coefficients between pooled and individual samples at each submission were 0.592(p<0.001) for HI $titer{\geq}9$ and 0.561(p<0.001) for ${\geq}10$, with common correlation coefficient of 0.576. This study indicated that pooled testing for the detection of IBV infection may be an alternative strategy when only the pooled results are of interest and the prevalence has not known exactly.

Diagnostic Significance of Combined Detection of Epstein-Barr Virus Antibodies, VCA/IgA, EA/IgA, Rta/IgG and EBNA1/IgA for Nasopharyngeal Carcinoma

  • Cai, Yong-Lin;Li, Jun;Lu, Ai-Ying;Zheng, Yu-Ming;Zhong, Wei-Ming;Wang, Wei;Gao, Jian-Quan;Zeng, Hong;Cheng, Ji-Ru;Tang, Min-Zhong
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.5
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    • pp.2001-2006
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    • 2014
  • The objective of this study was to investigate the diagnostic significance of EBV antibody combined detection for nasopharyngeal carcinoma (NPC) in a high incidence region of southern China. Two hundred and eleven untreated NPC patients, 203 non-NPC ENT patients, and 210 healthy controls were recruited for the study. The titers of VCA/IgA and EA/IgA were assessed by immunoenzyme assay, and the levels of Rta/IgG and EBNA1/IgA were determined by enzyme-linked immunosorbent assay. The levels of VCA/IgA, EA/IgA, Rta/IgG and EBNA1/IgA demonstrated no association with gender or age (p>0.05). The receiver operating characteristic curve and the area under the curve were used to evaluate the diagnostic value. The sensitivity of VCA/IgA (98.1%) and the specificity of EA/IgA (98.5%) were the highest. When a logistic regression model was used to combine the results from multiple antibodies to increase the accuracy, the combination of VCA/IgA+Rta/IgG, whose area under the curve was 0.99, had the highest diagnostic efficiency, and its sensitivity, specificity and Youden index were 94.8%, 98.0% and 0.93 respectively. The data suggest that the combination of VCA/IgA+Rta/IgG may be most suitable for NPC serodiagnosis.

Detection of Mutations to Zidovudine in the pol Gene of Human Immunodeficiency Virus-1 by Direct Sequencing (인면역결핍 바이러스 pol 유전자 염기서열 결정에 의한 지도부딘 (ZDV) 내성 돌연변이의 탐지)

  • Cho, Young-Keol;Lee, Hee-Jung;Sung, Heung-Sup;Kim, Yoo-Kyum;Kim, Young-Bong;Lee, Yong-Jin;Kim, Mi-Jung;Kim, Dae-Ghon;Won, Young-Ho;Cho, Goon-Jae
    • The Journal of Korean Society of Virology
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    • v.29 no.4
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    • pp.271-281
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    • 1999
  • The nested polymerase chain reaction (PCR) assay was used to determine the sequences of reverse transcriptase (RT) codons 41, 67, 70, 210, 215 and 219 of human immunodeficiency virus-1 (HIV-1) pol gene. Template DNA was obtained from uncultured peripheral blood mononuclear cells from 27 Korean HIV-1 infected patients treated with ZDV and Korean red ginseng. The second PCRs were done for 2 separated regions (RT codons $13{\sim}98$ and $152{\sim}259$) with $5\;{\mu}l$ of the first PCR productNucleotide sequences were determined by direct sequencing. In the 27 patients, CD4+ cell count decreased from $230{\pm}117/{\mu}l$ to $152{\pm}162/{\mu}l$ for $46{\pm}26$ months (Mo), and actual duration of ZDV intake was $72{\pm}16$ Mo. In the 16 patients who had been treated with ZDV therapy ${\ge}25$ Mo, the incidences of 70R, 215F/Y, and 41L were 61%, 28% and 22%, respectively and those of 67N, 210W and 219Q were 17%. The incidences of 215F/Y were 6.7% for group ${\le}12$ Mo treatment, 22.7% for group with 13 to 24 Mo, and 27.8% for group ${\ge}25$ Mo. There was no mutation in 9 patients. It might be associated with the interruption of ZDV therapy for more than 6 months in 6 patients. This study shows that the detection of mutation could be useful prognostic marker with other clinical and virological data, and very low mutation rate is dectected compared to overseas reports.

