• Journal of Food Hygiene and Safety
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• v.36 no.4
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• pp.289-297
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• 2021

In this study, multiplex PCR and real-time PCR were performed on Theragra chalcogramma (walleye pollock), Pangasianodon hypophthalmus (iridescent shark) and their processed foods, such as changnan-jeot and gaiyang-jeot (salted iridescent shark intestine). Species-specific primers for T. chalcogramma and P. hypophthalmus were designed, and genomic DNA was directly extracted from each sample to perform single PCR and multiplex PCR. As a result of PCR, in the case of single PCR, PCR bands of T. chalcogramma (297 bp) and P. hypophthalmus (132 bp) were identified, and in the case of multiplex PCR, it was confirmed that amplification occurred without cross-reaction between T. chalcogramma and P. hypophthalmus. As a result of checking the PCR sensitivity, the concentration of genomic DNA was detected up to 0.1 ng/µL in both single PCR and multiplex PCR. The real-time PCR results showed that the average Ct value of T. chalcogramma was 20.765±0.691, and the average Ct value of P. hypophthalmus sample was 35.719±1.828 in the T. chalcogramma species-specific primers. In the P. hypophthalmus species-specific primers, the average Ct value of the T. chalcogramma sample was 35.996±1.423, and the mean Ct value of the P. hypophthalmus sample was 20.096±0.793. These results demonstrated the significant differences in the efficiency, specificity and cross-reactivity of species-specific primers in real-time PCR. Based on these findings, 7 of changnan-jeot or gaiyang-jeot products were confirmed by multiplex PCR and real-time PCR, and valid results were confirmed in all samples.

• Journal of Dairy Science and Biotechnology
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• v.34 no.1
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• pp.43-49
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• 2016

Emulsion PCR (ePCR) has recently gained interest in the areas of food safety and biotechnology owing to its highly specific and sensitive performance in the amplification of target DNA. To facilitate the applications of ePCR to food safety and biotechnology, this paper describes the principles of ePCR and the factors that should be considered in designing ePCR. In addition, current research and applications related to ePCR are discussed.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.167-171
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• 2004

Reduction in the leakage of the amplified PCR product out of cell is required for effective in situ PCR. For this purpose, primers with complementary tail sequences at their 5' sides were utilized to synthesize high molecular weight PCR products, but it is time-consuming and causes deterioration of cellular appearance with many PCR cycles. Therefore, it is required to optimize the PCR condition with minimal PCR cycles. To achieve the pur-pose, primer polymers were made without the target DNA in tube from nonspecific amplification with tailed primers and treated onto the fixed Molt/LAV cells on the glass slide for the 20 cycle-in situ PCR, in which the appropriate target signals were observed for the possible use of primer polymers in in situ PCR.

• Journal of Korean Society of Environmental Engineers
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• v.29 no.8
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• pp.904-908
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• 2007

The protozoan pathogen Giardia lamblia has been major cause of waterborne enteric disease. In this study, we tried to identify G. lamlbia of human infectious species and to detect viable C. lamblia in river water samples including three sites of Han River mainstream and an its creek using PCR and RT-PCR technique. The PCR/RT-PCR methods were performed by using giardin primer based on the giardin gene targeting ventral disk of Giardia. Sensitivity testing in the DNA/RNA extraction and PCR/RT-PCR amplification steps showed that it was possible to detect a single cyst of G. lamblia and viable G. lamblia. The PCR/RT-PCR methods were compared with immunofluorescence(IF) assay by analyzing 48 samples collected from the mainstream water and the creek water. The mean concentration of the total cysts were 6.3 cysts/10 L(arithmetic mean, n = 48) and the positive detection rate were 62.5%(30/48). And the mean concentration of the cysts excluding empty cysts were 4.5 cysts/10 L and the positive detection rate were 52.1%(25/48). It resulted that 24 of 48 samples included Giardia lamblia by PCR assay and 10 of 48 samples included viable G. lamblia by RT-PCR assay. It resulted that the PCR/RT-PCR technique would be available to river water samples with low concentration of Giardia cysts. And it could support the Korean protozoan standard method, which provides useful information for species and viability.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.204-205
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• 2001

Milk is easily contaminated by pathogenic microorganisms and contains many ingredients that inhibit normal PCR. In this study, we developed a detection mothed for pathogenic microorganisms existing in milk by usting PCR. 'Sample pretreatment prior to PCR were compared to overcome the inhibition. A high PCR efficiency was achieved by SDS lysis pretreatment. without further purification of DNA for PCR.

