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

• Asian-Australasian Journal of Animal Sciences
• /
• v.28 no.4
• /
• pp.568-572
• /
• 2015

Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China.

• Journal of Veterinary Clinics
• /
• v.32 no.3
• /
• pp.219-223
• /
• 2015

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis or Johne's disease, an intestinal granulomatous infection in domestic and wild animals. The study aimed to develop and evaluate a panel of multiplex quantitative real-time polymerase chain reaction (mqPCR) assay for simultaneous detection of three MAP-specific genes (IS900, F57 and ISMAP02 genes). The analytic sensitivity (i.e., limit of detection, expressed as cells per 1 ml) was 150 for IS900, 1500 for F57, and 50 for ISMAP02. The specificity of the method was determined by testing 152 bovine fecal samples. Based on the test, it showed that the assay simultaneously detected the target genes in short period of time and at lower cost compared to laboratory routine tests. The test agreement between the assay and routine test was 94%. The discrepancy in the results was due to samples that were tested positive by the panel but negative by the routine tests, suggesting that the assay has higher sensitivity than the routine tests. In conclusion, the mqPCR assay could be a rapid and accurate testing tool for investigating paratuberculosis or Johne's disease cases in domestic and wild animals.

• Journal of Ginseng Research
• /
• v.34 no.1
• /
• pp.41-46
• /
• 2010

Expensive herbs such as ginseng are always a possible target for fraudulent labeling. New mountain ginseng strains have occasionally been found deep within mountain areas and commercially traded at exorbitant prices. However, until now, no scientific basis has existed to distinguish such ginseng from commonly cultivated ginseng species other than by virtue of being found within deep mountain areas. Polymerase chain reaction (PCR) analysis of the internal transcribed spacer has been shown to be an appropriate method for the identification of the most popular species (Panax ginseng) in the Panax ginseng genus. A single nucleotide polymorphism (SNP) has been identified between three newly found mountain ginseng (KGD4, KGD5, and KW1) and already established Panax species. Specific PCR primers were designed from this SNP site within the sequence data and used to detect the mountain ginseng strains via multiplex PCR. The established multiplex-PCR method for the simultaneous detection of newly found mountain ginseng strains, Korean ginseng, and foreign ginseng in a single reaction was determined to be effective. This study is the first report of scientific discrimination of "mountain ginsengs" and describes an effective method of identification for fraud prevention and for uncovering the possible presence of other, cheaper ginseng species on the market.

• Research in Plant Disease
• /
• v.23 no.4
• /
• pp.363-366
• /
• 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 Microbiology and Biotechnology
• /
• v.27 no.5
• /
• pp.909-915
• /
• 2017

Bifidobacterium bifidum BGN4 (BGN4) has many proven beneficial effects, including antiallergy and anticancer properties. It has been commercialized and used in several probiotic products, and thus strain-specific identification of this strain is very valuable for further strain-dependent physiological study. For this purpose, we developed novel multiplex polymerase chain reaction (PCR) primer sets for strain-specific detection of BGN4 in commercial products and fecal samples of animal models. The primer set was tested on seven strains of B. bifidum and 75 strains of the other Bifidobacterium species. The BGN4-specific regions were derived using megaBLAST against genome sequences of various B. bifidum databases and four sets of primers were designed. As a result, only BGN4 produced four PCR products simultaneously whereas the other strains did not. The PCR detection limit using BGN4-specific primer sets was $2.8{\times}10^1CFU/ml$ of BGN4. Those primer sets also detected and identified BGN4 in the probiotic products containing BNG4 and fecal samples from a BGN4-fed animal model with high specificity. Our results indicate that the PCR assay from this study is an efficient tool for the simple, rapid, and reliable identification of BGN4, for which probiotic strains are known.

• Korean Journal of Medicinal Crop Science
• /
• v.18 no.5
• /
• pp.329-337
• /
• 2010

'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.

• Journal of Veterinary Science
• /
• v.22 no.6
• /
• pp.87.1-87.12
• /
• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

• Applied Chemistry for Engineering
• /
• v.33 no.3
• /
• pp.235-241
• /
• 2022

According to the World Water Development Report 2015 released by the United Nations, drinking water is expected to decrease by 40% by 2030. This does not mean that the amount of water decreases, but rather that the water source is contaminated due to environmental pollution. Because microbes are deeply related to water quality, the analysis of microbe is very important for water quality management. While the most common method currently used for microbial analysis is microscopic examination of the shape and feature after cell culture, as the gene analysis technology advances, quantitative polymerase chain reaction (qPCR) can be applied to the microscopic microbiological analysis, and the application method has been studied. Among them, a reverse transcription (RT) step enables the analysis of RNA by RT-PCR. Integrated cell culture (ICC)-qPCR shortens the test time by using it with microbial culture analysis, and viability qPCR can reduce the false positive errors of samples collected from natural water source. Multiplex qPCR for improved throughput, and microfluidic qPCR for analysis with limited amount of sample has been developed In this paper, we introduce the case, principle and development direction of the qPCR method applied to the analysis of microorganisms.

• The Plant Pathology Journal
• /
• v.22 no.2
• /
• pp.164-167
• /
• 2006

An immunocapture reverse transcription-polymerase chain reaction (IC/RT-PCR) was developed for simultaneous detection of two pepper-infecting RNA viruses, Pepper mud mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV). The assay could be performed in a single tube for simultaneous and sensitive detection of these tobamoviruses. This detection system revealed thousand-fold increase in detection sensitivity compare to ELISA. This method could save time and reagent cost compare to common RT-PCR which needs several reactions and several procedures of viral RNA extractions for the same number of samples.