In the past four years, outbreaks of acute respiratory diseases associated with canine influenza H3N2 viruses in dogs and cats have been reported in South Korea and China. For prevention of disease from spread of the disease and for administration of timely medical treatments, including countermeasures for quarantine, use of a rapid and highly sensitive detection method are important to detection of the causative viruses. This study was conducted in order to develop a real time RT-PCR for the H3N2 subtype. It was based on primers targeting the highly homologous sequences of matrix, hemagglutinin, and neuraminidase genes. The detection limit of real time RT-PCR was 10 copies/ul with matrix and hemagglutinin genes, and 1 copy with neuraminidase genes, respectively. This real time RT-PCR was as sensitive as virus isolation in 52 clinical samples. The detection system developed in this study might provide more rapid and highly sensitive results than commercial rapid kits based on immunochromatographic assay.

Real-time PCR could help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging food-borne bacteria and diseases as identification and detection tools. The objective of this study was carried out to examine several critical parameters that must be optimized when converting from the ABI Prism 7000 SDS platform to the Cepheid SmartCycler Ⅱ so as to directly use the same primer and probe sequences. A lyophilized master mix-OmniMix HS bead, MgCl2 concentration, and PCR cycling conditions were evaluated so as to convert to a new platform, Smartcycler Ⅱ. The best optimal cycling conditions to detect Cronobacter sakazakii on SmartCycler Ⅱ were as follow: initial denaturation at 95℃ for 2 min followed by 45 cycles of 95℃ for 15 s, and 60℃ for 60 s using OmniMix HS bead contained 6 mM MgCl2 concentration. And the Ct value was 16.97 compared to 23.84 of Ct value in ABI Prism 7000 SDS. This result showed that when the several analytical parameters were taken the consideration for optimization, it could be performed assays between real-time PCR platforms. Also it is need of further study to develop the new single multiplex real-time PCR method for determining various Cronobacter spp. including three subspecies, too.

In this study, two duplex real-time PCR approach with melting curve analysis is presented for the detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus, which are important food-borne bacterial pathogens usually present in fresh and/or minimally processed vegetables. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the β-glucuronidase (uidA, E. coli), thermonuclease (nuc, S. aureus), hemolycin (hly, L. monocytogenes) and tetrathionate reductase (ttr, Salmonella spp.) genes. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic Tm values demonstrating the specific and efficient amplification of the four pathogens; 80.6 ± 0.9 ℃,86.9 ± 0.5 ℃, 80.4 ± 0.6 ℃ and 88.1 ± 0.11 ℃ for S. aureus, E. coli O157:H7, L. monocytogenes and Salmonella spp.,respectively. For all the pathogens, the two duplex, real-time PCR was equally sensitive to uniplex real-time PCR,using same amounts of purified DNA, and allowed detection of 10 genome equivalents. When our established duplex real-time PCR assay was applied to artificially inoculated fresh lettuce, the detection limit was 10³ CFU/g for each of these pathogens without enrichment. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.

We constructed a standard curve to quantify Listeria monocytogenes in ready-to-eat product, especially sausage samples, using real-time PCR. A standard curve was generated using serially diluted L. monocytogenes cells in distilled water. When cells were artificially inoculated in 10 g of sausage samples in 90㎖ buffered peptone water, the cell concentration of range was approximately 1.0×108 to 100 CFU/㎖. The standard curve of the serially diluted cells was linear for at least seven orders of magnitude from 103 to 109 CFU/㎖ of L. monocytogenes. When cells were diluted in sausages, the linearity range was from 104 to 108 CFU/㎖. The correlation coefficient (R2) of diluted cells was 0.9888 and the slope of the curve was —2.6621. The coefficient and slope of inoculated samples were 0.9916 and —2.747, respectively. The R2 value for serially diluted L. monocytogenes and artificially contaminated sausage samples were acceptable. The approach described in this study represents the potency of the quantification of L. monocytogenes in sausage samples by quantitative real-time PCR. It can be used in monitoring the presence and persistence of this pathogen in sausages.

