• Research in Plant Disease
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• v.24 no.4
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• pp.348-352
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• 2018

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed for the detection of Clover yellow vein virus (ClYVV), Peanut mottle virus (PeMoV), and Tomato spotted wilt virus (TSWV), which were recently reported to infect soybean and azuki bean in Korea. Species-specific primer sets were designed for the detection of each virus, and their specificity and sensitivity were tested using mixed primer sets. From among the designed primer sets, two combinations were selected and further evaluated to estimate the detection limits of uniplex, duplex, and multiplex RT-PCR. The multiplex RT-PCR assay could be a useful tool for the field survey of plant viruses and the rapid detection of ClYVV, PeMoV, and TSWV in leguminous plants.

• Journal of Genetic Medicine
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• v.9 no.1
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• pp.11-16
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• 2012

Purpose: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. Materials and Methods: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. Results: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. Conclusion: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

• Journal of Veterinary Clinics
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• v.29 no.5
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• pp.377-383
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• 2012

Bovine diarrhea is a major economical burden to the bovine industry in Korea. Since multiple infectious agents can be involved in bovine diarrhea, differential diagnosis is essential for effective treatment. Therefore, a panel of two multiplex real-time PCR assays which can simultaneously detect six major bovine enteric pathogens [i.e., bovine viral diarrhea virus (BVDV), bovine coronavirus (BCoV), group A bovine rotavirus (BRV), Salmonella spp., Escherichia coli (E. coli) $K99^+$, and Cryptosporidium parvum] was developed and applied to test 97 fecal samples collected from cattle farms in Korea. In addition, microscopic examination was also preformed on the samples to detect Coccidium oocyst. The estimated sensitivity of the multiplex PCR was 0.1 $TCID_{50}$ for BVDV, BCoV and group A BRV, 5 and 0.5 CFU for E. coli $K99^+$ and Salmonella, respectively, and 50 oocysts for Cryptosporidium. The amplification efficiency of the multiplex PCR ranged between 0.97 and 0.99 for each pathogen. Among 97 samples, 36 samples were positive for at least one of the 6 major pathogens and 6 samples were simultaneously positive for 2 pathogens by the multiplex PCR assay. Coccidium oocysts were also detected in 48 samples, which were all collected from over 1 month old calves. In conclusion, the multiplex real-time PCR panel can be a useful tool for fast and accurate diagnosis of calf diarrhea associated with BVDV, BCoV, group A BRV, E. coli $K99^+$, Salmonella, and/or Cryptosporidium and Coccidium may be an important target which needs to be included in the multiplex PCR panel in the future.

• Korean Journal of Clinical Laboratory Science
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• v.44 no.3
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• pp.155-165
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• 2012

The purpose of this study was to develop a multiplex PCR for the detection of aac(6')-Ib, aph(3')-Ia, and ant(2")-Ia; the genes that encode the most clinically relevant aminoglycoside modifying enzymes (AMEs) in Gram-negative bacteria. Clinical isolates of 80 E. coli and 23 K. pneumoniae from tertiary university hospital were tested by multiplex PCR. The most prevalent AME gene was aac(6')-Ib which was found in 22.3% of the isolates. Of the total 80 E. coli isolates, 1 isolate was found to contain both aph(3')-Ia and ant(2")-Ia simultaneouly. Of the total 23 K. pneumoniae isolates, 2 isolates were found to contain both aac(6')-Ib and aph(3')-Ia, and 1 isolate was found to contain both aac(6')-Ib and ant(2")-Ia simultaneously. Annual (2005~2009) analysis of isolates that contain the AME genes were of no correlation. The sensitivity and specificity of multiplex PCR in detecting AME genes was 94.4% (34 of 36 cases) and 100%, respectively. We suggest the multiplex PCR method we developed could be highly sensitive and specific in detecting the AME genes of E. coli and K. pneumoniae. This study could be the first published investigation in which the multiplex PCR method detects aac(6')-Ib, aph(3')-Ia, and ant(2")-Ia genes.

