• Biomedical Science Letters
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• v.27 no.1
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• pp.35-43
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• 2021

Human Sapovirus (HuSaV) is one of the major causes of acute gastroenteritis in humans, and it is used as a molecular diagnostic technique based on polymerase chain reaction (PCR) from humans, food, shellfish, and aquatic environments. In this study, the HuSaV diagnosis technique was used in an aquatic environment where a number of PCR inhibitors are included and pathogens, such as viruses, are estimated to exist at low concentration levels. HuSaV-specific primers are improved to detect 38 strains registered in the National Center for Biotechnology Information (NCBI). The established optimal condition and the composition, including the RT-nested PCR primers and SL® Non-specific reaction inhibitor, were found to have 100 times higher sensitivity based on HuSaV plasmid than the previously reported methods (100 ag based on HuSaV plasmid 1 ng/μL). Through an artificial infection test, the developed method was able to detect at least 1 fg/μL of HuSaV plasmid contaminated with total nucleic acid extracted from groundwater. In addition, RT-nested PCR primer sets for HuSaV detection can react, and a positive control is developed to verify false positives. This study is expected to be used as a HuSaV monitoring method in the future and applied to the safety response to HuSaV from water environments.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.6
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• pp.775-778
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• 2006

Sarabi cows (n = 136) from the Sarabi Breeding Station were genotyped at bovine lymphocyte antigen (BoLA)-DRB3.2 locus by a genotyping system that used the polymerase chain reaction and restriction fragment length polymorphism. Genomic DNA was extracted from whole blood samples. A two-step polymerase chain reaction was carried out in order to amplify a 284 base-pair fragment of target gene. Nested-PCR products were digested with three restriction endonuclease enzymes RsaI, BstYI and HaeIII. Digested fragments were analyzed by polyacrylamide gel electrophoresis. Twenty-six BoLA-DRB3.2 alleles were identified with frequencies ranging from 0.4 to 15.1%. Six new allele types observed in this study have not been reported previously. Identified alleles include: BoLA-DRB3.$2^*1$, $^*2$, $^*4$, $^*6$, $^*8$, $^*12$, $^*13$, $^*14$, $^*15$, $^*16$, $^*17$, $^*23$, $^*24$, $^*25$, $^*28$, $^*32$, $^*34$, $^*35$, $^*36$, $^*37$, $^*42$, $^*46$, $^*51$, $^*kba$, $^*laa$ and $^*vaa$. Their frequencies were found to be 0.4, 0.4, 0.7, 11.4, 1.1, 1.8, 2.9, 2.2, 4.4, 9.6, 1.1, 13.6, 0.4, 0.4, 1.1, 0.7, 0.4, 6.2, 2.2, 3.7, 1.1, 7.7, 1.5, 15.1, 2.6 and 7.3% respectively. The six most frequent alleles (DRB3.2 $^*6$, $^*16$, $^*23$, $^*46$, $^*kba$ and $^*vaa$) accounted for 64.7% of the alleles in the population of this herd. Numerous studies on this locus, covering different breeds, has revealed the existence of various alleles in this locus, and new investigations have introduced novel alleles. With respect to the high number of the observed alleles in this survey and the novelty of some alleles with no previous record of reporting, it is plausible to conclude that the BoLA-DRB3.2 locus is highly polymorphic in Iranian native Sarabi cows.

• Microbiology and Biotechnology Letters
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• v.51 no.1
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• pp.99-108
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• 2023

