• The Plant Pathology Journal
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• v.27 no.4
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• pp.385-389
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• 2011

A new reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the Potato leafroll virus (PLRV) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to address its advantages over RTPCR. RT-LAMP primers were designed from the open reading frame 3 (ORF3) sequence of PLRV. The RT-LAMP reactions were conducted without or with a set of loop primers. By real-time monitoring using Turbimeter, the RT-LAMP (with loop primers) detects PLRV in less than 30 min, compared to 120 min of RT-PCR. By adding fluorescent reagent during the reaction, final products of the RT-LAMP were fluorescently visualized under UV light or could be differentiated by naked-eye inspection under normal light. The RT-LAMP was extremely sensitive, about 2000-fold more sensitive than RT-PCR. This study presents great potential of the RT-LAMP for diagnosis and PLRV epidemiology because RT-LAMP method is speedy, sensitive, inexpensive, and convenient.

• Korean Journal of Clinical Laboratory Science
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• v.46 no.2
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• pp.64-67
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• 2014

Vancomycin-resistant Enterococci (VRE) are a leading cause of a nosocomial infection. While seven glycopeptide resistance genotypes have been found in Enterococci, vanA and vanB are the most common resistance genotypes. Aims of this study were to detect antibiotic susceptibilities of 23 Enterococcus spp, which broke out in a university hospital by the disk diffusion test, to investigate specific genes of vanA and vanB by conventional and real-time PCR. PCR for vanA and vanB was performed on 23 Enterococci, all 23 were positive for vanA type. This study reports the validation of a simple and rapid VRE detection method that can be easily incorporated into the daily routine of a clinical laboratory. Early detection of VRE strains, including those with susceptibility to Vancomycin, is of paramount clinical importance, as it allows a rapid initiation of strict infection control practices as well as a therapeutic guidance for a confirmed infection. The real-time PCR method is a rapid technique to detect vanA in Enterococci. It is simple and reliable for the rapid characterization of VRE.

• Research in Plant Disease
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• v.25 no.2
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• pp.89-93
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• 2019

Fire blight disease caused by the bacterium, Erwinia amylovora, was observed in apple and pear orchards in Korea in 2015. Since then, it has spread, sometimes over long distances to other orchards. Therefore, we examined the ability of E. amylovora to survive in soils and on the surfaces of common materials such as T-shirts, wrist bands, pruning shears, and rubber boots by both conventional PCR (cPCR) and quantitative PCR (qPCR) methods. E. amylovora was detected in all materials tested in this study and survived for sufficiently long periods to cause fire blight disease in new sites. Thus, based on the results of this study, sanitation protocols must be applied to equipment during orchard work.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.180-189
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• 2016

The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem-PCR) was applied to avoid interference between different primers in conventional multiplex-PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.

• Biomedical Science Letters
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• v.5 no.2
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• pp.191-200
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• 1999

In recent years continuously increasing number of tuberculosis (TB) cases due to the emergence of strains with multidrug resistance and AIDS is a significant global health problem. Therefore, more rapid and reliable diagnosis of TB may be one of the most urgent needs in efforts to eradicate the disease. The present study was designed to compare and assess the diagnostic values and efficiencies between the conventional methods (X-ray, AFB stain and culture) and PCR for pulmonary TB on 171 cases. Chest X-ray finding and clinical features revealed that 39 (22.8%) of 171 sputum specimens were pulmonary TB cases. The statistical data were taken on the basis of the definitive diagnosis: In X-ray, overall sensitivity, specificity, efficiency and false positive and false negative incidence was respectively 69.2%, 87.1％, 83.0%, 12.9%, and 30.8％； 79.5%, 95.5%, 91.8%,4.6％ and 20.5％ in AFB-stain; 56.4%, 99.2%,89.5%, 0.8% and 43.6% in culture; 82.1%, 96.2%, 93.0%, 3.8% and 17.9% in PCR. PCR got a highest sensitivity and efficiency as well as a lowest false negative incidence. Culture had a highest specificity with a lowest false positive incidence. These results imply that PCR assay is fast, sensitive and efficient method for diagnosis of pulmonary TB. However, combined use of the conventional methods with thorough quality control may offer more opportunities for detecting Mycobacterium tuberculosis and diagnosting TB although they have some limits.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.100-108
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• 2011

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 ($TCID_{50}$) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.

