• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1398-1403
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• 2016

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10¹ copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10² copies/20 g fresh lettuce, 9.7 × 10³ copies/20 g frozen strawberries, and 4.1 × 10³ copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.3
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• pp.1655-1659
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• 2013

Background: HER-2/neu is a proto-oncogene that encodes a transmembrane tyrosine kinase growth factor which is crucial for stimulating growth and cellular motility. Overexpression of HER-2/neu is observed in 10-35% of human breast cancers and is associated with pathogenesis, prognosis as well as response to therapy. Given the imperative role of HER-2/neu overexpression in breast cancer, it is important to determine the magnitude of amplification which may facilitate a better prognosis as well as personalized therapy in affected patients. In this study, we determined HER-2/neu protein expression by immunohistochemistry (IHC) concurrently with HER-2/neu DNA amplification by quantitative real time-polymerase chain reaction (Q-PCR). Materials and Methods: A total of 53 paired tissue samples from breast cancer patients were frozen-sectioned to characterize the tumour and normal tissues. Only tissues with 80% tumour cells were used in this study. For confirmation, Q-PCR was used to determine the HER-2/neu DNA amplification. Results: We found 20/53 (37.7%) of the tumour tissues to be positive for HER-2/neu protein overexpression using IHC. Out of these twenty, only 9/53 (17%) cases were in agreement with the Q-PCR results. The concordance rate between IHC and Q-PCR was 79.3%. Approximately 20.7% of positive IHC cases showed no HER-2/neu gene amplification using Q-PCR. Conclusion: In conclusion, IHC can be used as an initial screening method for detection of the HER-2/neu protein overexpression. Techniques such as Q-PCR should be employed to verify the IHC results for uncertain cases as well as determination of HER-2/neu gene amplification.

• Proceedings of the Korean Vacuum Society Conference
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• 2016.02a
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• pp.375.1-375.1
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• 2016

Polymerase chain reaction (PCR) has revolutionized genetics and become one of the most popular techniques in modern biological and medical sciences. It can be used not only as an in vitro DNA amplification method but also used in many bioassay applications. The PCR can be used to exponentially produce a large number of DNA copies from a small quantity of DNA molecules in a few hours. However, as unwanted DNA fragments are also often manufactured, the amplification efficiency of PCR is decreased. To overcome this limitation, several nanomaterials have been employed to increase the specificity of the PCR reaction. Recently, graphene has attracted a great interest for its excellent electron transfer, thermal and biocompatibility. Especially, gold nanoparticle-coated graphene oxide (GO/AuNPs) led to enhance electron and thermal transfer rate and low-charge transfer resistance. Therefore, we report the development of a demonstration for the PCR efficiency using a large-scale production of the GO and combination of gold nanoparticles. Because a thermal conductivity is an important factor for improving the PCR efficiency in different DNA polymerases and different size samples. When PCR use GO/AuNPs, the result of transmission electron microscopy and real-time quantitative PCR (qPCR) showed an enhanced PCR efficiency. We have demonstrated that GO/AuNPs would be simply outperformed for enhancing the specificity and efficiency of DNA amplification procedure.

• The Plant Pathology Journal
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• v.34 no.3
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• pp.163-170
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• 2018

Strawberry Fusarium wilt disease, caused by Fusarium oxysporum f. sp. fragariae, is the most devastating disease in strawberry production. The pathogen produces chlamydospores which tolerate against harsh environment, fungicide and survive for decades in soil. Development of detection and quantification techniques are regarded significantly in many soilborne pathogens to prevent damage from diseases. In this study, we improved specific-quantitative primers for F. oxysporum f. sp. fragariae to reveal correlation between the pathogen density and the disease severity. Standard curve $r^2$ value of the specific-quantitative primers for qRT-PCR and meting curve were over 0.99 and $80.5^{\circ}C$, respectively. Over pathogen $10^5cfu/g$ of soil was required to cause the disease in both lab and field conditions. With the minimum density to develop the wilt disease, the pathogen affected near 60% in nursery plantation. A biological control microbe agent and soil solarization reduced the pathogen population 2-fold and 1.5-fold in soil, respectively. The developed F. oxysporum f. sp. fragariae specific qRT-PCR protocol may contribute to evaluating soil healthiness and appropriate decision making to control the disease.

