• Research in Plant Disease
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• v.16 no.3
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• pp.247-253
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• 2010

Apple scar skin viroid (ASSVd) is one of the smallest viral pathogens infecting fruits, especially apple, and causes a significant damage to fruit trees. ASSVd usually induced the skin-dapple ring symptoms, but in 'Fuji' varieties, corked spot were occurred on the fruit skin in 2009. This new symptom will be of great helpful to diagnosis ASSVd in sight. ASSVd was surveyed in apple and pear from 2009 to 2010 in Korea, and ASSVd was identified in 20 out of 1,193 trees. The infection rate was 1.7%. To screen the infectivity of ASSVd among apple cultivars, real-time RT-PCR was applied followed by designing of ASSVd specific primers based on highly conserved regions of several ASSVd isolates including Korean isolate. NADH dehydrogenase subunit 5 (nad 5) gene, which is mRNA of the mitochondrial gene, was used for internal control. In this study, ASSVd infected apples were classified into 12 groups depending on different symptoms and symptom severity (scaring, rusting or malformation). Taken together, this study suggested that real-time PCR analysis was more sensitive to detect the low copy of ASSVd on early viroid infected apple skins than regular RT-PCR method.

• KSBB Journal
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• v.23 no.4
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• pp.303-310
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• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue-engineered products, and cell therapy. Manufacturing processes for the biologics have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine parainfluenza virus type 3 (BPIV3) is one of the common bovine pathogens and has widely been known as a contaminant of biologics. In order to establish the validation system for the BPIV3 safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BPIV3 contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPIV3 RNA was selected, and BPIV3 RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 2.8 $TCID_{50}/mL$. The real-time RT-PCR method was validated to be reproducible and very specific to BPIV3. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPIV3. BPIV3 RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect 7.8 $TCID_{50}/mL$ of BPIV3 artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BPIV3 contamination during the manufacture of biologics.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.270-274
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• 2013

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae was successfully amplified among examined genes and sequenced, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentration as low as $0.85ng/{\mu}l$ genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

• Korean Journal of Breeding Science
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• v.43 no.5
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• pp.448-456
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• 2011

We used suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) method to screen differentially expressed genes from red-fleshed kiwifruit 'Hongyang'. As a result, the 288 clones were obtained by subcloning PCR product and 192 clones that showed positive clones on colony PCR analysis were selected. All the positive clones were sequenced. After comparisons with the NCBI/Genbank database using the BLAST search revealed that 30 clones showed sequence similarity to genes from other organisms; 10 clones showed significant sequence similarity to known genes. Among these clones, 3 clones (AcF21, AcF42 and AcF106) had sequence homology to 1-aminicyclopropane-carboxylic acid (ACC)-oxidase (ACO) that known to be related to fruit ripening. The expression patterns of differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qReal-time PCR) analysis. All the data from qReal-time PCR analysis coincide with the results obtained from RT-PCR analysis. Three clones were expressed at higher levels in 'Hongyang' than 'Hayward'. AcF21 was highly expressed in the other genes at 120 days after full bloom (DAFB) and 160 DAFB of 'Hongyang'.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.117-123
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• 2011

The purpose of this study was to evaluate and compare different elution and concentration methods for optimization of human rotavirus (HRV) detection method using real-time RT-PCR and cell culture techniques. The leafy vegetable samples (lettuce, Chinese cabbage) were artificially inoculated with HRV. Viruses were extracted from the vegetables by two different elution buffers, buffer A (100 mM Tris-HCl, 50 mM glycine, 3% beef extract, pH 9.5) and buffer B (250 mM Threonine, 300 mM NaCl, pH 9.5), and the extracted viruses were concentrated by filtration and PEG precipitation sequentially. To determine infectivity of the viruses, the viruses recovered from the samples were infected to the MA-104 cells, and integrated cell culture real-time RT-PCR was performed at 1, 48, 72, 96, 120, 144, 168 h post-infection (p.i.). The elution buffer A was more efficient in extracting the virus from the produce samples tested than the buffer B, 29.54% and 18.32% of recoveries, respectively. The sensitivity of real-time RT-PCR method was markedly improved when the virus was concentrated by the filtration method. When the viruses were eluted and concentrated by buffer A and filtration, respectively, the average recovery rate was approximately 51.89%. When the viruses recovered from samples were infected to MA-104 cell, infectious HRV was detected within 48 h p.i. by ICC/real-time RT-PCR, whereas cytopathic effects were not observed until 72 h p.i. The optimized detection method evaluated in this study could be useful for rapid and reliable detection of HRV in fresh produce products and applied for detection of other food-borne viruses.

• Research in Plant Disease
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• v.27 no.3
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• pp.120-127
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• 2021

Plant viruses cause significant yield losses, continuously compromising crop production and thus representing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.

• International Journal of Oral Biology
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• v.36 no.1
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• pp.1-6
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• 2011

The purpose of this study was to develop species-specific real-time quantitative PCR (RT-qPCR) primers for use in the detection of Aggregatibacter actinomycetemcomitans. These primers were designed based on the nucleotide sequences of the RNA polymerase ${\beta}$-subunit gene (rpoB). We assessed the specificity of the primers against nine strains of A. actinomycetemcomitans, eight strains (three species) of the Haemophilus genus, and 40 strains of 40 other oral bacterial species. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC $33384^T$. Our data reveal that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 2 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these qRT-PCR primers are suitable for application in epidemiological studies.

• Journal of the Korea Institute of Military Science and Technology
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• v.22 no.4
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• pp.575-580
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• 2019

In biological warfare, it is important to identify biological agents for proper treatment. We focused on developing a real-time RT-PCR kit that can detect multiple species of biological agents. AccuPower(R) Biothreat Real-Time RT-PCR Kit(v3.0) could detect Bacillus anthracis, Yersinia pestis, Vibrio cholerae, Francisella tularensis, Salmonella typhi, Rickettsia prowazekii, Variola virus, Hantaan virus, Yellow fever virus, Brucella spp., Shigella dysenteriae in a single reaction. The results showed that the kit was verified to be able to detect at least 0.005 ng of nucleotide and 10,000 CFU/ml of bacteria. Therefore, the kit is expected to be used as a rapid and sensitive detection kit for 11 species of biological agents within 2 hours.

• Biomedical Science Letters
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• v.17 no.3
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• The Plant Pathology Journal
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• v.39 no.4
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.