• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.1
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• pp.35-41
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• 2003

The study was conducted to develope a rapid method for the detection of Yersinia enterocolitica in spring water and vegetables via multiplex polymerase chain reaction (PCR) technique using ail, yst, uirF and subgenus-specific Y16S primers. Specificity and sensitivity of multiplex PCR and application of best primers for the detection of Y. enterocolitica from spring water and vegetables were investigeted. Y. enterocolitica ATCC 27729 strains gave 356 bP and 200 bp (Y16S) and 134 bp (yst) bands. but Y. enterocolitica ATCC 9610 and ATCC 23715 strains gave 200 bp and 134 bp bands.In the meanwhile, non-pathogenic Yersinia species, such as Y. frederikseni, Y. inter-media, Y. kristenseni and Y. pseudotuberculosis gave only single 200 bp band, and other bacteria including Escherichia coli O157:H7 ATCC 25392, Shigella dysenteri. Staphylococcu aureus ATCC 25923 and Listeria mo-nocytogenes ATCC 19111 did not show any bands. Among primers, yst and Y16S primer showed the best sensitivity. Seven CFU/mL Y. enterocolitica cells could be detected with yst and Y16S primers and the sensitivity was significantly improved by the further 2nd PCR after 38 cycles of first PCR amplication. Spring water, cabbage and mushroom were inoculated with Y. enterocolitica to determine the sensitivity of multiplex-PCR for the rapid detection of Y. enterocolitica. Multiplex-PCR assay could detect 7 or 70 cells in spring water and vegetables using whole cell lysate with repeating PCR amplication.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.321-331
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• 2011

Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical-based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.

• Korean Journal of Veterinary Research
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• v.55 no.2
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• pp.105-110
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• 2015

A real-time PCR assay using hybridization probe (HybProbe) has been developed to detect Brucella (B.) melitensis strains. The primer and HybProbe sets were designed based on the gap gene of chromosome I with a specific single nucleotide polymorphism of B. melitensis. Specificity of the assay was confirmed by comparison to reference Brucella species and other related strains. In the melting curve analysis, B. melitensis generated a peak at $67^{\circ}C$ unlike those for other Brucella species observed at $61^{\circ}C$. Sensitivity of the assay for B. melitensis ranged from 20 ng to 200 fg of genomic DNA. The ability to identify 94 Mongolian B. melitensis isolates using the real-time PCR assay was identical to that of classical biotyping methods and differential multiplex PCR. These data showed that this new molecular technique is a simple and quick method for detecting B. melitensis, which will be important for the control and prevention of brucellosis.

• Journal of Applied Biological Chemistry
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• v.58 no.4
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• pp.383-387
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• 2015

A duplex polymerase chain reaction (PCR) detection method was developed to authenticate the use of cat and rabbit in food and to prevent unlawful distribution of illegally butchered meat in both domestic and imported food market. Species-specific primers were designed targeting mitochondrial cytochrome b gene. The sizes of PCR products were 191 bp for cat and 101 bp for rabbit, which were relatively small for better application of the detection method on processed foods. Specificities of primers were verified using 21 animal species including cat and rabbit. Limit of detection was examined by serial dilution of the sample DNA and confirmed as 0.005 ng for rabbit and 0.0005 ng for cat using Bioanalyzer. The developed duplex PCR method showed specificity and sensitivity in the identification of two target species.

• Fisheries and Aquatic Sciences
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• v.23 no.5
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• pp.13.1-13.7
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• 2020

Stock enhancement is considered to be a valuable approach for restoring fishery resources. Because no specific official institution in Taiwan is responsible for the production of fry, the released fry are purchased directly from the private sector. However, fishermen from the private industry have not established a genetic background, so the genetic composition for each batch of released fry is unclear. Mangrove red snapper (Lutjanus argentimaculatus), a prominent species released in Taiwan, was collected after its official release. One hundred and two field samples were compared with four batches of hatchery fry (n = 685) by using a microsatellite-based multiplex PCR assay. Four of the field samples (3.9%; 4/102) were revealed to be from a fish farm and most likely from a single batch. This study revealed that wild mangrove red snappers are genetically different from those originating from farms, and their origins can be traced through molecular markers, even without information on breeding stocks.

• The Plant Pathology Journal
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• v.40 no.1
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• pp.40-47
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• 2024

Garlic can be infected by a variety of viruses, but mixed infections with leek yellow stripe virus, onion yellow dwarf virus, and allexiviruses are the most damaging, so an easy, inexpensive on-site method to simultaneously detect at least these three viruses with a certain degree of accuracy is needed to produce virus-free plants. The most common laboratory method for diagnosis is multiplex reverse transcription polymerase chain reaction (RT-PCR). However, allexiviruses are highly diverse even within the same species, making it difficult to design universal PCR primers for all garlic-growing regions in the world. To solve this problem, we developed an inexpensive on-site detection system for the three garlic viruses that uses a commercial mobile PCR device and a compact electrophoresis system with a blue light. In this system, virus-specific bands generated by electrophoresis can be identified by eye in real time because the PCR products are labeled with a fluorescent dye, FITC. Because the electrophoresis step might eventually be replaced with a lateral flow assay (LFA), we also demonstrated that a uniplex LFA can be used for virus detection; however, multiplexing and a significant cost reduction are needed before it can be used for on-site detection.

