• Clinical and Experimental Reproductive Medicine
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• v.32 no.1
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• pp.17-26
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• 2005

Objective: Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. In this study, we evaluated the efficacy of PGD for Duchenne muscular dystrophy (DMD) cases by the fluorescent PCR with polymorphic linked markers and the conventional duplex-nested PCR methods. Methods: Biopsy of one or two blastomeres was done from the embryos fertilized by ICSI on the third day after fertilization. We performed two cases of PGD-DMD by the duplex-nested PCR for the causative mutation loci and the SRY gene on Y chromosome. The triplex fluorescent PCR for the mutation loci, the SRY gene and the polymorphic microsatellite marker on X chromosome was applied for two cases of PGD-DMD. Results: By the duplex-nested PCR, successful diagnosis rate was 95.5% (21/22), but we could not discriminate the female embryos whether normal or carrier in this X-linked recessive disease. However, the triplex fluorescent PCR method showed 100% (27/27) of successful diagnosis rate, and all female embryos (n=17) were distinguished normal (n=10) from carrier (n=7) embryos. Unaffected and normal embryos were transferred into mother's uterus after diagnosis. A healthy normal male was achieved after PGD with the duplex-nested PCR method and a twin, a male and a female, were delivered with triplex fluorescent PCR method. The normality of dystrophin gene was confirmed by amniocentesis and postnatal genetic analysis in all offsprings. Conclusion: The fluorescent PCR with polymorphic marker might be useful in improving the specificity and reliability of PGD for single gene disorders.

• Journal of Korean Society of Forest Science
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• v.96 no.4
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• pp.425-431
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• 2007

To investigate genetic diversity and PCR detection of Rhizina undulata, PCR detection and sequence analysis of rDNA ITS region of R. undulata in soil were analyzed and developed. The length of partial 18S rDNA from four R. undulata isolates were 1,375 nt. The sequence similarity of R. undulata isolates was 100%. The rDNA ITS regions of R. undulata isolates were 585 nt long. Nucleotide sequencing of the ITS regions showed that PDK-1, PTT-1 and PDJ-9 isolates had 100% sequence identity. But, PDS-5 isolate differed from the three isolates by two nucleotide substitution. R. undulata-specific primers designed by the sequence of ITS region were used in PCR detection of R. undulata. PCR products about 525 bp size, which is specific to R. undulata, were amplified from total DNAs of R. undulata isolates. To assay the sensitivity of PCR detection by R. undulata ITS-specific primer, purely cultured mycelial suspension of R. undulata was serially diluted and mixed with 100g of sterile sandy loam soil, respectively. And then, PCR products of total DNAs extracted from each mycelium-soil mixtures were analysed. The PCR protocol could detected up to 1ng mycelium of R. undulata within 100g of soil.

• Food Science and Preservation
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• v.16 no.6
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• pp.965-970
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• 2009

PCR technology has been widely used to detect and quantify microbial pathogens in foodstuffs, because the technique is rapid, sensitive, and selective. In this study, detection of contaminating pathogenic bacteria on shells of chicken eggs was performed using both a commercial multiplex polymerase chain reaction (PCR) kit and a viable count method employing a selective medium. The PCR kit was capable of detecting Campylobacter jejuni, Escherichia coli O157:H7, Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus, Listeria monocytogenes, Yersinia enterocolitica, Salmonella species, and Shigella species. Using the PCR method, five bacterial species were detected from 30 samples (33.3%) of 90 batches of eggs commercially available in a market. PCR products from B. cereus, S. aureus, L. monocytogenes, Y. enterocolitica, and E. coli O157:H7 were detected, and the numbers and frequencies of positive samples were 17 (18.8%), 12 (13.3%), 15 (16.6%), 16 (17.7%),and 4 (4.4%), respectively. None of any Salmonella species, C. jejuni, V. parahaemolyticus, or Shigella species was detected in this study. The results of PCR testing were confirmed using a typical viable count method employing a selective medium. We suggest that the multiplex polymerase chain reaction (mPCR) assay is a rapid and reliable method for detection of pathogenic bacteria contaminating eggs.

