• The Plant Pathology Journal
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• v.35 no.6
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• pp.654-661
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• 2019

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea's soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R² = 0.996, slope = -3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

• The Korean Journal of Mycology
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• v.36 no.2
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• pp.138-143
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• 2008

Botrytis cinerea, gray mold pathogen, causes serious losses in greenhouse tomato crop. In this study, a primer set was developed for identification and specific PCR detection of B. cinerea from tomato plants. The primer pair (BTF1/BTR1) was designed from polymorphic sequence region in pyruvate carboxylase gene (pyc) of B. cinerea. A PCR product (112 bp) was amplified on genomic DNA of 13 B. cinerea isolates from 10 different host plants, but not on those from 6 other Botrytis spp., 4 Botryotinia spp., 5 Sclerotinia spp. and 16 other genus of phytopathogenic fungi. The sensitivity limit of the primer set was 2 pg of genomic DNA of B. cinerea, approximately. The PCR assay using species-specific primer set was specifically able to detect the pathogen on naturally infected tomato plants and artificially infected plants. These results suggest that the sensitivity and specificity of this primer set can be applied in a rapid and accurate diagnosis of tomato disease caused by B. cinerea.

• Journal of Ginseng Research
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• v.43 no.1
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• pp.1-9
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• 2019

Background: Korean ginseng is an important cash crop in Asian countries. However, plant yield is reduced by pathogens. Among the Ilyonectria radicicola-species complex, I. mors-panacis is responsible for root-rot and replant failure of ginseng in Asia. The development of new methods to reveal the existence of the pathogen before cultivation is started is essential. Therefore, a quantitative real-time polymerase chain reaction method was developed to detect and quantify the pathogen in ginseng soils. Methods: In this study, a species-specific histone H3 primer set was developed for the quantification of I. mors-panacis. The primer set was used on DNA from other microbes to evaluate its sensitivity and selectivity for I. mors-panacis DNA. Sterilized soil samples artificially infected with the pathogen at different concentrations were used to evaluate the ability of the primer set to detect the pathogen population in the soil DNA. Finally, the pathogen was quantified in many natural soil samples. Results: The designed primer set was found to be sensitive and selective for I. mors-panacis DNA. In artificially infected sterilized soil samples, using quantitative real-time polymerase chain reaction the estimated amount of template was positively correlated with the pathogen concentration in soil samples ($R^2=0.95$), disease severity index ($R^2=0.99$), and colony-forming units ($R^2=0.87$). In natural soils, the pathogen was recorded in most fields producing bad yields at a range of $5.82{\pm}2.35pg/g$ to $892.34{\pm}103.70pg/g$ of soil. Conclusion: According to these results, the proposed primer set is applicable for estimating soil quality before ginseng cultivation. This will contribute to disease management and crop protection in the future.

• Journal of Microbiology
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• v.44 no.1
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• pp.92-97
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• 2006

We developed an mPCR assay for the simultaneous detection, in one tube, of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes using species-specific primers. The mPCR employed the E. coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B and L. monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405 and 210 bp, respectively. All PCR products were easily detected by agarose gel electrophoresis, and the sequences of the specific amplicons assessed. Potential pathogenic bacteria, in laboratory-prepared and four commercially available kimchi products, were using this mPCR assay, and the amplicons cloned and sequenced. The results correlated exactly with sequences derived for amplicons obtained during preliminry tests with known organisms. The sensitivity of the assay was determined for the purified pathogen DNAs from four strains. The mPCR detected pathogen DNA at concentrations ranging from approximately 0.45 to $0.05\;pM/{\mu}l$. Thus, this mPCR assay may allow for the rapid, reliable and cost-effective identification of four potentially pathogens present in the mixed bacterial communities of commercially available kimchi.

