• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1714-1727
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• 2015

This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex-PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

• BMB Reports
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• v.29 no.1
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• pp.45-51
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• 1996

Most expressed HLA loci exhibit a remarkable degree of allelic polymorphism, which derives from sequence differences predominantly localized to discrete hypervariable regions of the amino-terminal domain of the molecule. In this study, the HLA-DRB1 genotypes were determined in eighteen control cell lines and 112 unrelated Koreans using the PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primer) technique. 29 specific primer pairs in assigning the DRB1 gene were used. The results of control cells correlated well with the data which was previously reported. The heterozygosity and homozygosity of the DRB1 gene were 0.786 and 0.214, respectively. In a total of 41 different DRB1 alleles and 83 genotypes, the most frequent allele and genotype were DRB1*04 and DRB1*0901/1501, respectively. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-DRBI genotypes. Moreover, these results-allele and genotype frequency and heterozygosity of the HLA DRB1 gene-could be useful for database study before being applied to individual identification and transplantation immunity.

• The Plant Pathology Journal
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• v.20 no.2
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• pp.147-154
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• 2004

A simple and reliable procedure for RT-PCR detection of Apple stem pitting virus (ASPV), Cherry rasp leaf virus (CRLV), and Cherry necrotic rusty mottle virus (CNRMV) was developed. Two virus specific primer sets for each virus were found to specifically detect each virus among fourteen sets of designed oligonucleotide primers. Total RNAs extracted from healthy and from ASPV-,CRLV- and CNRMV-infected plant tissues were used to synthesize cDNA using oligo dT primer and then amplified by virus-specific primers for each virus. Each primer specifically amplified DNA fragments of 578 bp and 306 bp products for ASPV (prAS CP-C and prAS CP-N primers, respectively); 697 bp and 429 bp products for CRLV (prCR4 and prCR5-JQ3D3 primers, respectively); and 370 bp and 257 bp products for CNRMV (prCN4 and prCN6-NEG 1 primers, respec-tively) by RT-PCR. DNA sequencing of amplified DNA fragments confirmed the nature of each amplified DNA. Altogether, these results suggest that these virus specific primer sets can specifically amplify viral sequences in infected tissues and thus indicate that they can be used for specific detection of each virus.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.46 no.1
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• pp.53-56
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• 2001

Barley Yellow Dwarf Virus (BYDV), an aphid-borne luteovirus, is a major plant pathogenic disease causing a huge economic loss in the grain production of a wide range of Gramineae species throughout the world. It has been recently reported that BYDV also occurred frequently in wheat field of Korea. Here, we performed to develop the detection and classification methods of BYDV strains that were accomplished by reverse transcription-polymerase chain reaction (RT-PCR). Since there are high variations among BYDV strains, three pairs of primers were designed to detect BYDV strains such as PAV (Vic-PAV and CN-PAV) and MAV (primer A) simultaneously, specifically Vic-PAV(primer B), and MAV (primer C) based on the genomic RNA sequences of BYDV strains previously published. The validity of the primers was confirmed using several BYDV strains obtained from CIMMYT. Though three BYDV strains were able to be detected using primer A, PCR products were not distinguished between two PAV strains. It was possible to separate them with a restriction enzyme, EcoRI, whose restriction site was present in the amplified DNA fragment from Vic-PAV, but not from CN-PAV.

• Korean Journal of Veterinary Research
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• v.57 no.1
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• pp.37-42
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• 2017

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of $10^{2.0}\;TCID_{50}/mL$. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.821-831
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• 2022

