• Journal of Microbiology
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• v.44 no.1
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• pp.72-76
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• 2006

Plasma-derived products are produced from plasma via fractionation and chromatography techniques, but can also be produced by other methods. In the performance of nucleic acid amplification tests (NAT) with plasma-derived products, it is necessary to include an internal control for the monitoring of all procedures. In order to avoid false negative results, we confirmed the usefulness of the bovine viral diarrhoea virus (BVDV) for use as an internal control in the detection of hepatitis C virus (HCV) RNA in plasma-derived products. These products, which were spiked with BVDV, were extracted and then NAT was performed. Specificity and sensitivity were determined via the adjustment of primer concentrations and annealing temperatures. BVDV detection allows for validation in the extraction, reverse transcription, and amplification techniques used for HCV detection in plasma-derived products.

• 전기의세계
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• v.13 no.4
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• pp.25-29
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• 1964

This paper shows that higher amplification can be achieved by applying load current on the internal feedback winding of the magnetic amplifier under test. Since the magnetic flux of the control winding and internal feedback winding saturate the core more fully, the permeability tends to zero and load current increases to the value more than that of ordinary magnetic amplifier without feedback.

• Research in Plant Disease
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• v.21 no.3
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• pp.180-185
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• 2015

In the present work, a pair of primers specific to Tomato yellow leaf curl virus (TYLCV) was designed to allow specific amplification of DNA fragments from any TYLCV isolates using an extensive alignment of the complete genome sequences of TYLCV isolates deposited in the GenBank database. A pair of primers which allows the specific amplification of tomato ${\beta}$-tubulin gene was also analyzed as an internal PCR control. A duplex PCR method with the developed primer sets showed that TYLCV could be directly detected from the leaf crude sap of infected tomato plants. In addition, our developed duplex PCR method could determine PCR errors for TYLCV diagnosis, suggesting that this duplex PCR method with the primer sets is a good tool for specific and sensitive TYLCV diagnosis. The developed duplex PCR method was further verified from tomato samples collected from some farms in Korea, suggesting that this developed PCR method is a simple and reliable tool for rapid and large-scale TYLCV detections in tomato plants.

• Parasites, Hosts and Diseases
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• v.53 no.2
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• Korean Journal of Ecology and Environment
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• v.37 no.1 s.106
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• pp.122-129
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• 2004

In an effort to develop the biomarker for monitoring the contamination of xenoestrogen in the freshwater environment of Korea, reverse transcription-polymerasechain reaction (RT-PCR) analysis of vitellogenin (VTG) gene expression was optimized in Hearisarsus Iaseo, Based on the homology of the VTG cDNA sequences between the common carp and zebra fish, a set of PCR primers for VTG mRNA amplification for H; labo was designed. VTG mRNA level in livers from female and male fishes was analyzed by RT-PCR following single injection of 17 beta estradiol($E_2$ 10 mg $kg^{-1}$ B.W.). As an internal control, beta actin mRNA was amplified. One us of total liver RNA was subjected to RT-PCR. In female the amount of PCR productof VfC gradually increased in the range from 16 to 34 cycles of amplification. On the contrary, in control male, PCR product first detected at 32 cycles of amplification and linearly increased up to 40 cycles of amplification. In $E_2$ injected male liver, the VTC mRNA level was similar to that in the female. Taken together, this result suggests that liver of male H. labo expresses minute amount of VTG mRNA which are2-l6 equivalent of female and that induction of VTG mRNA occurs in male liver after estrogen treatment. In conclusion, the optimized protocol for RT-PCR analysis of VTG mRNA expression in liver of male H. labo will provide the environmental monitoring method for the xenoestrogen contamination in the rivers in Korea.

• Korean Journal of Environmental Biology
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• v.22 no.2
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• pp.329-335
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• 2004

To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

• Korean Journal of Environmental Biology
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• v.22 no.1
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• pp.206-212
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• 2004

In an effort to develop the tools for monitoring the contamination of xenoestrogen in the aquatic environment of Korea, reverse transcription-polymerase chain reaction (RT-PCR) analysis of vitellogenin (VTG) mRNA expression were optimized in Synechogobius hastus. Based on the partial VTG cDNA sequence VTG mRNA level in livers from male fishes was analyzed by RT-PCR. As an internal control beta actin mRNA was amplified. 3 ${\mu}g$ of total RNA was reverse transcribed in 20 $\mu$l reaction using murine leukemia virus 〔MuLV〕 reverse transcriptase. Subsequent PCR using the 1 ${\mu}g$ of cDNA resulted in linear increase in PCR product of VTG in female liver cDNA from 10 to 30 cycles of amplification. On the contrary, in male, PCR product first detected at 28 cycles of amplification and linearly increased during 38 cycles of amplification, suggesting that male S. hastus expresses minute amount of VTG mRNA which is $2^{-18}$ equivalent of female. In conclusion, the optimized protocol of VTG mRNA expression in the liver of male S. hastus will be promising the environmental monitoring the xenoestrogen contamination in the western coast and estuaries in Korea.

• Biomolecules & Therapeutics
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• v.7 no.4
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• pp.335-341
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• 1999

Antidiabetic activity and mechanism of Supungsungihyan(SPSGH) were examined in db/db mice, which is a spontaneously hyperglycemic, hyperinsulinemic and obese animal model. SPSGH and acarbose were administered orally for 4 weeks. Fasting and non-fasting serum glucose, glycated hemoglobin and trig-lyceride of SPSGH treated group were all reduced when compared with those of db/db control group. At 12th week after birth, SPSGH increased an insulin secretion although statistic significance was not seen. Total activities of sucrose, maltase and lactase in SPSGH treated group were not significantly different from those in db/db control. On the other hand, sucrase and maltase activities in acarbose treated groups were increased. Effect of SPSGH on mRNA expression of glucose transporter(GLUT-4) was also examined by RT-PCR and in vitro transcription with co-amplification of rat $\beta$-actin gene as an internal standard. Muscular GLUT-4 mRNA expression in SPSGH treated group was increased significantly. These results may suggest that SPSGH lowered blood glucose ascribing to upregulation of muscular GLUT-4 mRNA expression.

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

• BMB Reports
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• v.40 no.2
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• pp.226-231
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• 2007

Housekeeping genes are widely used as internal controls in a variety of study types, including real time RT-PCR, microarrays, Northern analysis and RNase protection assays. However, even commonly used housekeeping genes may vary in stability depending on the cell type or disease being studied. Thus, it is necessary to identify additional housekeeping-type genes that show sample-independent stability. Here, we used statistical analysis to examine a large human microarray database, seeking genes that were stably expressed in various tissues, disease states and cell lines. We further selected genes that were expressed at different levels, because reference and target genes should be present in similar copy numbers to achieve reliable quantitative results. Real time RT-PCR amplification of three newly identified reference genes, CGI-119, CTBP1 and GOLGAl, alongside three well-known housekeeping genes, B2M, GAPD, and TUBB, confirmed that the newly identified genes were more stably expressed in individual samples with similar ranges. These results collectively suggest that statistical analysis of microarray data can be used to identify new candidate housekeeping genes showing consistent expression across tissues and diseases. Our analysis identified three novel candidate housekeeping genes (CGI-119, GOLGA1, and CTBP1) that could prove useful for normalization across a variety of RNA-based techniques.