• Title, Summary, Keyword: nested-PCR

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Rapid Detection of Mycobacterium tuberculosis Complex in Tissues by Using the Nested PCR (Nested PCR을 이용하여 조직으로부터 Mycobacterium tuberculosis Complex 신속검출)

  • Park, Jung-Yeon;Yang, Byoung-Seon
    • Korean Journal of Clinical Laboratory Science
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    • v.47 no.4
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    • pp.313-317
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    • 2015
  • Due to the increase in incidence of infection of Mycobacterium tuberculosis complex (MTC), it is imperative that a rapid diagnosis accompanies the handling of MTC. This is due to the three to eight weeks it takes to culture Mycobacteria, and the lack of sensitivity of microscopic examination of AFB. Recently, nested PCR has been used to detect and diagnose mycobacteria. It is especially useful in complementing diagnosis by histological extra pulmonary. After culturing all the specimens and practicing the nested PCR, we did comparison analysis between nested PCR and culture. There were 76 specimens, 31 of which were positive. Of the 31 positive specimens in culturing, only 22 were positive in nested PCR. Of the 45 negative specimens, 36 were negative in nested PCR. As a result, Sensitivity was 71% and specificity was 80%. Furthermore, the positive predictive value was 71% and negative predictive value was 80%. These results indicate that nested PCR based techniques are sensitive, specific, and rapid methods for the detection of MTC.

Development of Nested-PCR Assay to Detect Acidovorax citrulli, a Causal Agent of Bacterial Fruit Blotch at Cucurbitaceae (박과 작물에 과일썩음병을 일으키는 Acidovorax citrulli 검출을 위한 nested-PCR 검사법 개발)

  • Kim, Young-Tak;Park, Kyoung-Soo;Kim, Hye-Seong;Lee, Hyok-In;Cha, Jae-Soon
    • Research in Plant Disease
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    • v.21 no.2
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    • pp.74-81
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    • 2015
  • The specific and sensitive nested-PCR method to detect Acidovorax citrulli, a causal agent of bacterial fruit blotch on cucurbitaceae, was developed. PCR primers were designed from the draft genome sequence which was obtained with the Next Generation Sequencing of A. citrulli KACC10651, and the nested-PCR primer set (Ac-ORF 21F/Ac-ORF 21R) were selected by checking of specificity to A. citrulli with PCR assays. The selected nested-PCR primer amplified the 140 bp DNA only from A. citrulli strains, and detection sensitivity of the nested PCR increased 10,000 times of $1^{st}$ PCR detection limit (10 ng genomic DNA/PCR). The nested PCR detected A. citrulli from the all samples of seed surface wash (external seed detection) of the artificially inoculated watermelon seeds with $10^1cfu/ml$ and above population of A. citrulli while the nested PCR could not detected A. citrulli from the mashed seed suspension (internal seed detection) of the all artificially inoculated watermelon seeds. When the naturally infested watermelon seeds (10% seed infested rate with grow-out test) used, the nested PCR detected A. citrulli from 2 seed samples out of 10 replication samples externally and 5 seed samples out of 10 replication samples internally. We believe that the nested-PCR developed in this study will be useful method to detect A. citrulli from the Cucurbitaceae seeds.

Identification of Cryptosporidium in Environmental Sample using Nested PCR-RFLP and DNA Sequencing (Nested PCR-RFLP 및 DNA Sequencing을 이용한 환경시료에서의 크립토스포리디움 동정)

  • Park, Sangjung;Jeong, Hyanghee
    • Journal of Korean Society on Water Environment
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    • v.24 no.6
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    • pp.817-822
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    • 2008
  • In order to identify various Cryptosporidium species in environment, nested PCR-RFLP and DNA sequencing method were used. The sensitivity of nested PCR-RFLP based on 18s rRNA gene was shown to 1 oocyst. Therefore, we applied nested PCR-RFLP method to environmental samples. As a result, only 4 samples out of 8 samples confirmed as Cryptosporidium parvum by standard method of Cryptosporidium were identified as Cryptosporidium parvum by nested PCR-RFLP and DNA sequencing method. The rest of 4 samples among 8 samples were identified as Cryptosporidium muris, Cryptosporidium bailey. Therefore, in addition to standard method of Cryptosporidium, supplementary verification through nested PCR-RFLP and DNA sequencing should be needed to give more accurate information about risk of Cryptosporidium.

