• Parasites, Hosts and Diseases
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• 제47권3호
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• pp.227-233
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• 2009

Local malaria transmission in the United Arab Emirates (UAE) came to an end in 1997. Nevertheless, UAE has been subjected to substantial importation of malaria cases from abroad, concerning both UAE nationals and immigrants from malarious countries with a total number of 2,119 cases in 2007. To evaluate a new DNA extraction technique using nested PCR, blood samples were collected from 132 individuals who presented to Infectious Diseases Department in Rashid Hospital, Dubai, and Central Department of Malaria Control with fever and persistent headache. Giemsa-stained blood films and ELISA test for malaria antibodies were carried out for detection of Plasmodium infection. Plasmodium infections were identified with the genus-specific primer set and species differentiation using nested PCR. A rapid procedure for diagnosis of malaria infections directly from dried blood spots using for the first time DNA extract from FTA Elute cards was evaluated in contrast to extraction techniques using FTA classic cards and rapid boiling technique. Our new simple technique for DNA extraction using FTA Elute cards was very sensitive giving a sensitivity of 100% compared to 94% using FTA classic cards and 62% in the rapid boiling technique. No complex preparation of blood samples was required prior to the amplification. The production cost of DNA isolation in our PCR assay was much less incomparable to that of other DNA extraction protocols. The nested PCR detected plasmodial infection and could differentiate P. falciparum from P. vivax, and also detected the mixed infection.

• 한국미생물·생명공학회지
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• 제51권1호
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• pp.99-108
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• 2023

대기 입자상물질(particulate matter, PM) 시료의 곰팡이 메타게놈 분석을 위해 DNA 추출 및 유전자 증폭 시 여러 문제가 발생한다. 본 연구에서는 PM 시료로부터 DNA를 추출하는 방법과 polymerase chain reaction (PCR)을 위한 프라이머 및 온도 조건의 최적화를 위하여 다양한 조건으로 실험하였다. 여러 조건에서 DNA 추출 여부를 비교 평가한 결과, bufffer와 proteinase K를 이용하여 20분 동안 화학적 세포 용해 처리와 bead beating 처리를 한 후 상용 DNA 추출 kit를 사용하면 DNA를 효율적으로 추출할 수 있었다. PCR 조건을 최적화하기 위해 ITS2 유전자 영역을 증폭할 수 있는 10개 조합의 프라이머를 이용하여 PCR을 수행한 결과, ITS3tagmix3/ITS4 조합의 프라이머로 annealing 온도 58℃로 하였을 때 증폭된 PCR 산물의 농도가 상대적으로 높았다. 이 조건에서도 PCR 산물의 농도가 낮은 경우에는 1차 PCR 산물을 주형 DNA로 사용하여 nested PCR을 수행하면 만족스러운 농도로 ITS2 유전자를 증폭할 수 있었다. 본 연구에서 도출한 조건으로 서울 대기 PM2.5를 포집한 필터 시료 15종을 대상으로 DNA 추출과 PCR을 수행한 결과 성공적으로 ITS2 유전자 증폭이 가능하였다. 본 연구에서 최적화한 방법은 대기 PM 시료의 곰팡이 메타게놈을 분석하고 해석하는 연구에 활용 가능하다.

• Mycobiology
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• 제38권1호
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• pp.33-38
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• 2010

We have successfully applied the nested PCR to detect Cylindrocarpon destructans, a major pathogen causing root rot disease from ginseng seedlings in our former study. The PCR assay, in this study, was used to detect the pathogen from soils. The nested PCR using internal transcribed spacer (ITS) 1, 4 primer set and Dest 1, 4 primer set maintained the specificity in soils containing various microorganisms. For a soil DNA extraction method targeting chlamydospores, when several cell wall disrupting methods were tested, the combination of lyophilization and grinding with glass beads, which broke almost all the chlamydospores, was the strongest. The DNA extraction method which was completed based on the above was simple and time-saving because of exclusion of unnecessary stages, and efficient to apply in soils. As three ginseng fields whose histories were known were analyzed, the PCR assay resulted as our expectation derived from the field information. The direct PCR method will be utilized as a reliable and rapid tool for detecting and monitoring C. destructans in ginseng fields.

