• Journal of Biomedical Engineering Research
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• v.44 no.6
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• pp.465-475
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• 2023

In this study, we demonstrated a silica nanofibrous membrane based on the electrospinning process and evaluated its DNA isolation and purification performance in PCR pretreatment. Generally, silica membranes made of non-woven fabric are used for PCR pretreatment, but this study aimed to improve the efficiency of the pretreatment process by developing a nanofiber-type silica membrane with high specific surface area and porosity. In order to manufacture a nanofiber-shaped silica film while maintaining the original physical properties of silica, nanofiber membranes produced by adding various concentrations of PEO (5 wt%, 8 wt%, and 10 wt%) to silica prepared by the sol-gel method were compared. In terms of nanofiber membrane production, the higher the PEO concentration, the more effective it was in producing nanofiber membranes. The produced silica nanofiber membrane was inserted to a pretreatment device used in commercial PCR equipment, and the pretreatment performance was compared and verified using Salmonella bacteria. When Salmonella was used, samples containing 5 wt% PEO showed superior PCR efficiency compared to samples containing 8 wt% and 10 wt% PEO. These results show that adding 5 wt% of PEO can effectively improve DNA purification and separation by producing a nanofiber-shaped silica film while maintaining the physical properties of silica. We expect that this study will contribute to the development of effective PCR pretreatment technology essential for various molecular biology applications.

• Journal of Pharmacopuncture
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• v.3 no.1
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• pp.35-51
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• 2000

The purpose of this study is to prove the analgesic effects of apitoxin and its mechanism via jaw-opening reflex(JOR) and measuring expression of mRNA in Phospholipase and Tryptophan hydroxylase(TPH) using RT-PCR. The experiments were carried out on Sprague-Dawley rats(300-400g) and mastocytoma(P-185 HTR) for JOR and RT-PCR, respectively. Rats anesthetized with thiopental sodium (80mg/kg) were used in the Tooth Pulp stimulation induced JOR. The amplitude of a digastric electromyogram (dEMG) was recorded during the stimulation at an intensity of 1.5 times the threshold for JOR. Apitoxin used in this experiment was diluted with normal saline by 1:1000. Apitoxin was injected intravenously into the test group while normal saline to the control group. However, it was injected directly into the cell of mastocytoma. We referred to base sequence registered in Genbank in designing primers for RT-PCR. The results were as follows; (1)Compared with control group, analgesic effect started to show right after Sprague-Dawely rats were treated with apitoxin($71.50{\pm}8.08$) and lasted for 50 minutes. (2)As a result of the experiment of RT-PCR, we witnessed significant changes in the degree of expression of phospholipase or rate-limiting enzyme of biosynthesis of prostaglandins with $10{\mu}g/ml$ apitoxin.($31.74{\pm}18.98%$, P<0.05) (3)As a result of the experiment of RT-PCR, we witnessed significant changes in the degree of expression of TPH or rate-limiting enzyme in biosynthesis of serotonin with $10{\mu}g/ml$ apitoxin.($131.37{\pm}16.87%$, P<0.05). These results suggest that $10{\mu}g/ml$ apitoxin have the most analgesic effects. This study showed that apitoxin has analgesic effects and held good for 50 minutes. The injection of apitoxin has brought out changes in the degree of expression of phospholipase and TPH. These results strongly suggest that analgesic mechanism by apitoxin is closely related to prostaglandins and serotonin.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.6
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• pp.806-811
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• 2017

A multiplex PCR system comprising 14 microsatellite markers was developed for genotyping analysis of the sea cucumber Stichopus japonicus. A total of 286 samples were used to evaluate genetic polymorphisms and forensic parameters of the microsatellite loci. In a single PCR reaction, all 14 loci were uniformly amplified and a total of 269 alleles were identified. The AJ19024 locus had the largest number of alleles (46), and its discriminatory power and exclusion power were 0.99 and 0.76, respectively. The fewest alleles (8) were present at the Psj2575 locus, which provided the lowest discriminatory power (0.81) and exclusion power (0.20). The mean number of alleles, mean heterozygosity, mean discrimination power and mean exclusion power per locus were 19.21, 0.70, 0.93, and 0.46, respectively. The combined matching probability for the 14 loci was $9.64{\times}10^{-19}$, and the combined power of exclusion was 0.999995. Thus, the forensic parameters evaluated in the present study demonstrated the utility of our multiplex PCR system for biological tracing methods, such as individual identification and paternity testing, in the sea cucumber.

• Biomedical Science Letters
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• v.13 no.4
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• pp.319-324
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• 2007

One-tube nested polymerase chain reaction (PCR) was evaluated for its efficacy in detecting Mycobacterium leprae in biopsy samples from leprosy patients. Primers were derived from the M leprae-specific element (RLEP) sequences which yield a 230 bp fragment. The specificity and the sensitivity of the one-tube nested PCR were compared with those of single PCR for detecting M leprae. The results showed that the one-tube nested PCR was about 100 times more sensitive than that of the single indicating the one-tube nested primer sets developed in this study can be an effective screening tool for the detection of M leprae in clinical diagnostic laboratories.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1592-1596
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• 2010

To verify the dominance of microorganisms in wastewater biological treatment, PCR-DGGE (denaturing gradient gel electrophoresis) was performed as a supplementary support method for screening of the dominant microorganisms from activated sludge. Results suggest that the dominant microorganisms in activated sludge are primarily responsible for strengthening its effectiveness as a biological treatment system, followed by the non-main dominant microorganisms, whereas the non-dominant microorganisms showed no effects. The degree of microbial abundance present on the profile of PCR-DGGE was in line with the treatment efficiency of augmented activated sludge with isolated cultures, suggesting that PCR-DGGE can be used as an effective supplementary method for verifying culturable dominant microorganisms in activated sludge of coking wastewater.

