Tuberculosis (TB) is a significant disease for both humans and animals worldwide. The genus Mycobacterium includes several species that cause TB disease in humans and other animals. Amongst the members of the Mycobacterium tuberculosis complex (MTC), M. tuberculosis is mainly a human pathogen, whereas M. bovis has a broad host range and is the principal agent responsible for TB in domestic and wild mammals. M. bovis also infects humans, causing zoonotic TB through ingestion, inhalation. M. bovis accounts for only a small percentage of the reported cases of TB in humans. In recent years, TB in farmed deer has become a disease as public health importance in several countries. Nowadays, there has been rapid outbreak of bovine TB in cattle and deer in Korea. Investigations are needed to elucidate the relative importance of M. bovis on TB incidence in humans, especially in Korea where bovine TB remains a problem. Also, the human sources as the cause of animal infection, M. tuberculosis from the farm workers also important for TB control of animals in Korea. Differentiation between the causative organisms may only be achieved by sophisticated laboratory methods involving bacteriological characteristics, biochemical properties, and routine resistance to pyrazinamide (PZA). M. bovis shows inherently resistant to PZA whereas M. tuberculosis is susceptible to PZA. In this study, we developed a multiplex-PCR assay based on a 12.7-kb fragment for the differential detection of M. bovis and M. tuberculosis. A total of 131 M. tuberculosis complex isolates were randomly obtained from cattle and deers that were PPD positive. they all yielded M. bovis. M. tuberculosis was not isolated from animals. and, a total of 25 M. tuberculosis complex isolates which is resistant to PZA were obtained from patient. PZA resistant MTC in humans was caused entirely by M. tuberculosis. The multiplex-PCR protocol was highly species-specific and time saving for identification of M. bovis and M. tuberculosis. This multiplex-PCR assay will be easily used as a routine monitoring tool in veterinary and medical laboratories.

The number of abandoned dogs is increasing with the worsening of the economy and the rising of feed value. It was becoming a serious social problem because of the disease transmission and destruction of natural ecosystems by abandoned dogs been wild animal. In order to solve these problems, companion dogs necessary to secure its own genetic information and to establish the systematic tracking system. Using multiplex-PCR method with 27 microsatellite marker (MS marker) divided 3 set, various alleles occurring to 6 dog breed (Labrador Retriever, German Shepherd, English Springer Spaniel, Belgian Malinois, Jindo Dog, PoongSan Dog) make use of markers to determine allele frequency and heterozygosity. MS marker FH2834 and FH2790 have only two allele and most were found in 13 alleles at FH3381 and FH3399. Average heterozygosity of MS marker is 0.534 and especially, heterozygosity represented the highest value of 0.765 at FH3381. So, it was recognized appropriate allele frequency for individual identification and paternity diagnosis in companion dogs. Using multiplex-PCR method with MS marker, various alleles occurring to dog breed make use of markers to deter mine individual identification and paternity diagnosis, traits associated biomarkers and breed-specific marker for faster, more accurate and ways to reduce the analysis cost. Based on this result, a scientific basis was established to the existing pedigree data by applying genetics additionally. Animal registration system is expected to be conducted nationwide in future. The method expects to very useful this system.

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.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema apis and Nosema ceranae in honeybee. Three sets of primers were selected from different genomic sequences to specifically amplify a 831 bp amplicon within the SSU rRNA gene, specific for both N. apis and N. ceranae (MSSR primer); a 375 bp amplicon within the SSU rRNA gene, specific for N. apis (NA primer); and a 1,131 bp amplicon within SSU rRNA gene, specific for N. ceranae (NC primer). Using the primers in conjunction (multiplex PCR) we were able to N. apis and N. ceranae and to differentiate between them. The sensitivity of this PCR assay was approximately 102 spores per milliliter. We proposed that the multiplex PCR was sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting N. apis and N. ceranae in honeybee.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema apis and Nosema ceranae in honeybee. Three sets of primers were selected from different genomic sequences to specifically amplify a 831 bp amplicon within the SSU rRNA gene, specific for both N. apis and N. ceranae (MSSR primer); a 375 bp amplicon within the SSU rRNA gene, specific for N. apis (NA primer); and a 1,131 bp amplicon with in SSU rRNA gene, specific for N. ceranae (NC primer). Using the primers in conjunction (multiplex PCR) we were able to N. apis and N. ceranae and to differentiate between them. The sensitivity of this PCR assay was approximately 102spores per milliliter. We proposed that the multiplex PCR was sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting N. apis and N. ceranae in honeybee.

In order to investigate the MDR gene by plasmid profile, multiplex-PCR and PFGE analysis, it was examined 20 Salmonella Typhimurium isolated from patients stool from 1999 to 2002. The plasmid profile analysis was shown one to five kinds of plasmid in 600bp~150Kb sized. Especially, PT104 isolates obtained has 3 plasmid identically. According to the result of multiplex-PCR, all PT104 isolates amplified only tet(G) and pse1, but aadA2 was not specialized. PFGE analysis of the fragments restricted by XbaⅠ evidently discriminated plasmid DNA among phage types.