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/, 1h) and (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to . However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

The infectious pathogens against honeybee (Apis mellifera) comprise a heterogeneous group of bacterial, viral, and fungal organisms including Paenibacillus larvae, Deformed Wing Virus (DWV) and Nosema apis. Many species like Paenibacillus larvae, Deformed Wing Virus (DWV) and Nosema apis have been isolated from a number of different continents, e.g. America, Asia and Europe, indicating its wide spread in whole nature. Little is known about the occurrence and distribution in the environment of these pathogens. For a more rapid, systematic and efficient monitoring of each pathogenic species against honeybee in the environment, PCR-based detection systems were developed that allows species-specific identifications of various pathogenic species with one reaction. These could be achieved by selecting specific primers from conserved regions of each species with speciesspecific DNA sequence variations. For the detection of any already known pathogen, well-developed PCR-detection system allows the specific detection of expected pathogenic species based on its specific nucleic acid sequence. Since each pathogenic species delivers a specific PCR-product of different size, bands can be distinguished very easily by simple gel electrophoresis. After the development of real-time PCR system, PCR-based specific detections of honeybee pathogens were dramatically improved their applications, from just detection to quantification of pathogens. These systems, quantitative PCR (qPCR) for the detection of honeybee pathogens, could be distinguished from previous PCR detection on the points of “real-time”, “easy” and “quantitative”. Moreover, very rapid PCR, so-called “Ultra-Rapid Real-Time PCR” were developed recently in field of pathogen-detection. Typical Honeybee pathogens such as Paenibacillus larvae, Israelli acute paralysis virus (IAPV) were successfully detected inner 7 minutes using 30 cycled Ultrarapid PCR. According to development of more rapid apparatus, even 30 cycled, 1 minute PCR seems to be possible. Ultla-Rapid PCR was currently attempted to apply for the direct detection system of all viral pathogens against honeybee from bee-samples and different environmental probes.

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.

본 연구는 Lightcycler (Roche)를 이용한 Real-Time PCR(LC-PCR)기법을 통하여 원유시료에서 신속, 정확하게 황색포도상구균을 검출하는 기법을 개발하고자 하였다. coagulase 전구체를 coding하는 113 bp의 coa 유전자의 증폭, melting curve 분석 및 DNA염기서열을 분석하여 황색포도상구균 특유의 유전자 검출하는 기법을 개발하였다. 또한 분리된 균주중 메치실린에 내성을 나타내는 균주를 검출하고자 penicillin-binding protein, PBP2a (mecA)를 coding 하는 209 bp의 mecA 유전자의 증폭, melting curve 분석 및 DNA염기서열을 분석하여 메치실린내성 황색 포도상구균을 real-time PCR 기법으로 검출하는 기술을 개발하였다. 본 실험에 따르면 647개의 원유시료중 6개의 시료에서 황색포도상구균이 검출되었으며 이중 2개의 시료에서 분리된 황생포도상구균이 메치실린내성 황색포도상구균임을 확인하였다. 또한 DNA 검출한계는 10 fg으로 기존 PCR에 비해 매우 감도가 우수한 것을 확인하였다. 또한 3개의 원유시료에서 돼지나 소의 삼출성 피부염의 원인균인 Staphylococcus chromogenes가 분리되었다.

Background : Development of new real-time PCR diagnosis method for simultaneous diagnosis of Cylindrocarpon destructans and Fusarium solani, causative fungi of ginseng root rot disease. C. destructans and F. solani are known to be the major pathogens of ginseng root rot disease. Root rot caused by these pathogens is a disease that is difficult to control because the disease progresses slowly and it is difficult to diagnose early and even when symptoms of plant seeding are present, the disease is already spread in the roots. Diagnostic methods to detect the presence or absence of ginseng roots rot fungi in soil before ginseng cultivation are currently being used as a method for controlling. However, commercialized soil extraction kits and PCR diagnostics have cost, diagnostic time, and single diagnostic problems, and need to develop new diagnostic methods. Methods and Results : Primers and probes in the beta-tubulin 2 gene were designed for species-specific detection. In silico analysis, the detection rate of C. destructans was 100% and the detection rate of F. solani was 95%. The multiplex real time PCR optimization conditions including the internal control were established. The analytical sensitivity using positive samples was 10 copies/㎕ for C. destructans and 10 copies/㎕ for F. solani. As a result of performance comparison test with conventional PCR diagnosis methods, it was confirmed that the developed multiplex real time PCR method has the same or better performance in terms of sensitivity. In the developed soil extraction kit, the extraction time was reduced and the extracted DNA quality was improved, compared to the used soil extraction kit. Conclusion : From the above results, we expect that the developed C. destructans / F. solani multiplex real time PCR diagnosis method and soil extraction kit will be useful for real-time monitoring of ginseng root rot pathogenic fungi in the soil of ginseng cultivation area and diagnosis of suitability of ginseng cultivation area.