• Proceedings of the National Institute of Ecology of the Republic of Korea
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• v.4 no.4
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• pp.154-158
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• 2023

The Asiatic black bear, Ursus thibetanus, is among the most threatened or endangered species in Asia. For its conservation and management, sex identification of U. thibetanus using non-invasive samples (e.g., hair and/or feces) is potentially valuable. In this study, a non-invasive molecular method for sex identification of U. thibetanus samples collected from various countries was first utilized, and it was based on polymerase chain reaction (PCR) amplification of the amelogenin gene via PCRs. Thirty-three bear DNA samples, extracted not only from blood (n=9) but also from hair (n=18) and feces (n=6), were used. We performed sex-specific PCR amplifications of the amelogenin gene using a primer set, SE47 and SE48. The primer set could successfully amplify a single X-specific band for females and both X- and Y-specific bands for males from all blood (100%) and hair (100%) samples. In addition, the primer set could distinguish the sex of bears in four out of a total of six fecal samples (approximately 67%). This study's findings suggest that this molecular method can be applied to sex identification of Asiatic black bears from various Asian regions using non-invasive samples, such as hair and feces.

• Korean Journal of Microbiology
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• v.39 no.1
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• pp.36-39
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• 2003

The rapid and reliable 16S rDNA multiplex polymerase chain reaction (PCR) assay was established to characterize bacterial etiologies of middle ear effusion. These etiologies included Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumonia, which were detected in middle-ear effusion (MEE) samples taken from patient with otitis media. A total of 39 MEE samples were aspirated from 26 patients. DNA was extracted from MEE samples, and PCR was done with DNA extracts by using the common primers, which is localized at C4 region in the 16S rDNA gene of all bacterial species, and species-specific primers: (i) Haemophilus-specific primer, (ii) Moraxella- specific primer, and (iii) Streptococcus-specific primer. Among 39 samples tested, 24 (61.5%) were positive for H. influenzae, 10 (25.6%) were positive for M. catarrhalis, 3(7.7%) were positive for S. pneumonia, and 11 (28%) were negative for 165 rDNA multiplex PCR reaction. Nine samples (28.6%) exhibited a mixed infection and were positive for both H. infuenzae and M. catarrhalis. We suggested that 16S rDNA multiplex PCR is a useful method to identify rapidly for rapid identification of the pathogenic bacteria and characterization of bacterial etiologies of middle ear effusion.

• BMB Reports
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• v.37 no.2
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• pp.213-222
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• 2004

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.

• Journal of the East Asian Society of Dietary Life
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• v.18 no.5
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• pp.753-759
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• 2008

The principal objective of this study was to develop the optimum oligonucleotide primers for the simple detection of Campylobacter in food samples. In order to achieve this goal, a variety of oligonucleotide primers were designed via the modification of 16S rDNA, ceuE and mapA sequences of Campylobacter. Through the subsequent analysis of the specificity and sensitivity of primers, two types of oligonucleotide primers, CB4 and CJ1, were selected for Campylobacter genus-specific and C. jejuni species-specific primers, respectively. The detection limit was found to be $10^0{\sim}10^1$ cells per reaction with the prepared cell suspension, however, the sensitivity in the meat samples was less, at $10^1{\sim}10^2$. We suggested that PCR inhibitors such as hemoglobin or immunoglobulin in pork or beef influenced.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.309-313
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• 2012

The purpose of this study is to simultaneously detect and identify specific genus of Lactobacillus distributed in vagina of healthy women in their twenties by using multiplex primer. Vaginal fluid samples were taken from 166 women who were healthy and did not have vaginosis symptoms caused by bacterial infection. Six species of lactic acid bacteria belong to Lactobacillus genus that are known to inhabit in healthy vagina were selected to make a species-specific multiplex primer. Multiplex primer I was specified to selectively detect L. iners, L. crispatus, L. gasseri and multiplex primer II was specified to selectively detect L. acidophilus, L. jensenii, L. fermentum. L. crispatus (77%) was most frequently detected, and L. acidophilus (57%) and L. jensenii (57%) were relatively higher than others. Although the proportion of L. iners (59%) was a little higher than those of L. acidophilus and L. jensenii, this should be further validated in healthy women's vagina to be sure since they often appear in women having bacterial vaginosis and/or during the antibiotic therapy. Conclusively, the multiplex PCR technique using the species-specific primer could a useful tool to predict variation of vaginal health condition and process of recovery from vaginal disease.

• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.909-915
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• 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.