• The Plant Pathology Journal
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• 제39권4호
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• 대한수의학회지
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• 제42권3호
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• pp.351-362
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• 2002

Newcastle disease (ND) is a highly contagious infection of poultry, Two pathology-based techniques, in situ RT-PCR and in situ hybridization (ISH) were applied to formalin-fixed, paraffin-embedded tissues from chickens naturally infected with velogenic ND virus (VNDV). Two pairs of primers and a probe for ISH and in situ RT-PCR, respectively, were selected from highly conserved region of matrix gene of NDV. The ISH experiment was carried out using MicroProbe$^{TM}$ capillary action system within 2 hours. In situ RT-PCR was performed using MicroProbe$^{TM}$ capillary action system and GeneAmp In Situ PCR system. With ISH and in situ RT-PCR, viral nucleic acid was detected in the central nervous system of chickens from infected with neurotropic velogenic Newcastle disease virus (NVNDV), whereas viral nucleic acid was detected in various organs or tissues of chickens from infected with viscerotropic velogenic Newcastle disease virus (VVNDV). In the NVND group, positive signals were characteristically defined in the cytoplasm of neuron, vascular endothelial cells, and perivascular mononuclear macrophages in the central nervous system. One of NVND group, chicken from one farm exhibited positive signals in the bronchial epithelium. The VVND group chickens showed positive reaction in the macrophages, vascular endothelium, and bronchiolar epithelium. Markedly, viral nucleic acid was detected in the macrophages of morphologically normal tissues which were peripheral or located in distant areas from lesions. The central nervous system of chickens infected with VVND virus had positive signals in the vascular endothelial cell, perivascular mononuclear macrophages and some neuron. The number and intensity of the positive cells by in situ RT-PCR were more and stronger, respectively, in comparison with those by ISH. Particularly, positive reaction was detected in macrophages infiltrating in cardiac muscle by in situ RT-PCR, but not obtained by ISH. Therefore, these results demonstrated that ISH is a rapid diagnostic method for detection of NDV and in situ RT-PCR can be used as an efficient method for detection of low viral load infection or subclinical viral infection of NDV.

• 한국임상수의학회지
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• 제24권2호
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• pp.77-81
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• 2007

본 연구는 개에서 심장사상충을 검출하기 위하여 표준검사를 적용하지 않은 상황에서 중합연쇄반응검사 (PCR)의 진단 능력을 평가하였다. 효소면역검사법 (ELISA)과 PCR 검사를 동시에 사용한 경우 PCR 검사의 민감도와 특이도는 두 검사의 조건부 독립을 가정한 상태에서expectation-maximization (EM) 알고리즘을 이용한 최대우도법과 Bayesian 기법으로 두 집단 검사 모형으로 분석하였다 2002-2004년 기간 중 심장사상충검사 결과를 기록한 의무기록에서 무작위로 266개 결과를 추출하여 133개씩 2회의 시험으로 배치하였다. 2회의 분석결과를 종합할 때 EM 알고리즘에서 PCR 검사의 민감도와 특이도는 각각 96.4-96.7%와 97.6-98.8%, Bayesian기법에서는 94.4-94.8h와 97.1-98%로 추정되었다. PCR 검사는 심장사상충을 스크리닝하는 도구로 유용하며, 표준검사를 적용하지 않은 상황에서 진단검사의 특성을 추론하는 방법으로 Bayesian 기법은 매우 유용함을 확인하였다.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.1-8
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• 2016

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, $RIDA^{(R)}$ Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

• 한국임상수의학회지
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• 제25권4호
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• pp.241-244
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• 2008

본 연구는 특이적인 임상증상이 없는 360두의 개를 대상으로 multiplex nested PCR 기법을 이용하여 Brucella spp. 및 Leptospira interrogans를 검출하였다. Brucella spp.는 총4두 (1.1%, 암컷 2두, 수컷 2두)가 검출되었고, Leptospira interrogans는 59두 (16.4%, 암컷31두, 수컷28두)에서 검출되었다. 결론적으로 Brucella spp.의 검출률은 낮은 반면 Leptospira interrogans는 높았다. 또한 multiplex nested PCR기법은 무증상의 개 혈액에서 Brucella spp. 및 Leptospira interrogans를 조기에 검출하는데 빠르고 편리한 기법으로 판단된다.

• 한국식품과학회지
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• 제41권3호
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• pp.350-353
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• 2009

땅콩(Arachis hypogaea)은 예민한 사람들에게 심한 알레르기를 일으킬 수 있다. Agglutinin은 땅콩에서 알레르기 유발 단백질의 하나로 알려져 있다. 식품중의 땅콩성분을 검출하기 위하여 agglutinin 유전자에 특이적인 primer pair를 이용하는 polymerase chain reaction(PCR) 방법을 개발하였다. PCR 반응은 땅콩에서 agglutinin DNA의 특정부분을 증폭시켰으나 11종의 다른 견과류, 두류 및 곡류(피스타치오, 아몬드, 해바라기씨, 잣, 호두, 대두, 검은콩, 강낭콩, 팥, 백미, 흑미)에 대해서는 반응하지 않았다. 이 PCR 방법으로 땅콩성분이 함유된 6종의 가공식품을 모두 확인할 수 있었으며 땅콩이 구성성분으로 표시되지 않은 13종의 다른 가공식품에 대해서는 모두 음성반응을 나타냈다. 본 방법은 정제된 땅콩 DNA를 100 pg까지 검출할 수 있었으며 대두 DNA에 땅콩 DNA가 0.1%까지 혼합된 경우도 검출이 가능하였다.