Background : Korean ginseng is an important cash crop in Asian countries. However, plant yield is reduced by pathogens. Among the Ilyonectria radicicola-species complex, I. mors-panacis is responsible for root-rot and replant failure of ginseng in Asia. It is essential to find a way to reveal the existence of the pathogen before starting cultivation. Therefore, qRT-PCR method is developed to detect and quantify the pathogen in ginseng soils. Methods and Results : In this study, species specific Histone H3 primer set is developed for the quantification of I. mors-panacis. The primer set was applied on DNA of other microbes to evaluate its sensitivity and selectivity on I. mors-panacis DNA. Sterilized soil samples artificially infected by the pathogen in different concentrations were used to evaluate the ability of the primer set to detect the pathogen population in the soil DNA. Finally, the pathogen was quantified in many natural soil samples. The designed primer set was found to be sensitive and selective to I. mors-panacis DNA. In artificially infected sterilized soil samples, the estimated template using qRT-PCR was positively correlated with the pathogen concentration in soil samples (R2=0.94), disease severity index (R2=0.99), and colony forming unit (R2=0.87). In the natural soils, the pathogen was recorded in the most of fields produce bad yields with the range of 5.82 ± 2.35 to 892.34 ± 103.70 pg/g of soil. Conclusion : According to the presented results, the proposed primer set is applicable for estimating soils quality before ginseng cultivation. This will help in the disease management and crop protection in the future.

Recently, the increased consumption of mixed-grain flour products have led to improved human health in busy modern life. For this reason, the verification of commercial food authenticity is one of important subjects. The development of DNA techniques such as real-time PCR has led to the increasing efficiency of illegal food product detection. Here, we have developed a comprehensive method for detecting the grain flour of various rice cultivars in commercial food products derived from different plant species. In the genetic variation analysis of different protein coding genes on various chloroplast genomes, we found the high numbers of segregating sites in rpoB and rpoC2 more than in other genes. Thus, we have attempted to develop chloroplast DNA (cpDNA) markers, which were Os_m_rpoB in rpoB, and Os_m1_rpoC2 and Os_m2_rpoC2 in rpoC2. To assess the applicability of three cpDNA markers, we have identified the appropriate statistical measurements of each marker in various mixed-grain flour samples derived from rice cultivars and different plant species by real-time PCR, In addition, the three cpDNA markers successfully applied for detecting of nonexistent rice flour in different commercial food products.

Watermelon (Citrullus lanatus) is one of the most economically important cucurbitaceous crop over the world. Screening of proper reference genes was needed to reverse transcription quantitative real-time PCR (qRT-PCR), and it is bausic step of many researches including gene expression analysis. However, the reference genes on watermelon has not yet been reported systematically. Therefore, eight candidates of reference genes were selected with reference to Arabidopsis or cucumber papers. They are β-Actin, elongation factor 1-α, glyceraldehy-3-phosphate-dehydrogenase, NADP-isocitrate dehydrogenase, leunig, polypyrimidine tract-binding protein1, ubiquitin-conjugating eznyme E2, and 18S ribosomal RNA. The expression levels of genes were evaluated by qRT-PCR under biotic stress (Colletotrichum orbiculare treatment), plant hormone treatment (100 μM ABA), and abiotic stresses such as drought, cold (4℃), salt (250 mM NaCl) stresses. We founded appropriate reference genes which did not induce or reduce gene expression levels under broad spectrum of stresses by qRT-PCR analysis. These results may provide proper information for the use of appropriate reference genes for gene expression studies in watermelon qRT-PCR analysis.

Gene-expression analysis is increasingly important in biological research, with real-time reverse PCR (RTPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive and reproducible measurements for specific mRNA sequence. Several genes are regulated in response to abitoic stresses, such as salinity, and their gene products function in stress response and tolerance. The design of the primers and TaqMan probes for real-time PCR assays were carried out using the Primer ExpressTM software 3.0. The PCR efficiency was estimated through the linear regression of the dilution curve. To understand the expression pattern of various genes under salt stressed condition, we have developed a unique public resource of 9 stress-related genes in poplar. In this study, real-time RT-PCR was used to quantify the transcript level of 10 genes (9 stress-related genes and 1 house keeping gene) that could play a role in adaptation of Populus davidiana. Real-time RT-PCR analyses exhibited different expression ratios of related genes. The data obtained showed that determination of mRNA levels could constitute a new approach to study the stress response of P. davidiana after adaptation during growth in salinity condition.