• 대한수의학회지
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• 제60권3호
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• pp.109-116
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• 2020

This study aimed to develop a semi-nested polymerase chain reaction assay for the direct detection of Mycoplasma synoviae (M. synoviae) from clinical samples using three newly designed oligonucleotide primers specific to the variable lipoprotein haemagglutinin (vlhA) gene and differentiate M. synoviae field strains based on a nucleotide deletion or the insertion of the proline-rich repeat (PRR) region of the vlhA gene. The developed semi-nested polymerase chain reaction (PCR) assay revealed positive results in 12 out of 100 clinical samples collected from chickens showing lameness and joint swelling. Six positive samples were selected randomly for sequencing, and sequence analysis revealed 96.3-100% nucleotide identities compared to the reference sequences. Phylogenetic analysis showed that sequences of the strains in this study were closely related to WVU1853 (Spain), CK.MS.UDL.PK.2014.2 (Pakistan), and F10-2AS (USA) strains, but they were distinct from the M. synoviae-H vaccine strain sequence. M. synoviae obtained from these samples were identified as types A and C with a length of 38 and 32 amino acids, respectively. These results indicated that the specific and sensitive semi-nested PCR could be a useful diagnostic tool for the direct identification of clinical samples, and the sequence analysis of the partial vlhA gene can be useful for typing M. Synoviae.

• Molecules and Cells
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• 제24권3호
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• pp.329-337
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• 2007

An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.

• Clinical and Experimental Reproductive Medicine
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• 제34권3호
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• pp.179-188
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• 2007

목 적: 본 연구에서는 삼핵산 반복서열 확장에 의해 발병하는 헌팅톤병, 척수소뇌성 운동실조증과 X-염색체 취약 증후군 등에 대한 착상전 유전진단을 시행하기 위한 전임상 검사에서 진단 방법을 최적화하는 과정을 통해 얻은 결과들에 대해 기술하고자 한다. 연구방법: 단일 림프구를 이용한 임상전 검사에서는 서로 다른 allele를 갖고 있는 환자의 단일 세포를 사용하였으며, 헌팅톤병과 척수소뇌성 운동실조증에서는 fluorescent semi-nested PCR 시행 후 fragment analysis를 수행하였다. X-염색체 취약 증후군의 경우 multiple displacement amplification (MDA) 방법을 이용한 whole genome amplification에서 얻어진 MDA 산물로 fluorescent PCR을 시도하였다. 결 과: 헌팅톤병의 경우 단일 림프구 시료 모두에서 CAG repeats 증폭에 성공하여 100.0%의 증폭성공률과 14.0% allele drop-out (ADO) rate를, 척수소뇌성 운동실조증의 경우 94.7%의 증폭성공률과 5.6%의 ADO rate을 나타내었다. X-염색체 취약 증후군의 경우 fluorescent semi-nested PCR 방법만으로는 단일 림프구 시료에서 CGG repeats이 증폭되지 않았지만, MDA 산물을 이용한 fluorescent PCR 결과 84.2%의 증폭성공률과 31.3%의 ADO rate을 얻을 수 있었다. 결 론: 본 연구를 통해 헌팅톤병과 척수소뇌성 운동실조증의 착상전 유전진단에는 fluorescent semi-nested PCR 방법의 적용이 가능함을 확인하였으며, X-염색체 취약 증후군의 경우에는 MDA를 이용한 fluorescent PCR 방법을 사용해야 함을 알 수 있었다. 유전자의 변이에 대한 분석이 쉽지 않은 단일 유전자 이상에 대한 착상전 유전진단의 경우 다양한 유전자 분석 방법을 이용한 단일 세포에서의 진단 방법의 최적화 연구가 필수적으로 선행되어야 할 것으로 사료된다.

• Journal of Animal Science and Technology
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• 제47권3호
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• pp.465-474
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• 2005

A two-step broad-range PCR method detecting gram-negative bacteria at the level as low as 2 CFU was developed by using primers of GNFI and GNRI and then semi-nested primer of GNF2 and GNRI. The nucleotide sequences of the primers were determined based on l6S rRNA gene. The DNA fragments of 1173 bp and 169 bp were amplified in one-step PCRs with primer sets of GNFI-GNRI and GNF2-GNRl, respectively, using template DNA from seven strains of gram-negative bacteria including Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas spp., and Acinetobacter baumaii but not from Achromobacter lyticus, Alca/igens faecalis, and five strains of gram-positive bacteria. DNA fragments of 180 bp were amplified from LTLT-pasteurized milk and UHf-pasteurized milk in the two-step PCR. The DNA fragments were amplified from LTLT-pasteurized milk which was added with Pseudomonas j/uorescens and subsequently heated at 65 $^{\circ}C$, 80 $^{\circ}C$, and 100 $^{\circ}C$ for 30 min but they were not amplified from the milk autoclaved at 121$^{\circ}C$ for 15 min. It was suggested in PCR that Pseudomonas fluorescens heated at 65 $^{\circ}C$ for 30 min in milk was more sensitive to DNase treatment than viable bacteria.

• 대한의생명과학회지
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• 제25권4호
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• pp.331-338
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• 2019

Human Aichivirus A1 (HuAiV-A1) is a waterborne human pathogenic virus classified as Picornaviridae and Kobuvirus. In this study, we developed a method that can detect about 35 minutes faster with the same detection sensitivity level than the previously reported HuAiV-A1 diagnostic RT-PCR primer. The RT-PCR primer sets developed in this study are capable of detecting HuAiV-A1 at a level of about 100 ag and formed 563 bp amplification product. In addition, the RT-nested PCR method was able to amplify 410 bp using the RT-PCR product as a template. The detection sensitivity of our method was 10 times higher than the method with the highest detection sensitivity to date. Therefore, the detection method of HuAiV-A1 developed in this study is expected to be used in the water environment in which a small amount of virus exists. Also, this detection method is expected to be used as HuAiV-A1 diagnostic technology in both clinical and non-clinical field.