Several challenges arise in DNA extraction and gene amplification for airborne fungal metagenome analysis from a particulate matter (PM) samples. In this study, various conditions were tested to optimize the DNA extraction method from PM samples and polymerase chain reaction (PCR) conditions with primer set and annealing temperature. As a result of comparative evaluation of DNA extraction under various conditions, chemical cell lysis using buffer and proteinase K for 20 minutes and bead beating treatment were followed by using a commercial DNA extraction kit to efficiently extract DNA from the PM filter samples. To optimize the PCR conditions, PCR was performed using 10 primer sets for amplifying the ITS2 gene region. The concentration of the PCR amplicon was relatively high when the annealing temperature was 58℃ with the ITS3tagmix3/ITS4 primer set. Even under these conditions, when the concentration of the PCR product was low, nested PCR was performed using the primary PCR amplicon as the template DNA to amplify the ITS2 gene at a satisfactory concentration. Using the methods optimized in this study, DNA extraction and PCR were performed on 15 filter samples that collected PM2.5 in Seoul, and the ITS2 gene was successfully amplified in all samples. The optimized methods can be used for research on analyzing and interpreting the fungal metagenome of atmospheric PM samples.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1146-1153
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• 2024

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.5
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• pp.527-533
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• 2017

We investigated the infection of nervous necrosis virus (NNV) in seven band grouper Hyporthodus septemfasciatus broodstocks, which have been reared in aquaculture farms in South Korea during 2012-2014. To investigate the prevalence of NNV within the broodstock, egg, sperm, and blood were sampled in the spawning season. The egg and sperm samples were subjected to a nested reverse transcription (RT) polymerase chain reaction (PCR) assay to detect NNV and were inoculated on SSN-1 cells to culture the virus. Blood samples were used to detect antibodies against NNV using enzyme linked immunosorbent assay (ELSIA). Positive values from ELISA were found in 39 of 162 samples (24%) in 2012, and 13 of 28 samples (46%) in 2014. Additionally, 4 of 34 broodstocks (11%) investigated in 2013-2014 were determined to be carriers from the nested RT-PCR and in vitro cultivation. The broodstocks in which antibodies against NNV were detected by ELISA, or in which NNV was detected by the nested RT-PCR assay, posed a risk of vertical transmission of NNV. Therefore, it is necessary to select virus-free broodstocks in seed production to reduce the possibility of the vertical transmission of NNV.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.493-496
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• 2016

Peach rosette mosaic virus (PRMV) is a plant virus that was first reported in 1933 by Peach. It can infect hosts including peach, grape, blueberry, dandelion, plum, cherry tree, and weeds. PRMV is non-reportable in Korea, but it is designated as a controlled virus requiring plant quarantine. In this study, for the rapid and specific detection of PRMV, we developed an assay using loop-mediated isothermal amplification (LAMP). Comparison between conventional polymerase chain reaction (PCR) methods (real time-PCR and nested PCR) and LAMP for the detection of PRMV revealed an equivalent level of sensitivity by all the tested methods. For the LAMP assay, outer primer sets were used to amplify a 264-bp PCR product, which was then digested using the restriction enzyme Pvu II (CAG/CTG), and the visualization of two digestion fragments (207 + 57 bp) indicated a positive reaction. The developed LAMP assay for PRMV is expected to enable the rapid monitoring of PRMV in plants.

• Journal of Oral Medicine and Pain
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• v.35 no.2
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• pp.101-110
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• 2010

Helicobacter pylori(H. pylori) is bacterial infection, with more than half of the world population infected and oral cavity is considered second reservoir of H. pylori infection. The purpose of this study was to evaluate role of oral cavity in H. pylori infection by comparison of the mode H. pylori infection in oral cavity and stomach. We recruited 100 subjects without systemic disease including gastrointestinal disease. Samples in oral cavity taken on gingival sulcus fluid(GSF) of lower left central incisor and 1st molar, area of buccal mucosa, dorsum of the tongue, palatal and saliva. We analyzed by Nested polymerase chain reaction(PCR) for oral infection and Urea Breath Test(UBT) for gastric infection. The results were as follows : 1. Among these 100 subjects, 36(36%) were positive by Nested PCR and 33(33%) were positive by UBT(p>0.05). 2. In detection rate of H. pylori in sites taken sample, 11(11%), 8(8%), 9(9%), 3(3%), 9(9%), 7(7%) were positive on GSF of lower left central incisor and 1st molar, area of buccal mucosa, dorsum of the tongue, palatal and saliva, respectively. Statical significance was observed in samples of GSF of lower left central incisor and area of dorsum of the tongue(p<0.05). 3. In comparison of the mode of H. pylori infection in oral cavity and stomach by analytic method, positive in oral cavity and stomach was 10(10%), negative in oral cavity and positive in stomach was 23(23%), positive in oral cavity and negative in stomach was 26(26%) and negative in oral cavity and stomach was 41(41%)(p>0.05). Conclusively, we can guess that oral H. pylori is not associated with gastric H. pylori infection and normal flora.

