• 식물병연구
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• 제27권1호
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• pp.38-44
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• 2021

Xylella fastidiosa is the most damaging pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, the loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assay were developed to mqsA gene of citrate-synthase (XF 1535) X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than current published tests for detection of X. fastidiosa when screened against 16 isolates representing the four major subgroups of the bacterium from a range of host species. No cross reaction with DNA from healthy hosts or other species of bacteria has been observed. The LAMP and PCR assays could detect 10-4 pmol and 100 copies of the gene, respectively. Hydroxynaphthol blue was evaluated as an endpoint detection method for LAMP. There was a significant color shift that signaled the existence of the bacterium when at least 100 copies of the target template were present.

• The Plant Pathology Journal
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• 제36권2호
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• pp.170-178
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• 2020

The Lily mottle virus (LMoV) impedes the growth and quality of lily crops in Lanzhou, China. Recently Arabis mosaic virus (ArMV) has been detected in LMoV-infected plants in this region, causing plant stunting as well as severe foliar symptoms, and likely posing a threat to lily production. Consequently, there is a need to develop simple, sensitive, and reliable detection methods for these two viruses to prevent them from spreading. Reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assays have been developed to detect LMoV and ArMV using two primer pairs that match six conserved sequences of LMoV and ArMV coat proteins, respectively. RT-LAMP assay results were visually assessed in reaction tubes using green fluorescence and gel electrophoresis. Our assays successfully detected both LMoV and ArMV in lily plants without the occurrence of viral cross-reactivity from other lily viruses. Optimal conditions for LAMP reactions were 65℃ and 60℃ for 60 min for LMoV and ArMV, respectively. Detection sensitivity for both RT-LAMP assays was a hundredfold greater than that of our comparative RT-polymerase chain reaction assays. We have also found this relatively rapid, target specific and sensitive method can also be used for samples collected in the field and may be especially useful in regions with limited or no laboratory facilities.

• Parasites, Hosts and Diseases
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• 제54권3호
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• pp.329-334
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• 2016

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.

• 한국축산식품학회지
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• 제34권6호
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• pp.799-807
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• 2014

Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be $85.56{\pm}0.07^{\circ}C$ for cattle, $84.96{\pm}0.08^{\circ}C$ for pig, and $85.99{\pm}0.05^{\circ}C$ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); $84.91{\pm}0.11^{\circ}C$ for goat and $83.90{\pm}0.11^{\circ}C$ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and $86.31{\pm}0.23^{\circ}C$ for chicken, $88.66{\pm}0.12^{\circ}C$ for duck, and $84.49{\pm}0.08^{\circ}C$ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from $10pg/{\mu}L$ to $100fg/{\mu}L$ levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.

• Pediatric Infection and Vaccine
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• 제22권2호
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• pp.106-112
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• 2015

목적: 장기간의 항생제 치료는 중이염 어린이 환자의 중이액으로부터 원인균이 배양되는 것을 방해한다. 본 연구는 배양 음성 중이액으로부터 분자적 진단에 의한 신속한 균 검출 가능성 여부를 확인하고자 하였다. 방법: 폐구균 lytA 유전자를 표적으로 하는 PCR과 LAMP로 민감도와 특이도를 비교 결정하고, 임상중이액에서의 폐구균 검출에 적용하였다. 결과: PCR 기법에 의한 폐구균 검출 최소한계는 약 $10^4$ 집락형성단위(CFU)이고, LAMP의 검출 최소한계는 10 CFU에서 결정되었다. 한편 두 가지 검사법 모두 Haemophilus influenzae 와 Moraxella catarrhalis 에 대해 $10^6$ CFU 이상에서도 DNA를 증폭하지 않았다. 22개의 배양음성 중이액 중에서 12개 검체가 LAMP-양성(54.5%, 12/22)으로 확인되었고, 이들 12개 LAMP-양성 검체 중, 3개의 검체만이 PCR-양성으로 확인되었다(25%, 3/12). 본 연구의 결과는 LAMP 기법의 폐구균 검출 해상력이 PCR 기법에 비교하여 4배 이상 높음을 보여준다(P<0.01). 결론: lytA -특이 LAMP 기법은, 중이액 내의 타 병원균과는 교차반응 없이 10 CFU 폐구균의 DNA를 검출할 수 있는 고해상 기술로서, 중이액 폐구균 검출 및 폐구균 백신의 보급에 따른 백신 효과 평가에 적용이 기대된다.

• Parasites, Hosts and Diseases
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• 제52권2호
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• pp.211-214
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• 2014

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia's 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia.

• 한국산업보건학회지
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• 제5권1호
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• pp.59-67
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• 1995

