• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1463-1470
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• 2010

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturingprocess for recombinant therapeutics.

• Biomedical Science Letters
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• v.22 no.3
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• pp.83-97
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• 2016

The measurement of viral load in HIV-1 infected patients is essential for the establishment of a therapeutic strategy. Several commercial assays have shown shortcomings in quantifying rare genotypes of HIV-1 such as minor groups of N and O. In this study, the HIV-1 RT-qPCR assay was developed. The primers and probe of HIV-1 were designed to target the pol gene and to increase the detection efficiency of various subtypes including group N and O. The HIV-1 quantitative RT-qPCR assay was assessed for its analytical performance and clinical evaluation. The LoD was determined to 33.9 IU/ml. The LoD of several subtypes including A, C, D, CRF_01AE, F, CRF_02AG, G and H, were determined to less than 40 IU/ml. The HIV-1 quantitative RT-qPCR assay was evaluated using the China National Reference Panel of HIV-1 RNA to determine the analytical performance. The results were all within the acceptable range. The clinical evaluation was performed at Hunan CDC in China. The clinical evaluation results were compared with those of the China domestic commercial kit. A significant correlation (fresh samples; $R^2=0.84$, P<0.001, frozen samples; $R^2=0.76$, P<0.001) between the two systems was observed for 64 fresh samples and 76 frozen samples with viral loads, and the Bland-Altman plot showed good agreement (98.4%, 96.1%, respectively). In conclusion, the HIV-1 quantitative RT-qPCR assay had comparable analytical performance with several commercial kits. The study provides basic data for the research of HIV-1 diagnosis and the development of P < HIV-1 molecular diagnostic assay.

• Korean Journal of Microbiology
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• v.55 no.1
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• pp.25-32
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• 2019

Influenza A (H1N1) virus caused a worldwide pandemic in 2009-2010 and still remains in seasonal circulation. Continuous surveillance activities are encouraged in the post pandemic phase to watch over the trend of occurrence every year, this is better to be done by a rapid and sensitive method for its detection. This study was conducted to detect proportions of occurrence of influenza A virus (H1N1) in patients with influenza-like illness. Samples from 500 patients with influenza or influenza-like clinical presentation were tested by real-time reverse transcription polymerase chain reaction (RT-PCR) and virus tissue culture. Among the total 500 participants, 193 (38.6%) were females and 307 (61.4%) males. Seventy-one patients (14.2%) were positive for H1N1 virus infection with real-time RT-PCR while 52 (10.4%) were positive by tissue culture. Non-statistically significant relation was found between age and gender with the positivity of H1N1. Sensitivity and specificity of real-time RT-PCR was 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%. This study showed that H1N1 virus was responsible for a good proportion of influenza during the post-pandemic period. Real-time RT-PCR provides rapidity and sensitivity for the detection of influenza A virus (H1N1) compared with virus isolation and thus it is recommended as a diagnostic tool.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.41-41
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• 2015

Oomycetes belong to the kingdom Straminipila, a remarkably diverse group which includes brown algae and planktonic diatoms, although they have previously been classified under the kingdom Fungi. These organisms have evolved both saprophytic and pathogenic lifestyles, and more than 60% of the known species are pathogens on plants, the majority of which are classified into the order Peronosporales (includes downy mildews, Phytophthora, and Pythium). Recent phylogenetic investigations based on DNA sequences have revealed that the diversity of oomycetes has been largely underestimated. Although morphology is the most valuable criterion for their identification and diversity, morphological species identification is time-consuming and in some groups very difficult, especially for non-taxonomists. DNA barcoding is a fast and reliable tool for identification of species, enabling us to unravel the diversity and distribution of oomycetes. Accurate species determination of plant pathogens is a prerequisite for their control and quarantine, and further for assessing their potential threat to crops. The mitochondrial cox2 gene has been widely used for identification, taxonomy and phylogeny of various oomycete groups. However, recently the cox1 gene was proposed as a DNA barcode marker instead, together with ITS rDNA. To determine which out of cox1 or cox2 is best suited as universal oomycete barcode, we compared these two genes in terms of (1) PCR efficiency for 31 representative genera, as well as for historic herbarium specimens, and (2) in terms of sequence polymorphism, intra- and interspecific divergence. The primer sets for cox2 successfully amplified all oomycete genera tested, while cox1 failed to amplify three genera. In addition, cox2 exhibited higher PCR efficiency for historic herbarium specimens, providing easier access to barcoding type material. In addition, cox2 yielded higher species identification success, with higher interspecific and lower intraspecific divergences than cox1. Therefore, cox2 is suggested as a partner DNA barcode along with ITS rDNA instead of cox1. Including the two barcoding markers, ITS rDNA and cox2 mtDNA, the multi-locus phylogenetic analyses were performed to resolve two complex clades, Bremia lactucae (lettuce downy mildew) and Peronospora effuse (spinach downy mildew) at the species level and to infer evolutionary relationships within them. The approaches discriminated all currently accepted species and revealed several previously unrecognized lineages, which are specific to a host genus or species. The sequence polymorphisms were useful to develop a real-time quantitative PCR (qPCR) assay for detection of airborne inoculum of B. lactucae and P. effusa. Specificity tests revealed that the qPCR assay is specific for detection of each species. This assay is sensitive, enabling detection of very low levels of inoculum that may be present in the field. Early detection of the pathogen, coupled with knowledge of other factors that favor downy mildew outbreaks, may enable disease forecasting for judicious timing of fungicide applications.