• Animal Bioscience
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• v.34 no.4
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• pp.734-742
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• 2021

Objective: The present study aimed to investigate the occurrence and species of coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS) in retail pork meat samples collected during nationwide monitoring. The staphylococcal isolates were characterized for antimicrobial and zinc chloride resistance and enterotoxigenic potential. Methods: A total of 260 pre-packaged pork meat samples were collected from 35 retail markets in 8 provinces in Korea for isolation of staphylococci. Antimicrobial and zinc chloride resistance phenotypes, and genes associated with the resistance phenotypes were determined on the isolates. Furthermore, the presence and distribution of 19 staphylococcal enterotoxin (SE) genes and enterotoxin-like genes among the pork-associated staphylococci were determined by multiplex polymerase chain reaction-based assays using the specific primer sets. Results: A total of 29 staphylococcal strains (29/260, 11.1%) were isolated from samples of retail pork meat, 24 (83%) of which were CoNS. The four CoNS species identified were S. saprophyticus (n = 16, 55%), S. sciuri (n = 3, 10%), S. warneri (n = 3, 10%), and S. epidermidis (n = 2, 7%). Among the 29 isolates, four methicillin-resistant CoNS (MR-CoNS; three S. sciuri and one S. epidermidis) and one methicillin-resistant CoPS (MR-CoPS; one S. aureus) were identified. In addition, a relatively high level of tetracycline (TET) resistance (52%) was confirmed in CoNS, along with a predominant distribution of tet(K). The most prevalent SEs were sep (45%), and sen (28%), which were carried by 81% of S. saprophyticus. Conclusion: These findings suggest that CoNS, especially S. saprophyticus strains, in raw pork meat could be a potential risk factor for staphylococcal food poisoning (SFP), and therefore, requires further investigation to elucidate the role of SEls in SFP and virulence of the pathogen. Our results also suggest that CoNS from raw pork meat may act as a source for transmission of antimicrobial resistance genes such as staphylococcal cassette chromosome mec and tet(K).

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.239-248
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• 2021

The emergence of multidrug-resistant Acinetobacter baumannii has partly increased treatment failure and patient mortality. Class D β-lactamases is an important mechanism of resistance to beta-lactam antibiotics in this species. This study aimed to investigate the relationship between the presence oxacillinase gene and genetic fingerprints of A. baumannii isolates from the intensive care unit of an Egyptian tertiary care hospital. One hundred and twenty A. baumannii clinical isolates were collected. Multiplex PCR was performed to detect genes encoding oxacillinases (OXA-23, OXA-24, OXA-51, OXA-58 and OXA-143). Molecular typing of all collected isolates was performed using random amplified polymorphic DNA (RAPD)-PCR assay. Out of 120 examined isolates, 92, 88 and 84% were resistant to ertapenem, imipenem and meropenem, respectively. The species-specific, commonly present OXA-51 gene was found in all isolates while OXA-23 showed a high prevalence of 88% of isolates. OXA-24 and OXA-143 genes were detected in 3% and 1% of isolates, respectively. No OXA-58 gene was detected. Five clusters consisting of 19 genotypes were detected using RAPD-PCR. Genotype A was the most prevalent, it was observed in 62% of the isolates followed by genotype B (12%). These results revealed that genotypes A and B are common in the hospital. Results also demonstrate that RAPD-PCR is a rapid and reliable method for studying the clonal similarity among A. baumannii isolated from different clinical specimens.

• Journal of Mushroom
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• v.15 no.3
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• pp.111-117
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• 2017

The mushroom species Cordyceps militaris has been studied and cultivated as a medicinal mushroom due to its multiple valuable biological and pharmaceutical activities. For breeding new strains of C. militaris, multiplex PCR assays were performed using primers specific for its mating type genes, MAT1-1 and MAT1-2. Mating types and mating status were confirmed, as evidenced by DNA bands at 233-bp and 191-bp for MAT1-1 and MAT1-2 respectively. The novel 'Dowonhongcho 2ho' was developed through mating; they were found to possess high-quality fruiting bodies when grown in artificial media. The stromata of the new strain were club-shaped, with a bright orange-red color, and measured 7.1 cm in length. They had an average cordycepin content of 0.33%. Compared to 'Dowonhongcho,' the new strain had a 7% higher yield, as well as firm fruiting bodies. The optimum temperature for mycelial growth was $20{\sim}25^{\circ}C$, and the optimum temperature for stroma development was $18{\sim}22^{\circ}C$. The fruiting bodies developed after 49.1 days from inoculation. The use of mating type molecular markers improved the breeding efficiency of the new strain 'Dowonhongcho 2ho.' Thus, they may be valuable for artificial cultivation and industrial-scale production of C. militaris with excellent characteristics.