• Journal of Food Hygiene and Safety
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• v.33 no.1
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• pp.50-57
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• 2018

Salmonella continue to be a major cause of food poisoning worldwide. The rapid detection method of food-borne Salmonella is an important food safety tool. A real-time polymerase chain reaction (PCR) has been used as a rapid method for the detection of pathogens. It has been recently reported that NBS LabChip real-time PCR is a novel, ultrafast, and chip-type-convenient real-time PCR system. We developed the assay method based on NBS LabChip real-time PCR for the rapid detection of Salmonella, which its reaction time was within 20 minutes. Two target genes (invA and stn) were selected to design target specific primers and probes. The new method was validated by checking specificity and sensitivity (limit of detection). This study included forty-two target and twenty-one non-target strains to assess the specificity. This assay was able to identify the 42 Salmonella strains correctly. The limit of detection (LOD) was $10^1copies/{\mu}L$ in Salmonella genomes DNA, while LOD incubated for 4 hr in the inoculated sausage sample ranged from $10^1CFU/g$ to $10^2CFU/g$ as an inoculated cell count. The assay developed in this study could be applied for the investigation of food poisoning pathogens.

• Journal of Animal Science and Technology
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• v.49 no.1
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• pp.1-8
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• 2007

This study was focused on discriminating the molecular sexes of red deer and elk by duplex polymerase chain reaction(PCR) using two primer sets. Sex differentiation of mammals is primarily dependent on the presence or absence of sex determining region Y(SRY) gene encoded on Y chromosome which plays a key role for male development. Zinc finger X-Y(ZFX-ZFY) gene, one of X-Y homology gene group was found on X- and Y- chromosomes, respectively. At first, the nucleotide sequences were characterized for the intron 9 flanking region of ZFX-ZFY genes. The intron 9 of ZFX and ZFY is 529-bp and 665-bp in length, respectively. A transposable element sequence similar to bovine SINE element Bov-tA was detected only in ZFY gene of Cervidae. Sexing analysis was conducted by duplex PCR assay for amplification of SRY and ZFX-ZFY genes. Two differentially amplified patterns were found: one for females has a common band amplified only from ZFX as a template, and another for males had three bands(a common ZFX and two male-specific ZFY and SRY). On the separate tests using each gene, the results was identical to those from duplex PCR assay. Moreover, the results from PCR assays provide also identical information to phenotypic investigation of individuals of red deer, elk as well as their hybridized progenies collected from two isolated farms. These results suggest that it may be a rapid and precise method for determining the sexes by duplex PCR amplification using Y-chromosome specific SRY and X- and Y- homologous ZFX-ZFY genes showing sexual dimorphism in red deer and elk without any other controls.

• Applied Biological Chemistry
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• v.48 no.1
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• pp.77-81
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• 2005

To monitor GM soybean in soybean processed foods, tofu and biji, we prepared tofu and biji containing 0%, 1%, 3%, 5% and 100% GM soybean, respectively. We examined epsps gene inserted in soybean by PCR and EPSPS protein expressed in soybean using western blotting and lateral flow strip test to compare the sensitivity of these methods. A PCR product of 123 bp inserted in GM soybean was detected in all tofu and biji containing 1%, 3%, 5% and 100% GM soybean with the exception of 0% samples; however, the size of 600 bp inserted in GM soybean was only detected in tofu containing 100% soybean and in biji containing 5% and 100% soybean. In the protein level, GM soybean product was only detected in tofu and biji containing 100% GM soybean by western blotting. In addition, only biji containing 100% GM soybean was detected by lateral flow strip test. We concluded that in order to detect GM soybean efficiently in processed food, the PCR method is more sensitive than immunological methods. With the PCR method, small size product with approximately 100 bp in PCR product is sensitive to detect GM soybean in processed foods.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.339-347
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• 2012

Viral safety is an important prerequisite for clinical preparations of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to several RNA viruses including reovirus (Reo), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus (BPIV) and there have been reports of such viral contaminations. Therefore, viral detection during the CHO cell process is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex reverse transcription (RT)-PCR assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect Reo, BVDV, and BPIV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for Reo, BVDV, and BPIV were selected, and a multiplex RT-PCR was optimized. The sensitivity of the assay for simultaneous amplification of all viral target RNAs was $7.76{\times}10^2\;TCID_{50}/ml$ for Reo, $7.44{\times}10^1\;TCID_{50}/ml$ for BVDV, and $6.75{\times}10^1\;TCID_{50}/ml$ for BPIV. The multiplex RT-PCR was proven to be very specific to Reo, BVDV, and BPIV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral RNA from CHO cells as well as culture supernatants. Therefore, it was concluded that the multiplex RT-PCR assay can be applied to detection of the adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.