• Parasites, Hosts and Diseases
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• 제56권1호
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• pp.25-32
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• 2018

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris-EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was $40parasites/{\mu}l$ for P. falciparum and $35.2parasites/{\mu}l$ for P. vivax, whereas for Sn-PCR the limit of detection was $1.6parasites/{\mu}l$ for P. falciparum and $1.4parasites/{\mu}l$ for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

• 한국식품과학회지
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• 제49권2호
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• pp.123-131
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• 2017

노로바이러스는 국내에서도 지속적으로 유행성 바이러스성 위장염을 일으키고 있다. 노로바이러스 저감화사업단(NOROTECL)의 1중 2세부과제에서는 농산물의 노로바이러스 오염 원인을 추적조사하여 노로바이러스 전파를 예방하고 저감화하기 위한 과제를 수행하였다. 2015년 1월부터 11월 사이에 노로바이러스와 관련이 있을 것으로 추정되는 80개 농산물, 80개 토양, 78개 인체분변, 3개 가축분변, 80개 농업용수, 80개 하천수 시료를 대상으로 노로바이러스, Male specific coliphage (MSC), 위생지표세균(Coliform, E. coli) 세 가지 검사를 통해 오염현황을 조사하였다. Semi-nested PCR과 DNA sequencing을 통해 18개의 Genogroup I과 3개의 Genogroup II 노로바이러스가 총 18개의 시료에서 발견되었다. Genogroup이 확정된 노로바이러스에 대하여 RT-PCR을 진행하였다. 대장균군과 대장균은 농산물에서 68%와 1%, 토양에서 88%와 7.5%, 인체분변에서 44%와 12.8%, 가축 분변에서 67%와 67%, 농업용수에서 74%와 30%, 하천수에서 96%와 51%의 검출율을 각각 나타냈다. MSC 결과에서는 14개의 시료가 양성으로 나타났다.

• 한국동물위생학회지
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• 제46권3호
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• pp.235-241
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• 2023

Noroviruses are a major cause of gastroenteritis in humans and animals worldwide. In 2021, canine norovirus (CNoV) infection was detected at an animal clinic in Gwangju area, South Korea. A semi-nested polymerase chain reaction was developed to amplify a 478 bp fragment of the RdRp gene of CNoV. The phylogenetic analysis of this fragment confirmed the strain to be genogroup IV.2 (Dog/GIV.2/gw/s377/2021/KOR), which exhibited the highest similarity to the feline NoV strain GIV.2/CU081210E/USA/2010 (accession no. NC_045762) with 95.1% nucleotide (nt) identity and 98.7% amino acid (aa) identity. These research findings indicate that the detected norovirus in dogs is genetically similar to a feline-origin norovirus, suggesting easy cross-species transmission among animals.

• Food Science and Biotechnology
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• 제18권2호
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• pp.407-412
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• 2009

In this study, a rapid and efficient concentrating procedure that can be used for detecting viruses in vegetables was developed. The Sabin strain of poliovirus type 1 was used to evaluate the efficiency of virus recovery. The procedure included: (a) elution with 0.25 M threonine-0.3 M NaCl pH 9.5; (b) polyethylene glycol (PEG) 8000 precipitation; (c) chloroform extraction; (d) 2$^{nd}$ PEG precipitation; (f) RNA extraction; (g) reverse transcription-polymerase chain reaction (RT-PCR) combined with semi-nested PCR. The overall recoveries by elution/concentration were 29.0% from cabbage and 13.7% from lettuce. The whole procedure usually takes 18 hr. The overall detection sensitivity was 100 RT-PCR units of genogroup II norovirus (GII NoV)/25 g cabbage and 100 RT-PCR units of GII NoV/10 g lettuce. The virus detecting method developed in this study should facilitate the detection of low levels of NoV in cabbage and lettuce.

• Food Science and Biotechnology
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• 제18권5호
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• pp.1150-1154
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• 2009

Low levels of virus contamination and naturally occurring reverse transcription-polymerase chain reaction (RT-PCR) inhibitors restrain virus detection in oysters. A rapid and efficient oyster-processing procedure that can be used for sensitive virus detection in oysters was developed. Poliovirus type 1 Sabin strain was used to evaluate the efficacy of virus recovery. The procedure included (a) acid-adsorption and elution with buffers (0.25M glycine-0.14 M NaCl, pH 7.5; 0.25M threonine-0.14M NaCl, pH 7.5); (b) polyethylene glycol (PEG) precipitation; (c) resuspension in Tween 80/Tris solution and chloroform extraction; (d) the second PEG precipitation; (e) viral RNA extraction with TRIzol and isopropanol precipitation; and (f) RT-PCR combined with semi-nested PCR. The overall recovery of elution/concentration was 19.5% with poliovirus. The whole procedure usually takes 19 hr. The overall detection sensitivity was 4 RT-PCR units of genogroup I norovirus (NoV) and 6.4 RT-PCR units of genogroup II Nov/25 g of oysters initially seeded. The virus-detecting method developed in this study should facilitate the detection of low levels of NoV in oysters.