• Parasites, Hosts and Diseases
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• v.52 no.3
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• pp.257-261
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• 2014

Toxoplasma 3 main clonal lineages are designated as type I, II, and III; however, atypical and mixed genotypes were also reported. This study was conducted for detection of Toxoplasma gondii genotypes in rats (Rattus rattus) in Riyadh region, Saudi Arabia. PCR test on T. gondii B1 gene was conducted on ELISA IgM positive samples for confirmation of the infection. However, genetic analysis of the SAG2 locus was performed to determine T. gondii genotypes using PCR-RFLP technique. PCR test on T. gondii B1gene showed that 22 (81.5%) out of the 27 ELISA IgM positive samples have T. gondii DNA. Genotypic analysis shows that, of the total 22 PCR positive samples, only 13 (59.1%) were of type II, 7 (31.8%) were of type III, and 2 (9.1%) were of an unknown genotype. It is obvious that the prevalence of both type II and III is high in rats. No reports have been available on T. gondii genotypes among rats in Riyadh region, and only little is known about its seroprevalence in rats. Future studies on T. gondii genotypes in rats using multi-locus markers is needed in Riyadh region, Saudi Arabia for better understanding of T. gondii pathogenesis and treatment in humans and animals.

• Journal of agriculture & life science
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• v.44 no.5
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• pp.75-79
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• 2010

Apple, one of the most important fruit crops world-widely, is required for rapid, cost-effective and sensitive virus detection for its better productivity. RT-PCR is extensively employed for apple virus diagnosis, but the technique requires complete tissue homogenization which is time consuming and laborious. In this study, heating-based RNA extraction method was developed and proven to effectively work for stem tissues slightly better than for leaf tissues. In RT-PCR, almost identical results were generated from the use of RNA extracts from both tissues.

• Tuberculosis and Respiratory Diseases
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• v.44 no.5
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• pp.1001-1010
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• 1997

Background : PCR technique is useful in diagnosis of pulmonary tuberculosis. But, its sensitivity and specificity is some different among several studies. Our aim is compare our PCR results with other's previous PCR results in AFB smear negative patients. Methods : PCR were performed in patients that their disease were suspected as active pulmonary tuberculosis and that their initial serial sputum AFB smear results were negative. Total number of patients studied by PCR technique was 177. Also, we analyzed the data only in patients whose bronchial washing fluid AFB smear was negative. And the primer had been used was IS 6110. Results : In our retrograde study, the number of patients who are diagnosed as having active pulmonary tuberculosis, inactive pulmonary tuberculosis and nontuberculous pulmonary disease was 99, 28, 50, respectively. In the sputum study, the sensitivity of PCR is 41.5% (27 PCR positive cases/65 active TBc cases). And the sensitivity of TB culture is 53.8% (35 TB culture positive cases/65 active TBc cases). In the bronchial washing specimen study, the sensitivity of PCR is 53.8% (21 PCR positive cases/39 active TBc cases). And the sensitivity of TB culture is 43.6% (17 TB culture Positive cases/39 active TBc cases). The specificity of PCR in our study is 94.9%. (74 PCR negative cases/78 inactive TBc or nontubereulous cases) In the cases of patients who were never takened anti-TBc medication, the sensitivity of PCR (45.6%--25 positive cases/55 cases) is some lower than culture (58.2%--32 positive cases/55 cases). In the cases of patients who had been takened anti-TBC medication. the sensitivity of PCR (60%--18 positive cases/30 cases) is some superior than culture (50%--15 positive cases/30 cases). Conclusion : We think that PCR results in cases of sputum AFB smear negative patients is nearly same as culture. And PCR is especially useful in patients who had been takened anti-TBc medication on admission.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7621-7628
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• 2013

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.

• Korean Journal of Environment and Ecology
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• v.24 no.3
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• pp.318-324
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• 2010

The aim of this study is to examine feeding habits of the Korean water deer(Hydropotes inermis argyropus) from its rumen contents using a PCR-DGGE method. For this study, rumen contents were collected from water deer causalities by natural death or road-kill in two different sites(Cheorwon, Gangwon province and the Eastern part of Jeonnam province). DNA was extracted from rumen contents of a total of 44 individuals. Two primers, rbcLZ1aF(GC) and rbcL19bR, were used for PCR amplifications of ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) gene. Among 44 samples, twenty-nine samples were successfully amplified by PCRs. The 29 PCR products of partial rbcL gene were applied for PCR-DGGE. Totally, six families of plants were detected from the diet analyses. Five families of plants were found in Cheorwon, Gangwon province, but only three families of plants were found in the Eastern part of Jeonnam province. The PCR-DGGE method will provide us with a potential tool to study feeding habits of ungulates including water deer, even though our results failed to identify the prey plants at the level of species.