• Journal of Plant Biotechnology
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• v.49 no.1
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• pp.30-38
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• 2022

Solanum brevicaule is one of the tuber-bearing wild Solanum species. Because of its resistance to several important pathogens infecting potatoes during cultivation, it can be used for potato breeding. However, the fact that S. brevicaule used in this study has an EBN value of two causes the sexual reproduction barriers between the species and cultivated potatoes. In this study, specific markers for discriminating S. brevicaule from other Solanum species were developed on the basis of the results of sequence alignments with the whole chloroplast genomes of S. brevicaule and seven other Solanum species. The chloroplast genome of S. brevicaule was completed by next-generation sequencing technology described in other recent studies. The total sequence length of the chloroplast genome of S. brevicaule is 155,531 bp. Its structure and gene composition are similar to those of other Solanum species. Phylogenetic analysis revealed that S. brevicaule was closely grouped with other Solanum species. BLASTN search showed that its genome sequence had 99.99% and 99.89% identity with those of S. spegazzinii (MH021562) and S. kurtzianum (MH021495), respectively. Sequence alignment identified 27 SNPs that were specific to S. brevicaule. Thus, three PCR-based CAPS markers specific to S. brevicaule were developed on the basis of these SNPs. This study will facilitate in further studies on evolutionary and breeding aspects in Solanum species.

• Korean Journal of Environmental Biology
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• v.20 no.2
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• pp.109-117
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• 2002

Detection of protozoan parasites Perkinsus sp. and P. atlanticus was developed in this study using a specific polymerase chain reaction (PCR) to diagnose the presence of those organisms that causes extensive mortalities of marine shellfishes. The PCR was conducted together with fluid thioglycollate medium (FTM) method and 2 M NaOH lysis method. For the test, Manila clams, Ruditapes philippinarum, were collected from four coastal locations in Korea including Wando Island, Gimnyeong, Sungsan and Sogwipo in Jeju. In addition, trophozites of Perkinsus sp. cultivated in vitro and the granular ark clam, Tegillarca granosa, taken from Gangjin on the south coast of Korea, were used as positive and negative controls, respectively. Expected DNA bands were detected in the samples from Wando Island, Sungsan and the in vitro cultured Perkinsus sp. when the probes specific for the genus Perkinsus and P. atlanticus were used. The samples were also positively diagnosed by the FTM and 2 M NaOH methods. In contrast, the Manila clams from Gimnyeong and Sogwipo, and the granular arks clams from Gangjin showed no detectable signs of infection with the PCR, the FTM method and the 2 M NaOH lysis method. On the other hand, being amplified by p. atlanticus specific primer, it is suggested that the protozoan parasite Perkinsus sp. found in the Korean Manila clam is P. atlanticus. Finally the PCR- based assay developed in the present study can be used in detection of Perkinsus infection and discrimination of Peykinsus species in quarantine stations or laboratories due to the high sensitivity and specificity as well as its rapid detection.

