• Journal of fish pathology
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• v.17 no.3
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• pp.159-169
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• 2004

Bacterial wihte enteritis ocurred by infection of V. ichthyoenteri is a devastating disease in olive flounder (Paralichthys olivaceus) hatcheries in Korea. Since white enteritis has been a problem in aquqtic industries, necessity of a rapid detection method is increased. In an attempt to develop rapid PCR method the detection of V. ichthyoenteri, we examined the 16S-23S rRNA intergenic spacer region(ISR) of V. ichthyoenteri and developed species-specific primer for V. ichthyoenteri. The intergenic spacers were amplified by primers complementary to conserved region of 16S and 23S rRNA genes. The intergenic spacer region between the 16S and 23S rRNA genes of V. ichthoenteri were investigated by PCR fragment length typing and DNA sequencing. Analysis of the ISR sequences showed that V. ichthyoenteri contains one types of polymorphic ISRs. The size of ISRs ranged 348bp length and not contains tRNA genes. Mutiple alignment of representative sequences from different Vibrio species revealed several domains of high sequence variability, and allowed to design species-specific primer for detection of Vibrio ichthyoenteri. PCR. The specific of the primer was examined using genomic DNA prepared from 19 different Vibrio species, isolated 18group Vibrio species. The results showed that the PCR reaction using species-specific primer designed in this study can be used to detect V. ichthyoenteri.

• Journal of Food Hygiene and Safety
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• v.37 no.1
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• pp.9-14
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• 2022

The purpose of this study was to analyze the hazard of fungi in Garaetteok (Korean rice cake) by isolating and identifying of fungi contaminated with Garaetteok and investigating the possibility of mycotoxin production. Garaetteok used in this study were the ones that were returned back to the manufacturers in Jeollanam-do due to the presence of foreign matters presumed to be fungi. The fungi foreign matter was collected and inoculated on Potato dextrose agar, Malt extract agar, and Czapek yeast extract agar, and then cultured at 25℃ for 7 days. The micro-structure was observed under an optical microscope for the colonies in which pure isolation was confirmed. The gene sequencing of the product of amplified PCR was analyzed using the ITS primer. Colony-1 and 2 maintained the same properties in each tray, confirming that they were purely isolated. Budding cells were observed from the Colony-1, thus, it was determined to be yeast. Colony-2 was determined to be a fungus that belongs to Fusarium spp. as fusiform conidia were observed. As a result of gene sequencing, a total of 76 cases of fungi of Fusarium spp. were found, among which Fusarium solani was the most observed cases (53 cases). From the morphological and genetic identification, Colony-2 was identified as Fusarium spp., specifically, Fusarium solani. The fungi found in Fusarium spp. produce mycotoxins such as nivalenol, zearalenone, and fumonisin, which may cause vomiting, diarrhea, and cancer. Conclusively, the results confirm the possibility of mycotoxin production by Fusarium spp. isolated from Garaetteok. Consequently, when an unknown fungus was found, it is necessary to isolate and identify the fungus, determine whether it is a mycotoxin producing species, and strengthen relative administrative measures, accordingly.

• Journal of Industrial Technology
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• v.28 no.A
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

• Korean Journal of Clinical Laboratory Science
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• v.40 no.1
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• pp.1-5
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• 2008

Thirty two of bathroom water samples from public bathroom in Seoul areas were examined using acid-fast staining, Lowenstein-Jensen (L-J) medium culture and PCR-restriction fragment length polymorphism (PCR-RFLP). In 6.25% (2/32) bathroom water samples, acid-fast bacilli were detected by AFB stain, and in 21.9% (7/32) bathroom water samples, acid fast bacilli grew on L-J media. Of them, six acid-fast bacilli were identified as Mycobacterium avium, and the other AFB as Mycobacterium szulgai by PCR-RFLP. These results are suggested that accidental nontuberculosis mycobacterial infection to a weakness person will be possible in public area.

• Korean Journal of Clinical Laboratory Science
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• v.38 no.3
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• pp.158-165
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• 2006

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.190-198
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• 2021

Human Aichivirus (Aichivirus A; AiV-A) is a positive-sense single-strand RNA non-enveloped virus that has been detected worldwide in various water environments including sewage, river, surface, and ground over the past decade. To develop a method with excellent sensitivity and specificity for AiV-A diagnosis from water environments such as groundwater, a combination capable of reverse transcription (RT)-nested polymerase chain reaction (PCR) was developed based on existing reported and newly designed primers. A selective method was applied to evaluate domestic drinking groundwater samples. Thus, a procedure was devised to select and subsequently identify RT-nested PCR primer sets that can successfully detect and identify AiV-A from groundwater samples. The findings will contribute to developing a better monitoring system to detect AiV-A contamination in water environments such as groundwater.

• Journal of fish pathology
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• v.34 no.1
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• pp.31-38
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• 2021

The objective of this study was to determine comparative biochemical characteristics and RAPD (random amplified polymorphic DNA) profiles of 18 strains of Edwardsiella tarda isolated from cultured olive flounder (Paralichthys olivaceus) and eel (Anguilla spp) that showed diseases between 1996 and 2010 in Korea. In terms of biochemical properties, they showed four patterns with differences in citrate degradation and production of H₂S and indole. All strains were identified as E. tarda. Characteristics of isolates were not classified according to their host. As a result of PCR with E. tarda-specific primer, EDtT, the same band of about 270 bp was detected in all 18 isolates. However, no specific band was detected in type strains of E. tarda or Edwardsiella ictaluri. As a result of RAPD PCR performed with primer No. 5 and No. 6 of a Ready-To-Go-RAPD kit, the band profile showed clear differences among isolates of olive flounder, isolates of eel, and E. tarda and E. ictaluri type strains. It was possible to organize their characteristics according to the origin of host with possibility to develop tools for pathogen monitoring.

• Korean Journal of Ecology and Environment
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• v.33 no.3 s.91
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• pp.206-212
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• 2000

Since some cyanobacterial isolates under selective culturing conditions are lacking of characteristic specialized cells or showing altered morphology, the morpho-taxonomic criteria are not accurate enough to discriminate between species. Instead of morphological parameters, a method based on the single or the combination of repetitive oligonucleotides in a single PCR, repetitive oligonucleotides-primed PCR (ROP-PCR), was applied to generate DNA profiles for members of the cyanobacterial genera Anabaena and Oscillatoria, both of which are responsible for causing poisonous blooms in various freshwater systems. ROP-PCR performed on 10 isolates of the cyanobacteria with ERIC and REP sequences from gram-negative bacteria, STRR1A and LTRR sequences derived from cyanobacterial genome, and eukaryotic repetitive sequences, led to the identification of distinct genotypes, and provided specific and repeatable DNA fingerprints for cyanobacterial isolates. Grouping analysis of cyanobacterial isolates showed a signifiant difference depending on the primer used in PCR.

• Journal of Life Science
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• v.24 no.4
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• pp.361-369
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• 2014

Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.19-25
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• 2006

This study was undertaken to develop PCR primers for the identification and detection of Streptococcus mutans (by)using species-specific forward and universal reverse primers. These primers targeted the variable regions of the 16S ribosomal RNA coding gene (rDNA). The primer specificity was tested against 11S. mutans strains and 10 different species (22 strains) of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of S. mutans ATCC $25175^T$. The data showed that species-specific amplicons were obtained from all the S. mutans strains tested, which was not observed in the other species. The direct and nested PCR could detect as little as 2 pg and 2 fg of the chromosomal DNA from S. mutans ATCC $25175^T$, respectively. This shows that the PCR primers are highly sensitive and applicable to the detection and identification of S. mutans.