American plum line pattern virus (APLPV), a member of the genus Ilarvirus in the family Bromoviridae, is one of the plant quarantine pathogens in Korea. In this study, 15 candidate primer sets were designed and examined to develop a reverse transcription polymerase chain reaction (RT-PCR) assay for plant quarantine inspection of APLPV. Using APLPV-infected and healthy samples, the primer sets were assessed for APLPV detection. To confirm the occurrence of nonspecific reactions, six ilarviruses (Apple mosaic virus, Asparagus virus 2, Blueberry shock virus, Prune dwarf virus, Prunus necrotic ringspot virus, and Tobacco streak virus) and 10 target plants (Prunus mume, P. yedoensis, P. persica, P. armeniaca, P. dulcis, P. tomentosa, P. avium, P. glandulosa, P. salicina, and P. cerasifera) were examined. Finally, two primer sets were selected. These primer sets could generate the expected amplicons even with at least 1 ng of the total RNA template in concentration-dependent amplifications. In addition, a positive clone was developed for use as a positive control in the abovementioned RT-PCR assay.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.25-32
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• 2015

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.6
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• pp.1016-1024
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• 2000

The study was conducted to develop a rapid method for the detection of Listeria monocytogenes in foods via polymerase chain reaction (PCR) technique using hemolysin gene (hlyA) primers. Specificity and sensitivity of PCR, optimal conditions for PCR and application of hlyA gene primers for the detection of L. monocytogenes from milk and beef were investigeted. Each of the 20 L. monocytogenes strains gave a single 713 bp band, but other Listeria sup. and other bacteria did not show any bands. As few as 1 pg of L. monocytogenes DNA or 2.4$\times$10$^4$L. monocytogenes cells could be detected with hlyA gene primers. PCR product was most improved at 20~30 cycle in terms of removal of tailing and sensitivity. Also, the sensitivity was significantly improved by the further 10~15 cycle after 20 cycle PCR amplication. Milk (10 mL) and beef (10 g) samples were inoculated with L. monocytogenes at the concentrations ranging from 0 to 10$^{7}$ CFU/mL or g to determine the best sensitivity of PCR for the rapid detection of L. monocytogenes. PCR assay could detect 2 cells in milk with repeating PCR amplication and 2.6$\times$10$^2$cells in beef sample after 24 hr enrichment growth at 35$^{\circ}C$ in LEB.

• Research in Plant Disease
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• v.23 no.4
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• pp.363-366
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• 2017

Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV) and Barley yellow dwarf virus (BYDV) have been identified as an important causative agents for an economically important disease of winter barley in Korea. In this study, a multiplex reverse transcription polymerase chain reaction (mRT-PCR) method was used for the simultaneous detection. Three sets of virus-specific primers targeted to the capsid protein coding genes of BaMMV, BaYMV and BYDV were used to amplify fragments that were 594 bp, 461 bp, and 290 bp, respectively. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by multiplex RT-PCR. The optimum primer concentrations and RT-PCR conditions were determined for the multiplex RT-PCR. The mRT-PCR assay was found to be a better and rapid virus diagnostic tool of specific barley diseases and potential for investigating the epidemiology of these viral diseases.

• Journal of Life Science
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• v.21 no.7
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• pp.947-952
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• 2011

A number of epidemiological studies have identified human papillomavirus (HPV) types 1, 2, 3, 4, 7, 10, 27, 57, and 65 in cutaneous common warts. However, identification of the HPV subtype by conventional polymerase chain reaction (PCR) is time consuming with its multi-step laboratory process. In this study, we aim to develop a specific one-step multiplex polymerase chain reaction method which capably identifies six different HPV genotypes related to common warts. By HPV DNA sequence analysis, 6 pairs of specific primers were designed from the intergenic regions of genes L1 to E6, and from genes E2 to L2. DNA sequence analysis with the L1 gene sequence of the sample was performed to measure the specificity of multiplex PCR. HPV-1, -2, -3, -4, -27, and -57 were identified without cross amplification in 109 out of 129 samples. The sensitivity and specificity of our set of primers in detecting HPV were 85% and 99.5%, respectively. For the 20 samples where HPV type was not identifiable by our batch of primer sets, multiplex PCR with an additional set of HPV primers was done, where 7 were found positive for HPV-7 or -65. Our results demonstrate that the newly designed multiplex PCR can rapidly detect the specific HPV subtype involved in common warts with high accuracy.