• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.205-205
• /
• 2004

최근에 개발된 real time RT-PCR 방법은 소량의 시료에서 특정 유전자의 mRNA 발현 양상을 분석하는데 효율적으로 이용되고 있다. 특히, 난자 또는 배아와 같이 성숙과 발생단계에 따라 유전자의 발현 양상이 현저하게 변화되는 경우에는, 각각의 시료에서 발현 양상이 크게 변하지 않는 대조군으로 사용할 수 있는 유전자를 이용한 비교, 분석이 중요하다. 본 연구에서는 생쥐의 난자와 초기 배아를 이용한 real time RT-PCR에서 mRNA의 추출방법과 형광 probe의 사용 유무 그리고 대조군으로 사용되고 있는 유전자들에 대한 검증을 시행하였다. (중략)

• Biomedical Science Letters
• /
• v.16 no.2
• /
• pp.127-131
• /
• 2010

Mycobacterium leprae detection is difficult even with molecular biological techniques due to the low sensitivity of current methodologies. In this report, real-time PCR targeting the M. leprae-specific repetitive element (RLEP) sequence was developed as a new diagnostic tool and evaluated using clinical specimens. For this, M. leprae DNAs were extracted from skin biopsy specimens from 80 patients and analyzed by real-time PCR using TaqMan probe. Then, the detection efficiency of the real-time PCR was compared with that of standard PCR. In brief, the rate of positive detection by the standard PCR and real-time PCR was 32.50% and 66.25%, respectively. The results seemed to clearly show that the TaqMan real-time PCR developed in this study may be a useful tool for sensitive detection of M. leprae from clinical specimens.

• Journal of Environmental Science International
• /
• v.18 no.12
• /
• pp.1331-1338
• /
• 2009

Marine dinoflagellate Alexandrium minutum producing paralytic shellfish toxins is responsible for paralytic shellfish poisoning (PSP). To investigate its temporal distributions in Chinhae Bay where PSP occurs annually, SYBR Green I based A. minutum-specific real-time PCR probe was developed on the LSU rDNA region. Assay specificity and sensitivity were tested against related species, and its specificity was further confirmed by sequencing of field-derived samples. Ten months field survey in 2008 (a total 100 surface water samples) by using the real-time PCR probe showed that A. minutum was detected at very low densities of 1-4 cells $L^{-1}$ in May and June being spring in Chinhae Bay, Korea.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.8
• /
• pp.1453-1458
• /
• 2008

For quantitative PCR assay of Salmonella enterica serovar Typhimurium in food samples, a real-time PCR method was developed, based on DNA genome equivalent. Specific primers and probe designed based on the STM4497 gene of S. Typhimurium LT2 showed the specificity to S. Typhimurium. Threshold cycle (Ct) values of real-time PCR were obtained from a quantitative standard curve with genomic DNA of Salmonella Typhimurium. In addition, the recovery of S. Typhimurium inoculated artificially to chicken samples with $4.5{\times}10^5$ to 4.5 CFU/ml was evaluated by using real-time PCR and plate-count methods. Result showed that the number of cells calculated from the real-time PCR method had good correlation with that of the plate-count method. This real-time PCR method could be applicable to the detection and quantification of S. Typhimurium in food samples.

• Parasites, Hosts and Diseases
• /
• v.56 no.5
• /
• pp.419-427
• /
• 2018

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, $FAM^{TM}$, $HEX^{TM}$, $Cy5^{TM}$, and CAL Fluor $Red^{(R)}$ 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was $2{\times}10$ copies for C. parvum and for C. cayetanensis, while it was $2{\times}10^3$ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

• The Korean Journal of Malacology
• /
• v.27 no.4
• /
• pp.387-393
• /
• 2011

The objective of this study was to develop a real-time PCR method for the rapid detection and quantification of the protozoan pathogen Perkinsus olseni using a TaqMan probe. For the standard, genomic DNA was extracted from $10^5$ in vitro-cultured P. olseni trophozoites, and then 10-fold serial dilutions to the level of a single cell were prepared. To test the reliability of the technique, triplicates of genomic DNA were extracted from $5{\times}10^4$ cells and 10-fold serial dilutions to the level of 5 cells were prepared. The standards and samples were analyzed in duplicate using an $Exicycler^{TM}$ 96 real-time quantitative thermal block. For quantification, the threshold cycle ($C_T$) values of samples were compared with those obtained from standard dilutions. There was a strong linear relationship between the $C_T$ value and the log concentration of cells in the standard ($r^2$ = 0.996). Detection of DNA at a concentration as low as the equivalent of a single cell showed that the assay was sensitive enough to detect a single cell of P. olseni. The estimated number of P. olseni cells was similar to the original cell concentrations, indicating the reliability of P. olseni quantification by real-time PCR. Accordingly, the designed primers and probe may be used for the rapid detection and quantification of P. olseni from clam tissue, environmental water, and sediment samples.

• Fisheries and Aquatic Sciences
• /
• v.11 no.4
• /
• pp.205-208
• /
• 2008

The mixotrophic dinoflagellate Cochlodinium polykrikoides was reported to be linked to major fish kills in Korea and Japan since the 1990s. Rapid and sensitive detection of microalgae has been problematic because morphological identification of dinoflagellates requires light microscopic and scanning electron microscopic observations that are time consuming and laborious compared to real-time PCR. To address this issue, a real-time PCR probe targeting the ITS2 rRNA gene was used for rapid detection and quantification of C. polykrikoides. PCR inhibitors in water column samples were removed by dilution of template DNA for elimination of false-negative reactions. A strong association between cell quantification using real-time PCR and microscopic counts suggests that the real-time PCR assay is an alternative method for cell estimation of C. polykrikoides in environment samples.

• Korean Journal of Veterinary Research
• /
• v.44 no.4
• /
• pp.655-662
• /
• 2004

The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.39 no.10
• /
• pp.1522-1527
• /
• 2010

This study was conducted to find out the minimal time needed for detection of Salmonella spp. which exist at very low concentration in foods by using real-time PCR. The sal-F and sal-R sequences were used as primers and sal-P was used as a probe. The detection limit of Salmonella spp. was $3.77{\times}10^2\;cfu/mL$ in buffered peptone water (BPW). Microbial growth was monitored after artificially inoculated Salmonella spp. into BPW. The obtained growth curve was well fitted with the equation, y＝$0.0127x^2$+0.5927x-0.4317 ($R^2$=0.99), if assuming that 1 cell exists in 25 g sample (0.04 cfu/mL). The microbial concentration will be reduced to 10 fold by adding BPW during sample treatment, so actual initial concentration at the starting point of enrichment is 0.004 cfu/mL. At this condition, real-time PCR detection would be possible only when microbial concentration increase occurs to exceed the detection limit (377 cfu/mL). The time needed for microbial increase was calculated from the growth curve equation as 7 hours and 20 minutes. Therefore the total time required for detection was less than 10 hours including the PCR operating time.

• Korean Journal of Plant Resources
• /
• v.21 no.3
• /
• pp.210-215
• /
• 2008

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 $Express^{TM}$ 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.