• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.1-9
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• 2009

The genetic monitoring method was developed for the rapid detection of the PGPR and biocontrol agent, B. subtilis AH18 in red-pepper field soil by multiplex PCR using sid, aec and cel gene primers. The monitoring of B. subtilis AH18 in the soil was carried by amplified a 2,3-dihydro-2,3-dihydroxy benzoate dehydrogenase [EC: 1. 3. 1. 28]gene (sid - 794 bp : EF408238) which is a key enzyme of siderophore synthesis, an auxin efflux carrier gene (aec - 1,052 bp : EF408239) and a cellulase gene (cel - 1,582 bp : EF070194). The natural un sterilized soil was inoculated with B. subtilis AH18 to determine the sensitivity ($1.8\times10^5$ cfu/g) of multiplex PCR for the rapid dectection and then the strain was monitored successfully in rhizosphere or non-rhizosphere soil of red-pepper cultural soil. At 3 weeks after the treatment, density of the strain was monitored more abundantly in rhizosphere soil.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.1-8
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• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• Korean Journal of Veterinary Research
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• v.42 no.4
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• pp.495-504
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• 2002

Streptococcus suis is an important swine pathogen in nearly all countries with an extensive pig industry. It is associated with meningitis, arthritis, endocarditis, septicaemia, bronchopneumonia and sudden death. Attempts to control the disease are still hampered the lack of effective vaccines and sensitive diagnostic tools. A PCR method which can be used for the detection of virulent strains of serotype 2, which is most prevalent serotype, and serotype 1 was developed. However, serotype 1, 2, 7 and 9 strains are frequently isolated from diseased pigs. In European countries, S suis serotype 2 is the most prevalent type isolated from diseased pigs, followed by serotype 9 and 1. In Japan, capsular serotype 2 was also the most prevalent serotype, followed by capsular serotype 7. Most of S suis isolated from diseased pigs belong to a limited number of capsular serotype, often those between 1 and 9. We investigated the distribution of S suis serotype 1, 2, 7 and 9 from 740 pig lungs at abattoir in Jeolla and Chungcheong by rapid multiplex PCR assay. Fifty of 740 lung samples, 6.8%, were S suis postitive and identified S suis were divided by 38% (19/50) in serotype 2, 2% (1/50) in serotype 7 and 4% (2/50) in serotype 9. The distribution of S suis serotype in Korea was similar to other countries. Moreover, the multiplex PCR assay may be an useful diagnostic tool for the detection of pigs carrying serotype 1, 2, 7, 1/2, 9 and 14 strains in epidemiological and transmission studies and facilitate control and eradication programs.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.90-96
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• 2015

Garlic generally becomes coinfected with several types of viruses belonging to the Potyvirus, Carlavirus, and Allexivirus genera. These viruses produce characteristically similar symptoms, they cannot be easily identified by electron microscopy (EM) or immunological detection methods, and they are currently widespread around the world, thereby affecting crop yields and crop quality adversely. For the early and reliable detection of garlic viruses, virus-specific sets of primers, including species-specific and genus-specific primers were designed. To effectively detect the twelve different types of garlic viruses, primer mixtures were tested and divided into two independent sets for multiplex polymerase chain reaction (PCR). The multiplex PCR assays were able to detect specific targets up to the similar dilution series with monoplex reverse transcription (RT)-PCR. Seventy-two field samples collected by the Gyeongbuk Agricultural Technology Administration were analyzed by multiplex RT-PCR. All seventy two samples were infected with at least one virus, and the coinfection rate was 78%. We conclude that the simultaneous detection system developed in this study can effectively detect and differentiate mixed viral infections in garlic.

• Journal of Life Science
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• v.20 no.6
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• pp.955-959
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• 2010

Differential diagnosis of the taeniid proglottids between Taenia asiatica and T. sagniata is a daunting task due to their close morphological similarity. However, to correctly diagnose them on time is important in managing infected patients, as well as for reducing serious complications such as cysticercosis. Currently, DNA-based methods for the dissection of genomic information of parasites are being employed to make accurate and rapid diagnoses in the field of parasitology. In this study, multiplex PCR was established and exploited to identify exact species of taeniid adult worms recovered from Korean people. To discriminate one from the other other, primers-Ta4978F, Ts5058F, Tso7421F, and Rev7915- were used for the multiplex PCR, which provided swift and precise identification of the taeniid worms being observed. Also,having instituted PCR methodology, we ascertained that easiness would be achieved to reassess and re-evaluate Korean endemic data on human taeniid cestodes.