• Biomedical Science Letters
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• v.5 no.1
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• pp.101-107
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• 1999

Bovine immunodeficiency virus (BIV) which was grouped into the Lentivirinae of family Retroviridae, was known to be causing many immunodeficiency syndromes among cows. The BIV was studied worldwide during last several years for its importance in cattle industries but nothing was reported in Korea until now Thus we initially tried to study the existence of BIV in cattle around the Daegu·Kyungpook area by PCR related molecular techniques. As a prerequisite investigation for detecting Korean-type BIV, we had focused our aim into BLV infected cows because the BLV infected cows tend to show BIV infection with 5％ ranges. Hence we randomly sampled fresh bloods from 248 cows and bulls near the Daegu·Kyungpook area and collected peripheral blood monocytes (PBMC) from the sample bloods. After extracting genomic DNA from the PBMC, we subjected it to PCR and Soluthern blot analysis for BIV/BLV detection. Overall, 66.9% (81/121) of the cow PBMC samples turned out to be BLV positive by PCR and the result was reconfirmed by Southern blot analysis. The value was two times higher than the previously reported results of BLV infection in Korea. The significant difference was mainly due to 1) applying highly specific methods for BLV detection such as PCR 2) that BLV was continuously spreaded in the Daegu Kyungpook area without any notice during last ten years. We also tested the BLV positive samples with the same techniques for BIV detection. And we found some BIV positives among the lot 3C samples by PCR, which had showed 100% BLV positive.

• Korean Journal of Environmental Biology
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• v.20 no.1
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• pp.109-109
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• 2002

Detection of protozoan parasites Perkinsus sp. and P. atlanticus was developed in this study using a specific polymerase chain reaction (PCR) to diagnose the presence of those organisms that causes extensive mortalities of marine shellfishes. The PCR was conducted together with fluid thioglycollate medium (FTM) method and 2 M NaOH lysis method. For the test, Manila clams, Ruditapes philippinarum, were collected from four coastal locations in Korea including Wando Island, Gimnyeong, Sungsan and Sogwipo in Jeju. In addition, trophozites of Perkinsus sp. cultivated in vitro and the granular ark clam, Tegillarca granosa, taken from Gangjin on the south coast of Korea, were used as positive and negative controls, respectively. Expected DNA bands were detected in the samples from Wando Island, Sungsan and the in vitro cultured Perkinsus sp. when the probes specific for the genus Perkinsus and P. atlanticus were used. The samples were also positively diagnosed by the FTM and 2 M NaOH methods. In contrast, the Manila clams from Gimnyeong and Sogwipo, and the granular arks clams from Gangjin showed no detectable signs of infection with the PCR, the FTM method and the 2 M NaOH lysis method. On the other hand, being amplified by p. atlanticus specific primer, it is suggested that the protozoan parasite Perkinsus sp. found in the Korean Manila clam is P. atlanticus. Finally the PCR- based assay developed in the present study can be used in detection of Perkinsus infection and discrimination of Peykinsus species in quarantine stations or laboratories due to the high sensitivity and specificity as well as its rapid detection.

• Journal of the Korea Organic Resources Recycling Association
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• v.28 no.4
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• pp.5-13
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• 2020

This study was conducted to evaluate effects of compressed expansion rice hull (CERH) as bulking agent on physicochemical properties of composting poultry manure (PM) and of its fertilization on lettuce and pak choi growth. Treatments were designed as follows; sawdust treatment (90% PM + 10% sawdust; SP), peatmoss treatment (90% PM + 10% peatmoss; PP), and CERH treatments [PCR1 (90% PM + 10% CERH 1.3 mm) and PCR2 (90% PM + 10% CERH 3.0 mm)]. Physicochemical properties such as temperature, water content, pH, and total carbon of composted poultry piles for 31 days were unaffected by various bulking agents. However, total nitrogen content in compost pile was higher in PP and PCR1 than that of SP or PCR2. After composting for 31 days, content ranges of N, P2O5, and K2O in the composting PM piles were 19.1~19.7%, 47.6~51.6%, 2.76~3.65%, and 2.53~2.90%, respectively. As compared to SP treatment, dry weight of lettuce treated with PP and PCR1 increased by more than 10%, but only in PP for pak choi. These results indicated that CERH 1.3 could be used as bulking agents for composting PM on behalf of peatmoss or sawdust.

• The Plant Pathology Journal
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• v.26 no.2
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• pp.207-214
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• 2010