• 생명과학회지
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• 제8권2호
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• pp.126-130
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• 1998

Vibrio unlnificus ATCC 27562의 16S rRNA 유전자를 PCR법과 제한효소절단법으로 분석 하여 얻은 결과는 다음과 같다. 1. 고안된 한쌍의 primer로 PCR을 시행하여 얻은 산물은 약 1.3Kb 였다. 2. PCR산물을 여섯가지의 제한 효소로 절단하여 얻은 단편들은 아래와 같다. BamH I: 어떤 restriction fragment도 만들지 않았다. Alu I : 약 400bp와 200bp의 두가지 fragment를 생산하였다. Sau3A I : 약 70bp에서 450bp 사이에 세가지 fragment를 생산하였다. Hind III : 약 800bp와 500bp의 약간 큰 두가지 fragment를 생산하였다. Sal I : 약 500bp와 750bp의 두가지 fragment를 생산하였다. Sma I : 약 800bp와 470bp의 두가지 fragment를 생산하였다.

• 한국양봉학회지
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• 제32권2호
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• pp.99-109
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• 2017

DWV, IAPV, KBV, SBV, BQCV, kSBV, SBPV and Paenibacillus larvae, Mellisococcus plutonius, Lysinibacillus fusiformis, Klebsiella oxytoca의 뒤영벌 병원체들에 대한 다중 실시간 중합효소 연쇄반응법(PCR)을 개발하였다. 하나의 시료에서 추출된 핵산은 11종 PCR들에 같은 시간 및 조건으로 사용될 수 있으며, 각 병원체 특이 표적 DNA가 PCR 기질로 1000분자가 존재한다면, 해당 특이 PCR 증폭산물들은 정성적, 정량적으로 20분안에 성공적으로 증폭되었다. 우리가 제안하는 이 다중 PCR 검출법이 뒤영벌의 국제교역을 위한 검역검사에 사용되기를 기대한다.

• 한국동물위생학회지
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• 제28권1호
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• pp.1-21
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• 2005

Pullorum disease and Fowl typhoid are kind of poultry specific disease for poultry. The peculiar character of these poultry specific diseases is that it can be infected by transmitting vertically and horizontally, also it is hard to be discovered by clinical sign, and pathology or immunology. So, to develop the PCR method which distinguishes these two genetically similar diseases of separated the specific DNA fragment from each strain and use it for differential diagnosis by subtraction PCR method. Standard strain of S gallinarum and S pullorum, and field isolation strain were verified by biochemistry, It confirmed existence of plasmid by using the PFGE. Then, Isolated DNA from it and used it as materials for the experiment. After cutting genomic DNA of two strains by using Sau 3Al, It ligated primer to tester DNA for PCR amplification and separated specific DNA fragment bacteria with method of subtraction PCR. And, It confirmed that it is a piece of unique DNA in every bacteria using base sequence of separated DNA fragment. 1. The six specific DNA fragment were separated from the DNA of S gallinarum and S pullorum by the subtraction PCR method. 2. In the result of comparison after setting base sequence of each fragment, each separated base sequence of DNA fragment they did not correspond to each other 3. As the result of each DNA fragment is derived from the each strain of DNA, and there was no homology of genomic DNA level in mutual. 4. The fragment originated in plasmid and includes S pullorum did not separate. 5. In the result of searching base sequence in Genebank, it partially shows homology in Salmonella enterica, S typhimurium, S dublin, Escherichia coli, Shigella flexneri, Yersinia pestis, Klebsiella pneumoniae. 6. Primer design by S gallinarum DNA 2, 3 fragment used PCR, They are positive reaction in only S gallinarum at 276, 367 bp position.

• Journal of Microbiology and Biotechnology
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• 제27권10호
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• pp.1778-1782
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• 2017

To identify and discriminate the bacterial species at the subspecific level, rep-PCR is a reliable genomic fingerprinting tool. Fourteen strains of bacteria were isolated from different food sources, identified as Leuconostoc citreum using 16S rRNA gene sequencing, and amplified using rep-primers (REP, ERIC, and $(GTG)_5$). Fingerprinting patterns generated bands in the range of 300-6,000 bp with REP, 150-6,000 bp with ERIC, and 200-1,700 bp with $(GTG)_5$ primers. In UPGMA dendrogram analysis, 14 strains were clustered into three clades (I, II, and III) with all the primers, thus differentiating them at the molecular level. The present study revealed the differentiation of L. citreum strains using rep-PCR.