• 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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• 제31권6호
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• pp.1628-1634
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• 1999

Listeria monocytogenes의 식품 속에서 신속하고 특이적인 검출을 위하여, listeriolysin O gene에 의한 primer, LM 1과 LM 2를 선택하여 PCR을 수행하였다. L. monocytogenes의 DNA 추출이나 cell lysis 없이 intact whole cell을 직접 이용하여 PCR을 하였으며 $10^{2-6}$ CFU 수준의 균체 배양액으로부터 L. monocytogenes에 특이적인 702 bp의 PCR 증폭 산물을 확인하였다. 우유, 닭고기, 김치 등의 식품에 L. monocytogenes를 접종하여 증균배양 전후 균의 PCR에 대한 감도와 생균수를 비교한 결과, 실험된 식품 속에서의 L. monocytogenes의 검출 감도는 순수 배양액에서의 경우에 비해 약 1/10로 둔화되었으나 역시 특이적인 검출이 가능하였다. Primer LM 1과 2를 이용한 본 실험조건에서의 L. monocytogenes의 PCR에 의한 검출은 약 4시간으로 확인이 가능하였으며, 기존의 배양 방법에 비해, 특이성이나 검출 속도 면에서 식품위생 실무에 적용하기 위한 높은 잠재력을 보여 주었다.

• Tuberculosis and Respiratory Diseases
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• 제43권1호
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• pp.30-37
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• 1996

연구배경: 본 연구는 결핵의 조기검출을 위하여, PCR을 이용하는데 있어서의 중요한 문제점의 하나인 임상검체로부터 결핵균을 분리 방법을 개선하여, 교차오염의 가능성을 최소화하고 PCR에 의한 결핵균의 DNA검출을 보다 쉽고 안전하게 실시하여 대량의 검체를 처리해야하는 일상 임상검사법으로 정착시키고자 하는데 그 목적이 있다. 방법: 다양한 방법으로 DNA를 검출하여 동일한 방법으로 PCR을 수행하여 이차 PCR 산물의 전기영동 결과를 AFB도말 및 배양검사 결과와 함께 비교하여 감수성, 특이성, 양성예측도 및 음성예측도의 항목으로 비교분석하였다. 실험 1은 Proteinase K 처리후 phenol로 추출하는 방법과 Chelex 100 이온교환 수지를 이용한 InstaGene법을 100예에서 비교하였으며, 실험 2에서는 Microwave 처리후 원침 상청액을 직접 시용하는 방법과 Chelex 100 이온교환 수지를 이용한 InstaGene 법을 98예에서 비교하였다. 결과: 세 DNA 분리법으로 실시한 PCR결과에서 모두 특이성과 양성예측도가 매우 낮았다. 실험 1에서 Proteinase K 법에 의한 경우 보다 Insta Gene을 이용한 경우에 약 20% 높은 감수성과 10%~20% 높은 음성예측도를 보이고 있으며, 특이성은 2%~8% 낮고, 양성예측도는 2.5%~4% 높은 것으로 나타났다. 실험 2에서는 Microwave법과 Insta Gene을 이용한 경우에 거의 비슷한 결과를 보였다. 결론: 결핵진단시 PCR을 위한 객담검체에서의 DNA 분리시에 Microwave법과 $InstaGene^{TM}$ DNA분리 kit가 매우 효율적이며, 특히 InstaGene법이 교차오염의 가능성이 거의 없고, 처리 시간이 짧으며, 조작이 매우 간단하며 매우 경제적인 것으로 나타났다. 다만 특이성을 더욱 높일 수 있도록 연구가 추가되어야 할 것이며 일반 인구집단에서 충분한 임상검체를 대상으로 연구가 추가되면 InstaGene법의 유용성이 더욱 확실히 검증 될 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제54권3호
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• pp.253-259
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• 2016

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, $20{\mu}l$ in 1 sample, was optimal, and the parasite density as low as $2p/{\mu}l$ for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.

• Journal of Preventive Medicine and Public Health
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• 제45권4호
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• pp.235-243
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• 2012

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6929-6933
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• 2013

Background: Multiple etiologies have been hypothesized for prostate cancer, including genetic defects and infectious agents. A recently reported gamaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) has been reported to be detected in prostate cancer. However, this virus has not been detected in similar groups of patients in other studies. Herein, we sought to detect XMRV in prostate cancers and benign controls in Sanandaj, west of Iran. Materials and Methods: In a case-control study, genomic DNA was extracted from formalin fixed and paraffin embedded prostate tissues from a total of 163 Iranian patients. We developed a conventional and a nested PCR assay using primers targeting to an env specific sequence of XMRV. PCR assays were carried out on 63 prostate cancers and 100 benign prostate hyperplasias. Results: Beta-actin sequences were successfully detected in the DNA extracts from all prostate tissues, confirming DNA extraction integrity. We did not detect XMRV in samples either from prostate cancers or benign prostate hyperplasias using XMRV specific primers. Conclusions: We conclude that in our population XMRV does not play a role in genesis of prostate cancer.