• Korean Journal of Microbiology
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• v.44 no.1
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• pp.49-55
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• 2008

White Spot Syndrome Virus (WSSV) is one of the most virulent viral agents in the penaeid shrimp culture industry. In this study, WSSV in a Fenneropenaeus chinensis shrimp farm and an adjacent seawater were concentrated using a membrane filtration and quantified using the quantitative real-time PCR (QRT-PCR) method with newly designed primers and Taqman probe. Sensitivity of primers and probe was proven by WSSV standard curve assay in QRT-PCR. In order to demonstrate the relationship between WSSV and environmental parameters, physicochemical and biological parameters of the farm and influent seawaters were monitored from June to September, 2007. The abundance of WSSV ranged 3,814-121,546 copies per 1 liter of seawater, which was correlated with fecal enterococci ($r^2=0.9$, p=0.02), chlorophyll ${\alpha}$ ($r^2=0.8$, p=0.03) and $BOD_5$ ($r^2=0.8$, p=0.07). Subsequently, it is concluded that the QRT-PCR method using Taqman probe established in this study was efficient to clarify the quantification of WSSV in seawaters. Statistical analyses of environmental parameters obtained in this study also showed that the abundance of WSSV was correlated with several biological parameters rather than physicochemical parameters.

• Research in Plant Disease
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• v.14 no.3
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• pp.233-237
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• 2008

Tomato yellow leaf curl virus (TYLCV), a newly reported Geminivirus from tomato, generated recently large economic losses in Korea. Development of a fast and precise genetic diagnosis technique for detecting TYLCV which Agricultural research and extension services can utilize easy and handy is very important to prevent yield losses. Virion Capture (VC)/PCR is a simple, accurate and economical genetic detection method without any works or commercial kits for the extraction of the nucleic acid from the infected plants. Primers of twenty two for detection of TYLCV were designed and tested with extracted total DNA or crude sap from tomato leaf infected with TYLCV and healthy plant. Nine primers for total DNA using conventional PCR and another 9 primers for VC/PCR were selected eventually. Primers of six having same specificity were selected from the two methods and tested with other Geminivirus, Tobacco leaf curl virus (TLCV) by VC/PCR. Finally specific primers of four were selected for detection of TYLCV using VC/PCR, and Deng (540, 541), a degenerate primer for Geminivirus reported in 1996, was also developed for VC/PCR.

• Biomedical Science Letters
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• v.19 no.2
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• pp.153-157
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• 2013

Although culture is the gold standard method to identify mycobacteria, its use in tuberculous lymphadenitis (TBL) is limited due to formalin fixation of the submitted specimens. We evaluated the performance of quantitative real-time PCR (q-PCR) for Mycobacterium Tuberculosis (MTB) in granulomatous lymphadenitis using formalin-fixed paraffin-embedded (FFPE) tissues. From 2000 to 2010, a total number of 117 cases of lymph node samples with granulomatous inflammation which were surgically removed and fixed in formalin were studied. Hematoxylin & Eosin (H&E) and Ziehl-Neelsen-stained (ZN) slides were reviewed. qPCR using Real TB-Taq$^{(R)}$ was performed for all cases to identify Mycobacterium tuberculosis. Thirteen non-tuberculous lymphadenopathy cases were used as negative control. Cervical lymph nodes were more frequently affected (60%, 70/117) than other sites. ZN stain for acid fast bacilli was positive in 19 (16.24%) cases. qPCR for tuberculosis was positive in 92 (78.63%) cases. Caseous necrosis was found in 103 (88.03%) cases. While the ZN stain and qPCR were both negative in all control cases, the qPCR showed a significantly higher positive rate (78.63% vs. 16.24%) compared to ZN stain in histologically diagnosed TBL. Quantitative real-time PCR proves to be more sensitive than ZN stain for diagnosis of tuberculous lymphadenitis.