• The Plant Pathology Journal
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• v.32 no.5
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• pp.414-422
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• 2016

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

• Clinical and Experimental Pediatrics
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• v.55 no.11
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• pp.424-429
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• 2012

Purpose: Methods for quick and reliable detection of Streptococcus pneumoniae are needed for the diagnosis of pneumococcal disease and vaccine studies. This study aimed to show that sequential multiplex polymerase chain reaction (PCR) is more efficient than conventional culture in achieving S. pneumoniae -positive results. Methods: Nasopharyngeal (NP) secretions were obtained from 842 pediatric patients admitted with lower respiratory infections at Severance Children's Hospital in Korea between March 2009 and June 2010. For identification and serotype determination of pneumococci from the NP secretions, the secretions were evaluated via multiplex PCR technique with 35 serotype-specific primers arranged in 8 multiplex PCR sets and conventional bacteriological culture technique. Results: Among the results for 793 samples that underwent both bacterial culture and PCR analysis for pneumococcal detection, 153 (19.3%) results obtained by PCR and 81 (10.2%) results obtained by conventional culture technique were positive for S. pneumoniae. The predominant serotypes observed, in order of decreasing frequency, were 19A (23%), 6A/B (16%), 19F (11%), 15B/C (5%), 15A (5%), and 11A (4%); further, 26% of the isolates were non-typeable. Conclusion: As opposed to conventional bacteriological tests, PCR analysis can accurately and rapidly identify pneumococcal serotypes.

• Parasites, Hosts and Diseases
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• v.48 no.4
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• pp.297-301
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• 2010

Recently, emerging waterbome protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from $10^1$ to $10^2$ oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium paNum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

• Korean Journal of Veterinary Service
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• v.37 no.4
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• pp.247-252
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• 2014

The economic impact of swine mycoplasma infection is high. An accurate diagnosis is often difficult and time consuming. We report the development and validation of an effective multiplex polymerase chain reaction (PCR) assay that detects Mycoplasma (M.) hyopneumoniae and M. hyorhinis. The multi detection of M. hyopneumoniae and M. hyorhinis primer set were employed to detect mycoplasma species and typing of the species was performed on the basis of sequence analysis of the PCR product. The target nucleic acid fragments were specifically amplified by M. hyopneumoniae and M. hyorhinis PCR with 16S ribosomal DNA primers. Single and mixed Mycoplasma species DNA templates were used to evaluate the specificity of the multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of M. hyopneumoniae and M. hyorhinis.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.829-832
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• 2008

A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect 4 varieties of genetically modified (GM) cotton. The event-specific primers were used to distinguish the 4 varieties of GM cotton (MON1445, MON15985, MON88913, and LLcotton25) using multiplex PCR. The acyl carrier protein 1 (Acp1) gene was used as an endogenous reference gene of cotton in the PCR detection. The primer pair Acp1-AF/AR containing a 99 bp amplicon was used to amplify the Acp1 gene and no amplified product was observed in any of the 13 different plants used as templates. This multiplex PCR method allowed for the detection of event-specific targets in a genomic DNA mixture of up to 1% GM cotton containing MON1445, MON15985, MON88913, and LLcotton25.

• Biomedical Science Letters
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• v.13 no.1
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• pp.71-73
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• 2007

For the rapid detection of objective pathogenic bacteria from colon biopsy specimens, multiplex PCR (polymerase chain reaction) method was developed. The major objective bacteria in this study were Shiga-like toxin producing E. coli O157:H7, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Shigella spp. Salmonella spp. and Yersinia spp.. To detect simultaneously 7 kinds of pathogenic bacteria in single reaction tube, multiplex PCR system was executed using 6 sets of primers used in single PCR system for the respective bacteria. The results in this research might be applied for the detection of pathogenic bacteria form colon biopsy samples.