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Effects of extruded pellet and moist pellet feed on health conditions of olive flounder Paralichthys olivaceus (배합사료 및 습사료 투여가 양식 넙치, Paralichthys olivaceus의 건강도에 미치는 영향)

  • Cho, Mi-Cho;Oh, Yun-Kyeong;Park, Shin-Hoo;Lee, Hae-Young;Kang, Yong-Jin;Park, Myoung-Ae
    • Journal of fish pathology
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    • v.22 no.3
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    • pp.283-291
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    • 2009
  • This study was conducted to evaluate the effects of a commercial extruded pellet (EP) and raw fish moist pellet (MP) diet on disease prevalence and serum chemistry of olive flounder Paralichthys olivaceus grown in two commercial-scale aquaculture farms from July to December in 2008. The contents of serum GOT, GPT and glucose in fish fed EP diet (EP group) were higher than those of fish fed the MP diet (MP group). There were no distinct differences in survival rates and mean detection rates of fish pathogens among fish group fed the experimental diets. However, the mean detection rate of fish pathogens in MP group was higher than that of EP group from July to October which are high water temperature season. The dominant pathogens isolated in EP group were Dactylogyrus sp., E. tarda and red sea bream iridovirus (RSIV). On the other hand, Trichodina sp., Streptococcus spp., viral nervous necrosis virus (VNNV) were dominant in MP group.

Single-tube nested reverse transcription-polymerase chain reaction for simultaneous detection of genotyping of porcine reproductive and respiratory syndrome virus without DNA carryover contamination (DNA 교차오염 방지기능이 있는 single-tube nested reverse transcription-polymerase chain reaction을 이용한 돼지생식기호흡기증후군바이러스 유전형 감별진단)

  • Jeong, Pil-Soo;Park, Su-Jin;Kim, Eun-Mi;Park, Ji-Young;Park, Yu-Ri;Kang, Dae-Young;Cha, Hyun-Ouk;Lee, Kyoung-Ki;Kim, Seong-Hee;Park, Choi-Kyu
    • Korean Journal of Veterinary Service
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    • v.39 no.2
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    • pp.107-116
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    • 2016
  • In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.

Molecular epidemiologic trends of norovirus and rotavirus infection and relation with climate factors: Cheonan, Korea, 2010-2019 (노로바이러스 및 로타바이러스 감염의 역학 및 기후요인과의 관계: 천안시, 2010-2019)

  • Oh, Eun Ju;Kim, Jang Mook;Kim, Jae Kyung
    • Journal of Digital Convergence
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    • v.18 no.12
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    • pp.425-434
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    • 2020
  • Background: Viral infection outbreaks are emerging public health concerns. They often exhibit seasonal patterns that could be predicted by the application of big data and bioinformatic analyses. Purpose: The purpose of this study was to identify trends in diarrhea-causing viruses such as rotavirus (Gr.A), norovirus G-I, and norovirus G-II in Cheonan, Korea. The identified related factors of diarrhea-causing viruses may be used to predict their trend and prevent their infections. Method: A retrospective analysis of 4,009 fecal samples from June 2010 to December 2019 was carried out at Dankook University Hospital in Cheonan. Reverse transcription-PCR (RT-PCR) was employed to identify virus strains. Information about seasonal patterns of infection was extracted and compared with local weather data. Results: Out of the 4,009 fecal samples tested using multiplex RT-PCR (mRT-PCR), 985 were positive for infection with Gr.A, G-I, and G-II. Out of these 985 cases, 95.3% (n = 939) were under 10 years of age. Gr.A, G-I, and G-II showed high infection rates in patients under 10 years of age. Student's t-test showed a significant correlation between the detection rate of Gr.A and the relative humidity. The detection rate of G-II significantly correlated with wind-chill temperature. Conclusion: Climate factors differentially modulate rotavirus and norovirus infection patterns. These observations provide novel insights into the seasonal impact on the pathogenesis of Gr.A, G-I, and G-II.