• Korean Journal of Veterinary Service
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• v.37 no.4
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• pp.253-261
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• 2014

Porcine circovirus disease (PCVD) is a major problem of swine industry worldwide, and diagnosis of PCV2, causal agent of PCVD, has been doing in clinical laboratories of pig disease by polymerase chain reaction (PCR) methods. But the PCR analyses have a serious problem of misdiagnosis by contamination of DNA, in particular, from carryover contamination with previously amplified DNA or extracted DNA from field samples. In this study, an uracil DNA glycosylase (UNG)-based direct PCR (udPCR) without DNA extraction process and DNA carryover contamination was developed and evaluated on PCV2 culture and field pig samples. The sensitivity of the udPCR combined with dPCR and uPCR was same or better than that of the commercial PCR (cPCR) kit (Median diagnostics, Korea) on PCV2-positive serum, lymph node and lung samples of the pigs. In addition, the udPCR method confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PCV2 DNA from previous udPCR. In clinical application, 170 pig samples (86 tissues and 84 serum) were analysed by cPCR kit and resulted in 37% (63/170) of positive reaction, while the udPCR was able to detect the PCV2 DNA in 45.3% (77/170) with higher sensitivity than cPCR. In conclusion, the udPCR developed in the study is a time, labor and cost saving method for the detection of PCV2 and providing a preventing effect for DNA carryover contamination that can occurred in PCR process. Therefore, the udPCR assay could be an useful alternative method for the diagnosis of PCV2 in the swine disease diagnostic laboratories.

• Journal of Pharmacopuncture
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• v.12 no.1
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• pp.13-20
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• 2009

Objective : This study developed species-specific PCR and PCR-RFLP to detect the adulteration of Fel ursi products with cattle and pig bile juices. Methods : All the primers for PCR and PCR-RFLP in this study were designed based on nucleotide sequences of cytochrome b genes in the mitochondria. Results : The species-specific PCR amplified a DNA fragment of 214, 214, 295, and 167 bp from Fel ursi product, bear fur, cattle bile juice, and pig bile juice, respectively. The survey using the speciesspecific PCR indicated that some of commercial Fel ursi products were adulterated with cattle and pig bile juices. PCR-RFLP using the restriction endonucleases, HaeIII and HinfI enabled differentiation among Fel ursi product, cattle bile juice, and pig bile juice. Bear furs from two animals showed variations in PCR-RFLP patterns with HaeIII. Discussion : The detection methods of the species-specific PCR and PCR-RFLP could be useful in eliminating adulterated Fel ursi products from the market.

• Genomics & Informatics
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• v.19 no.3
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• pp.34.1-34.6
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• 2021

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.788-797
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• 2001

The usefulness of three PCR methods were evaluated for the epidemiological typing of Staphylococcus aureus: an enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic element PCR (REP-PCR), and 16S-23S intergenic spacer PCR (ITS-PCR). The analysis was performed using a collection of S. aureus strains comprised of 6 reference and 79 isolates from patients with various diseases. Among the 85 S. aureus strains tested, 6 references and 6 isolates were found to be susceptible to methicillin, whereas the remaining 73 isolates were resistant to it. PCR methods are of special concern, as conventional phenotypic methods are unable to clearly distinguish among methicillin-resistant S. aureus (MRSA) strains. The ability of the techniques to detect different unrelated types was found to be as follows: ERIC-PCR, 19 types; REP-PCR, 36 types; and ITS-PCR, 14 types. On the basis of combining the ERIC, REP, and ITS fingerprints, the 85 S. aureus strains were grouped into 56 genetic types (designated G1 to G56). The diversities for the 85 S. aureus strains, calculated according to Simpson\`s index, were 0.88 for an ERIC-PCR, 0.93 for a REP-PCR, and 0.48 for an ITS-PCR, and the diversity increased up to 0.97 when an ERIC-PCR and REP-PCR were combined. The above discrimination indices imply that the genetic heterogeneity of S. aureus strains is high. Accordingly, this study demonstrates that DNA sequences from highly conserved repeats of a genome, particularly a combination of ERIC sequences and REP elements, are a convenient and accurate tool for the subspecies-specific discrimination and epidemiologic tracking of S. aureus.

• Biomedical Science Letters
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• v.6 no.1
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• pp.55-63
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• 2000

This study has been carried out to investigate the sensitivity of polymerase chain reaction (PCR) method over conventional acid-fast bacilli (AFB) staining and/culture methods for the detection of Mycobacterium tuberculosis from trace body fluid and paraffin-embedded tissues (PET) specimens. A total of 65 cases were employed for the AFB staining and culture test, and a total of 50 cases were subjected to PCR and histopathological analysis. Among the specimen showing negative reaction to AFB staining, 12.1% were positive to PCR and 3.7% of the specimen representing negative result to culture test showed positive reaction to PCR. In addition, 20.0% of the specimen with AFB negative showed positive reaction to PCR. From these results, it could be concluded that PCR method overwhelms AFB staining and culture tests in sensitivity and specificity to M tuberculosis detection.