• Mycobiology
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• v.31 no.2
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• pp.68-73
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• 2003

Roots of Glycine max and Miscanthus sinensis and soil samples were collected from various field sites at Goesan, Chungbuk in Korea. Microscopic observations of the roots indicated high colonization rates of both arbuscular mycorrhizal fungi(AMF) and other fungi. The partial small subunit of ribosomal DNA genes were amplified with the genomic DNA extracted from their roots by nested polymerase chain reaction(PCR) with universal primer NS1 and fungal specific primers AML Restriction fragment length polymorphism(RFLP) was analyzed using the combinations of three restriction enzymes, HinfI, AluI and AsuC21. Nucleotides sequence analysis revealed that ten sequences from Miscanthus sinensis and one sequence from Glycine max were close to those of arbuscular mycorrhizal fungi. Also, 33% of total clones amplified with NS31-AM1 primers from M. sinensis and 97% from G. max were close to Fusarium oxysporum or other pathogenic fungi, and they were successfully distinguished from AME Results suggested that these techniques could help to distinguish arbuscular mycorrhizal fungi from root pathogenic fungi in the plant roots. Especially, DNA amplified by these primers showed distinct polymorphisms between AMF and plant pathogenic species of Fusarium when digested with AsuC21.

• Korean Journal of Veterinary Service
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• v.35 no.4
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• pp.333-337
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• 2012

This study was carried out to investigate the prevalence of bovine leukemia virus (BLV) infection and to compare the results of enzyme-linked immunosorbent assay (ELISA) and nested polymerase chain reaction (nPCR) in dairy farms in northern Gyeonggi province from August through December 2011. A total of 625 dairy cattle from 14 dairy farms were tested for antibodies against BLV using commercially available ELISA test kit. The overall seroprevalence of BLV infection was 76.3%. The seroprevalence of diary cattle according to age was the highest at 61~72 months (88.0%, P<0.001). Two hundred fifty one dairy cattle from 7 diary farms were tested ELISA and nPCR. The kappa value of BLV between ELISA and nPCR was 0.765. The results indicate that BLV infection spread widely in dairy farms and the nPCR is rapid method for the early detection of BLV infection.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3603-3606
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• 2013

Background: To investigate the infection status and predominant genotype distribution of human papillomavirus (HPV) infection among Chinese patients with mucopurulent cervicitis (MPC) or cervical cancer (CC) in Hangzhou. Methods: Initially, 217 cases of healthy cervix controls (n=50), acute MPC (n=89), and CC (n=78) were included; samples were collected between January 1, 2010, and January 30, 2013. Cervical specimens were screened for HPV using a nested polymerase chain reaction assay and DNA sequencing. Results: Overall prevalence of HPV infection was 16.7% in the control group, 51.9% in the MPC group, and 84.4% in the CC group. The predominant genotype detected in all 3 groups was the oncogenic variant HPV 16 (55.8%, 17.3%, and 6.3% in the CC, MPC and control specimens, respectively), HPV58 was the second most predominant HPV type in CC (9.1%), MPC (8.6%), and control group (4.2%). Most or all of the genotypes were oncogenic in the three groups. Conclusions: Infection with HPV was found to be prevalent among Chinese women with MPC or CC and oncogenic variants were in the majority. Therefore, peoples who suffered MPC with HPV DNA positive should be regularly followed-up, for prevention and early treatment of cervical cancer.