Listeria monocytogenes and L. ivanovii are important food-pathogens for human and animal. The diagnostic of Listeria in food using culture medium requires time and laborwork, because there are many other nonpathogenic species like L. innocua, L. welshimeri, L. seeligeri and L. grayi in Genus Listeria. For these reasons, Lismix multiplex PCR method was developed as a rapid method for the detection and identification of Listeria. In this study we developed a practical system of Lis-mix PCR detection for the application to food samples and new developed Siw-mix III PCR system. Using this Lis-mix PCR system overall 69 listerial strains were successful species-identified and confirmed. Also, the Siw-mix III PCR system allows the species-specific identification among L. ivanovii, L. welshimeri and L seeligeri in a single PCR.

In this study, we report that the development of a multiplex PCR method using species-specific primers for the simultaneous detection of Poaceae family members, including adlay, barley, maize, rice and wheat, based on the sequence polymorphism of the DNA-directed RNA polymerase beta'' chain (rpoC2) genes Species-specific primers were constructed with common forward primer and each reverse primers which have differences on the basis of sequences. Each primer pairs could amplify PCR products of 443 bp for rice, 346 bp for barley, 278 bp for adlay, 221 bp for wheat and 96 for maize, respectively, from the five chloroplast DNAs. The series of template DNA concentrations were identified by the sensitivity of multiplex PCR. The band of products were clearly amplified from the DNA concentration range of 0.01 to 10 ng/μL. In addition, the species-specific primers were examined for the detection of seven commercial flour mixed products. The combination of the sensitivity of a multiplex PCR with the specificity of the primers for the detection of species would allow to be applied in analyses of processed foods.

The fruits of Schisandra chinensis have been used as an edible ingredient and traditional medicine in Korea. Due to morphological similarities of dried mature fruits, the correct identification of S. chinensis from other closely related Schisandrae species is very difficult. Therefore, molecular biological tools based on genetic analysis are required to identify authentic Schisandrae Fructus. Random amplifed polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were used to develop an easy, reliable and reproducible method for the authentication of these four species. In this paper, we developed several RAPD-derived species specific SCAR markers and established a multiplex-PCR condition suitable to discriminate each species. These genetic markers will be useful to distinguish and authenticate Schisandrae Fructus and four medicinal plants, S. chinensis, S. sphenanthera, S. repanda and K. japonica, in species level.

PCR-based assays with co-dominant or dominant allele-specific STS (sequence tagged sites) markers and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) have been used in this study to discriminate Glu-1 and Glu-3 alleles in Korean wheat cultivars. Three alleles were identified at each high molecular weight glutenin (Glu-1) homoeologous locus and 10 alleles were identified at low molecular weight glutenin (Glu-3) homoeologous locus in Korean wheat cultivars. At Glu-1 loci, wheat cultivars contained Glu-A1a, b and c alleles amplified a 1093, 1063 and 920bp fragments, respectively, Glu-B1b, c and f alleles discriminated with 707(Glu-B1b and f) and 662bp(Glu-B1c) fragments and Glu-D1a, d and f alleles amplified a 576(Glu-D1d) and 612bp(Glu-D1a and f) fragments, respectively. At Glu-3 loci, wheat cultivars contained Glu-A3c, d and e alleles amplified a 573, 967 and 158bp fragments, respectively. Glu-B3d, g, h and i alleles gave a 662, 853, 1022 and 621bp, respectively and Glu-D3a, b and c alleles amplified a 884(Glu-D3a and b) and 338bp(Glu-D3c) fragments. A multiplex-PCR assay was also established which permitted the discrimination of the major Glu-1 and Glu-3 to determine glutenin compositions in a single PCR reaction and agarose gel assay because these systems could be useful to select DNA-based identification of wheat lines carrying good breadmaking performance in Korean wheat breeding programs. Three multiplex-PCR for Glu-1 and Glu-3, Glu-A1c + Glu-B1acf + Glu-D1d, Glu-A3ac + Glu-A3d + Glu-A3e and Glu-B3d + Glu-B3fg + Glu-B3h were established in this study.

The rhizomes and herbal medicines originating from Acorus gramineus, A. calamus, A. tatarinowii, and A. gramineus var. pusilus, show significant similarity, and the correct identification of species is very difficult. Random Amplified Polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were used to develop a reliable method for identification of these four species. Several distinct SCAR markers were developed from species-specific RAPD amplicons for each species. Furthermore, a useful molecular marker was established for multiplex-PCR, in order to the four species could be distinguished concurrently. These markers allow efficient and rapid identification of closely-related Acorus species and will be useful for standardization of herbal medicines.

'Angelicae Pubescentis Radix' (APR) is an important oriental medical preparation. In Korea, Aralia continentalis has been recognized as the source plant of APR. Aralia cordata, which is difficult to distinguish from A. continentalis, and Heracleum moellendorffii, which is frequently used in lieu of A. continentalis, are traded in Korean herbal markets. In contrast, in China, Angelica pubescens is recognized as the source plant of APR. In this study, we devised a method not only to discriminate A. contientalis from A. cordata, but also to discriminate both A. contientalis and A. cordata from H. moellendorffii and A. pubescens. Based on the discrepancy in the sequences of specific regions of ITS, we designed a Cont F/ Cont R primer set to amplify a 173 bp PCR band that appears only in A. continentalis. Additionally, we designed an Ara F/ Ara R primer set to amplify a 278 bp PCR band that appears in both A. continentalis and A. cordata. Using these primer sets and the ST R primer to confirm the PCR amplification results, we developed a simple multiplex PCR method for differentiating A. continentalis from A. cordata and to concurrently differentiate both A. continentalis and A. cordata from other APR herbs.