현재 국내에서 원자흡광법에 의한 혈중연 분석은 분석선 283.3 nm에서의 $D_2$ 보정 방식을 이용한 흑연로 원자흡광법이 주로 이용되고 있으며, 일반적인 시료중 납에 관한 분석은 217.0 nm에서의 $D_2$ 보정 방식이 보편화되어 있다. 그러나 이들 방식은 바탕 보정에 제한성 때문에 새로운 바탕 보정법에 대해 관심을 가지게 되었다. 그러던 중 1980년대 말부터 혈중연 분석자들은 연 분석에 있어 Zeeman effect 보정방식이 보다 좋은 결과를 나타낸다 하여 이 방법에 대하여 관심을 갖게 되었다. 따라서 본 연구는 국내 대부분의 혈중연 분석기관들이 보유하고 있는 $D_2$ 보정방식 (217.0과 283.3 nm)의 혈중연 측정결과를 편광 Zeeman effect 보정방식의 측정 결과와 비교함으로서 현재 사용 중인 기기 들의 측정결과의 타당성을 검토하기 위하여 시도하였으며 다음과 같은 결과를 얻었다. Zeeman형의 바탕보정 방식을 사용하는 기기의 결과를 편의상 1.00으로 하고, $D_2$ 형 보정 장치의 217.0 nm와 283.3 nm에서의 결과를 짝비교 (paired t-test)를 하였을 때 혈중연 농도가 $20.0{\mu}g/dl$ 이하인 경우에 0.92와 0.90으로 Zeeman형보다 낮은 값으로 분석되었으며 통계적으로 유의하였다.(P<0.001). $20.1-40.0{\mu}g/dl$인 군에서는 $D_2$ 보정방식의 결과간에 차이는 없었다. Zeeman 및 $D_2$ 보정방법에서는 혈중연 증가에 따른 바탕보정장치의 변동이 적었고 혈중연 이외의 다른 금속 즉, 철, 구리, 아연에서는 바탕보정장치에 관계없이 철은 역상관인 것으로 나타났으며, 구리와 아연은 정상관을 갖는 것으로 나타났다. 연구 결과로 미루어보아 두방법간의 차이가 없으므로 혈중연 분석에 있어서 Zeeman형 바탕보정 장치를 사용하거나 $D_2$ 바탕보정 방식(217.0, 283.3nm)의 기기를 사용하여도 무난할 것으로 생각된다.

• 대한화학회지
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• 제54권2호
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• pp.215-221
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• 2010

살모넬라는 음식과 식수에서 흔히 나오는 중요한 병원체로 세계 곳곳에서 급성 위장염과 같은 감염증을 일으키며, 일반적으로 인간의 혈청형 임상종으로는 Salmonella enterica의 혈청형인 S. Typhimurium과 S. Enteritidis가 있다. 일반적인 검출 방법으로 살모넬라를 기본으로 하여 선택적인 배양으로 샘플을 수집하였고 살모넬라를 일으키는 군체의 특징을 생화학과 혈청학상인 테스트를 하였으나 이러한 방법들은 일반적으로 시간이 걸리고 높은 감도를 보이지 않았다. 최근, 등온증폭반응법과 real-time PCR법을 이용하여 높은 감도, 특이성으로 현재 병원성 박테리아에 빠르게 수행할 수 있게 되었다. 본 연구에서는 등온증폭반응과 real-time PCR법을 사용하여 S. Typhimurium과 S. Enteritidis의 검출하였다. 선택적인 타겟 유전자로, invA를 살모넬라종의 염기서열에 특이적으로 임의복제 하였다. 등온증폭반응과 real-time PCR은 살모넬라종으로부터 임의의 염기서열을 증폭하여 검출하였고, invA는 S. Typhimurium과 S. Enteritidis의 두 가지 종을 모두 검출하였다. 이러한 등온증폭반응과 real-time PCR법으로 S.Typhimurium과 S. Enteritidis의 검출 가능성을 보였으며, 살모넬라 종에 대한 특이성, 민감성을 갖춘 유용한 검출방법을 제시하였다.

• 대한수의학회지
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• 제51권1호
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• pp.15-20
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• 2011

In this study, we isolated 12 of Brucella (B.) spp. from cattle, which have been positive in Rose Bangal test and tube agglutination test in Gyeongbuk province in 2009. According to AMOS PCR analysis, isolated 12 strains were identified as B. abortus. Murine derived macrophage, RAW 264.7 cells, were infected with isolated 12 strains or reference strain (B. abortus 544), and bacterial internalization were characterized. According to these results, we divided the isolated strains into the following three groups: class I, lower internalization than that of B. abortus 544; class II, similar internalization to that of that of B. abortus 544; class III, higher internalization than that of B. abortus 544 within RAW 264.7 cells. Furthermore, intracellular growth, bacterial adherent assay, LAMP-1 colocalization, virulence in mice and surface protein pattern were characterized. From these results, representative strains of class III showed lower LAMP-1 colocalization, higher adherent efficiency, higher virulence in mice than those of B. abortus 544, and showed different pattern of surface proteins. These results suggest that B. abortus field strains, isolated from cattle in Korea, possess various virulence properties and higher internalization ability of field strain may have an important role for its virulence expression.

• 한국의류학회지
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• 제21권4호
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• pp.750-756
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• 1997

The purpose of the study was to investigate a transmittance rate of UVB (Ultraviolet B) through summer fabrics and a protection rate of summer fabric from UVB. The subjects were randomly selected 159 fabrics from Korean common summer fabrics. The protection rates of 159 fabrics from UVB were measured by UVB lamp and UVB sensor, and 14 fabrics among these fabrics were selected for an assay of MTT(3-(4, 5-dimethylthiazol-2-yl) -2, 5 -diphenyltetrazolium). The protection rate of fabrics from cell toxicity of UVB was measured by investigating the difference of the amount of cell toxic substance on between fabrics covered with and without HeLa cell The average protection rate of 159 fabrics from UVB was 95.08%. As result findings, three negative correlations were found between: 1) the transmittance rate of UVB and the amount of MTT on fabrics (y=0.0373+0.O0518 x, r=-0.9323, p<0.001); 2) the air permeability of fabrics and the amount of MTT (r: -0.79, p< 0.01); 3) the air permeability of fabrics and the protection rate of fabrics from UVB (r=0.89, p<0.01). However, there was no effect of thickness of fabrics on the protection rate from UVB and the amount of MTT.