• Food Science and Biotechnology
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• v.16 no.1
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• pp.104-109
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• 2007

A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect three varieties of genetically modified (GM) canola. The construct-specific primers were used to distinguish the following three varieties of GM canola; GT73, MS8xRF3, and T45, using multiplex PCR. The FatA (fatty acyl-ACP thioesterase) gene was used as an endogenous canola reference gene in the PCR detection. The primer pair Canendo-FIR containing a 105 bp amplicon was used to amplify the FatA gene and no amplified product was observed in any of the 15 different plants used as templates. The GT73-KHUF1/R1 primer recognized the 3'-flanking region of GT73, resulting in an amplicon of 125 bp. The Barstar-F1/MS8xRF3-R primer recognized the junction region of bars tar and the NOS terminator introduced into MS8xRF3, resulting in a 162 bp amplicon, and the T45-F2/R2 primer recognized the junction region of PAT and the 35S terminator introduced into T45, resulting in an amplicon of 186 bp. This multiplex PCR allowed for the detection of construct-specific targets in a genomic DNA mixture of up to 1% GM canola containing GT73, MS8xRF3, and T45.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.205-211
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• 2013

The swine is one of the most widespread mammalian throughout the whole world. Presently, many studies concerning microsatellites in swine, especially domestic pigs, have been carried out in order to investigate general diversity patterns among either populations or breeds. Until now, a lot of time and effort spend into a single PCR method. But simple and more rapid multiplex PCR methods have been developed. The purpose of this study is to develop a robust set of microsatellites markers (MS marker) for traceability and individual identification. Using multiplex-PCR method with 23 MS marker divided 2 set, various alleles occurring to 5 swine breed (Berkshire, Landrace, Yorkshire, Duroc and Korea native pig) used markers to determine allele frequency and heterozygosity. MS marker found 4 alleles at SW403, S0227, SWR414, SW1041 and SW1377. The most were found 10 alleles at SW1920. Heterozygosity represented the lowest value of 0.102 at SWR414 and highest value of 0.861 at SW1920. So, it was recognized appropriate allele frequency for individual identification in swine. Using multiplex-PCR method, MS markers used to determine individual identification biomarker and breed-specific marker for faster, more accurate and lower analysis cost. Based on this result, a scientific basis was established to the existing pedigree data by applying genetics additionally. Swine traceability is expected to be very useful system and be conducted nationwide in future.

• Korean Journal of Veterinary Research
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• v.52 no.2
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• pp.75-82
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• 2012

We have developed and optimized a multiplex polymerase chain reaction (mPCR) for simultaneous detection of Brucella, Salmonella and Leptospira with high sensitivity and specificity. Three pairs of oligonucleotide primers were designed to specifically amplify the targeted genes of Salmonella, Leptospira and Brucella species with sizes of 521, 408 and 223 bp, respectively. The mPCR did not produce any nonspecific amplification products when tested against 15 related species of bacteria. The sensitivity of the mPCR was 100 fg for Brucella and 1 pg for both Salmonella and Leptospira species. In the field application, kidney, liver and spleen were collected from wild rats and stray cats and examined by mPCR. The high specificity and sensitivity of this mPCR assay provide a valuable tool for diagnosis and for the simultaneous and rapid detection of three zoonotic bacteria that cause disease in both humans and animals. Therefore, this assay could be a useful alternative to the conventional method of culture and single PCR for the detection of each pathogen.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.58 no.2
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• pp.142-148
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• 2013

Bacterial diseases in soybean are bacterial pustule by Xanthomonas axonopodis pv. glycines, wildfire by Pseudomonas syringae pv. tabaci, bacterial blight by Pseudomonas savastanoi pv. glycines and bacterial brown spot by Pseudomonas syringae pv. syringae in Korea. It is difficult to identify each disease by early symptoms in fields, because the initial symptoms of these diseases are very similar to each other. In this study, we developed multiplex PCR detection method for rapid and accurate diagnosis of bacterial diseases. The glycinecin A of X. axonopodis pv. glycines, the tabtoxin of P. syringae pv. tabaci, the coronatine of P. savastanoi pv. glycines and the syringopeptin of P. syringae pv. syringae have been reported previously. These bacteriocin or phytotoxin producing genes were targeted to design the specific diagnostic primers. The primer pairs for diagnosis of each bacterial diseases were selected without nonspecific reactions. The studies on simultaneous diagnosis method were also conducted with primarily selected 21 primers. As a result, we selected PCR primer sets for multiplex PCR. Sizes of the amplified PCR products using the multiplex PCR primer set consist of 280, 355, 563 and 815 bp, respectively. This multiplex PCR method provides a efficient, sensitive and rapid tool for the diagnosis of the bacterial diseases in soybean.

• Food Science of Animal Resources
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• v.30 no.4
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• pp.590-596
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• 2010

Meat and meat products are a potential source of food-borne pathogens, including Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7, and Bacillus cereus. A sensitive and specific PCR assay for the detection of these pathogens in meat and meat products was developed in this study, as part of a broader effort to reduce the potential health hazards posed by these pathogens. Initially, PCR conditions were standardized with purified DNA. Under standard conditions, the detection level for PCR was as low as 10 pg of purified bacterial DNA. After overnight growth of bacteria in a broth medium, as few as $10^2$ CFU of bacteria were detected by PCR assay. The primers employed in the PCR assay were found to be highly specific for individual organisms, and evidenced no cross-reactivity with heterologous organisms. Additionally, the multiplex PCR assays also amplified some target genes from the four pathogens, and multiplex amplification was obtained from as little as 10 pg of DNA, thus illustrating the excellent specificity and high sensitivity of the assay. In conclusion, this PCR-based technique provides a sensitive and specific method for the detection of S. aureus, Salmonella spp., E. coli O157:H7, and B. cereus in meat and meat products.