• Fisheries and Aquatic Sciences
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• v.7 no.3
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• pp.122-129
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• 2004

Harmful Cochlodinium polykrikoides is a notorious harmful algal bloom (HAB) species that is causing mass mortality of farmed fish along the Korean coast with increasing frequency. We analyzed the sequence of the large subunit (LSD) rDNA D1-D3 region of C. polykrikoides and conducted phylogenetic analyses using Bayesian inference of phylogeny and the maximum likelihood method. The molecular phylogeny showed that C. polykrikoides had the genetic relationship to Amphidinium and Gymnodinium species supported only by the relatively high posterior probabilities of Bayesian inference. Based on the LSU rDNA sequence data of diverse dinoflagellate taxa, we designed the C. polykrikoides-specific PCR primer set, CPOLY01 and CPOLY02 and developed PCR detection assays for its sensitive, accurate HAB monitoring. CPOLY01 and CPOLY02 specifically amplified C. polykrikoides and did not cross-react with any dinoflagellates tested in this study or environmental water samples. The effective annealing temperature $(T_{p})$ of CPOLY01 and CPOLY02 was $67^{\circ}C$. At this temperature, the conventional and nested PCR assays were sensitive over a wide range of C. polykrikoides cell numbers with detection limits of 0.05 and 0.0001 cells/reaction, respectively.

• Korean Journal of Veterinary Service
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• v.24 no.4
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• pp.335-341
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• 2001

Swine dysentery caused by Brachyspira hyodysenteriae, an anaerobic, beta-hemolytic spirochete, is a severe mucohemorrhahic diarrheal disease that primarily affects pigs during the growing and finishing period. The current standard laboratory procedure to culture and identify B hyodysenteriae takes 3 to 7 days. This report present a rapid PCR for detection B hyodysenteriae in a single reaction using DNA from swine intestinal samples. The PCR produced a specific 421bp PCR product with template DNA purified from B hyodysenteriae, and the accuracy for detection of B hyodysenteriae by PCR results compared with those of conventional method was 100% in intestinal specimens. Nonspecific bands were not detected with B innocens, a nonpathogenic common inhabitant spirochete, including other enteric bacterial organisms. This procedure could detect as little as 50 pg of template DNA for B hyodysenteriae.

• Parasites, Hosts and Diseases
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• v.37 no.4
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• pp.289-292
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• 1999

BALB/c mice infected with a high virulent strain of Trypanosoma brucei gambiense IL3707 were treated intraperitoneally (ip) with either Melarsoprol （Mel-B) or PSG(+) buffer as controls. The mice were subsequently monitored regularly for parasites by direct microscopic examination of their tail blood or buffy coat and by polymerase chain reaction (PCR). Mel-B was found to be an effective drug for treatment against T.b. gambiense because at the end of the first treatment schedule, all treated mice were negative for parasites even by PCR, while all the control animals were positive. Three of the five Mel-B treated mice, while parasitologically negative, were PCR positive between 53 and 80 days post infection (DPI), indicating that they still harbored an infection. All treated mice were subsequently negative for parasites even by PCR at 88 DPI. A combination of conventional microscopic examination and PCR offers a good prediction of cure following treatment of trypanosomosis.

• Proceedings of the National Institute of Ecology of the Republic of Korea
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• v.1 no.1
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• pp.83-89
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• 2020

Methods for detecting the presence of genetically modified (GM) crops are evolving to comply with legislation and to enhance monitoring by biotechnology companies and regulators. In order to cover a broad range of detection methods for a new GM crop, conventional multiplex PCR methods are required. Based on the genetic information on three GM alfalfa varieties (J101, J163, and KK179), which were recently approved in South Korea, we developed a fast, reliable, and highly specific multiplex polymerase chain reaction (PCR) method with basic PCR equipment and inexpensive reagents. To validate and verify the newly developed multiplex PCR method, we applied a limit of detection assay and random reference material analysis. We also monitored the unintentional environmental release of GM alfalfa in South Korea by performing the multiplex PCR analysis with 91 feral alfalfa specimens collected from 2000 to 2018. Our methodology is a sensitive, simple, quick, and inexpensive tool for detecting and identifying three GM alfalfa varieties.