• Food Science of Animal Resources
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• v.36 no.4
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• pp.499-507
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• 2016

In this study, traditional culture method and 16S rRNA gene analysis were applied to reveal the composition and diversity of lactic acid bacteria (LAB) of fermented cow milk, huruud and urum from Baotou and Bayannur of midwestern Inner Mongolia. Also, the quantitative results of dominant LAB species in three different types of dairy products from Baotou and Bayannur were gained by quantitative polymerase chain reaction (q-PCR) technology. Two hundred and two LAB strains isolated from sixty-six samples were identified and classified into four genera, namely Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, and twenty-one species and subspecies. From these isolates, Lactococcus lactis subsp. lactis (32.18%), Lactobacillus plantarum (12.38%) and Leuconosto mesenteroides (11.39%) were considered as the dominated LAB species under the condition of cultivating in MRS and M17 medium. And the q-PCR results revealed that the number of dominant species varied from samples to samples and from region to region. This study clearly shows the composition and diversity of LAB existing in fermented cow milk, huruud and urum, which could be considered as valuable resources for LAB isolation and further probiotic selection.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.30-37
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• 2011

This ultra real-time PCR (UPCR) based diagnosis system for avian influenza A virus (AIV) subtype was designed. The target primer in this study was derived from H5N1 subtype-specific 133 bp partial gene of hemagglutinin (HA), and was synthesized by using PCR-based gene synthesis on the ground of safety. UPCR was operated by Mini-Opticon Q-PCR Quantitative Thermal Cycler using aptamer-based molecular beacon, total 10 ${\mu}l$ of reaction mixture with extraordinarily short time in each steps in PCR. The detection including UPCR and analysis of melting temperature was totally operated within 15 min. The AIV-specific 133 bp PCR product was correctly amplified until 5 molecules of HA gene as minimum of templates. This kind of PCR was drafted as UPCR in this study and it could be used to detect not only AIV subtype, but also other pathogens using UPCR-based diagnosis.

• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1228-1237
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• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• Korean Journal of Veterinary Service
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• v.41 no.1
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.555-559
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• 2013

Vibrio cholerae O1 and O139 are the major serotypes associated with illness, and some V. cholera non-O1 and non-O139 isolates produce cholera toxin. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the species-specific detection and quantitation of V. cholera using a primer pair based on an outer membrane lipoprotein lolB gene for the amplification of a 195 bp DNA fragment. The qPCR primer set for the accurate diagnosis of V. cholera was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Korean Journal of Pharmacognosy
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• v.45 no.2
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• pp.168-173
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• 2014

The aim of this study was to evaluate the anti-helicobacter activity of the ethanol extract of Sohamhyoongtang (Coptidis Rhizoma, Pinelliae Tuber and Trichosanthis Semen) and Coptidis Rhizoma total alkaloids, which is one of the components of Sohamhyoongtang. Crude ethanol extract of Sohamhyoongtang (ESHHT) and Coptidis Rhizoma total alkaloids (CRTA) were used for this experiment. Five different types of H. pylori (including H. pylori 26695) were used as test strain. To determine anti-helicobacter activity, minimum inhibitory concentration (MIC) was determined by agar dilution method. The effect of ESHHT and CRTA on the gene expression of H. pylori was investigated by quantitative realtime-PCR (qRT-PCR). MICs of ESHHT against five H. pylori strains were $250{\sim}500{\mu}g/ml$ and MICs of CRTA against five H. pylori strains were $50{\sim}200{\mu}g/ml$. Four representative virulence genes of H. pylori, cagA, ureA, ureB and ureI were tested as target genes for qRT-PCR. According to the qRT-PCR results, both ESHHT and CRTA markedly repressed the expression of cagA gene of H. pylori 26695 (6.91 and 20 folds respecively). These results showed that the ESHHT and CRTA demonstrated antihelicobacter properties, suggesting their potential use in gastritis or duodenal ulcer.