Detection of Ralstonia solanacearum with Nested PCR and DNA Enzyme-Linked Immunosorbent Assay (Nested PCR과 DNA Enzyme-Linked Immunosorbent Assays를 이용한 Ralstonia solanacearum의 검출)

  • Ko, Young-Jin;Cho, Hong-Bum
    • Korean Journal of Microbiology
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    • v.43 no.3
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    • pp.179-185
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    • 2007
  • In this study, we used the method of guanidin isothiocyanate and boiling with Chelex-100 resin to extract genomic DNA of Ralstonia solanacearum from soil. It is more efficient than general protocols to remove inhibitory compounds in soil and R. solanacearum on. Then, we applied polymerase chain reaction and DNA enzyme-linked immunosorbent assay (ELISA) to identify and detect pathogen. The fliC gene of R. solanacearum was selected for specific detection of pathogen and primer sets were designed. Among the primer sets, two specific and sensitive primer sets, RsolfliC(forward: 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. $Sch\ddot{o}nfeld$ et al.) and RS_247 (forward: 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3 designed by this study), were designed to perform nested PCR. Nested PCR primer was labeled with biotin for hybridization between nested PCR product and probe to analyze with DNA ELISA.

Detection of Marine Birnavirus (MBV) from Rockfish Sebastes schlegeli Using Reverse Transcription and Nested PCR

  • Joh, Seong-Joon;Kim, Doo-Won;Kim, Jeong-Ho;Heo, Gang-Joon
    • Journal of Microbiology
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    • v.38 no.4
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    • pp.260-264
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    • 2000
  • Reverse transcription (RT)-PCR and nested PCR methods (2-step PCR) were tested for their ability to detect marine birnavirus (MBV) in cultured rockfish, Sebastes schlegeli. One set of primers for RT-PCR was designed, based on a gene of infectious pancreatic necrosis virus (IPNV), and another set of primers for nested PCR was designed based on the VP2/NS junction region of MBV. This 2-step PCR method was specific for MBV and sensitivity was heightened when nested PCR was combined to RT-PCR. This 2-step PCR method was useful for detecting MBV not only in diseased fish, but also in asymptomatic fish. These results indicate that this 2-step PCR method is useful for detecting MBV in rockfish.

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Detection of Mycobacterium leprae by Nested PCR Targeting M. leprae-Specific Repetitive Element (RLEP) Sequence

  • Wang, Hye-Young;Kim, Yeun;Bang, Hye-Eun;Kim, Hyun-Chul;Cho, Sang-Nae;Lee, Hye-Young
    • Biomedical Science Letters
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    • v.13 no.1
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    • pp.33-38
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    • 2007
  • The aim of this work was to validate a rapid and an accurate method for detecting Mycobacterium leprae in clinical specimens using nested PCR targeting M. leprae-specific repetitive element (RLEP) sequence. The primers were derived from the RLEP sequence which yield a 272 bp outer product and a 230 bp inner product. The specificity and the sensitivity of the nested PCR were compared with those of single PCR for detecting M. leprae using DNAs isolated from reference strain and various species of Mycobacterium. The results showed that the sensitivity of the nested PCR was about 100 to 1,000 times higher than that of the single PCR and also showed that both the single and the nested PCR were highly specific to M. leprae. Subsequently, the usefulness of the single and nested PCR was evaluated with clinical samples isolated from leprosy patients. The number of positive detections by the single and the nested PCR with a total of 20 specimens from leprosy patients were 9 (45%) and 20 (100%), respectively. The results clearly showed that nested PCR has highest sensitivity in detecting M. leprae from clinical specimens. Therefore, nested primers targeting RLEP sequence developed in this study seems to be useful to detect the presence of M. leprae.

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Detection of Pseudomonas tolaasii causing brown blotch disease in water from oyster mushroom cultivation farms by PCR (PCR을 이용한 느타리버섯 재배사 물로부터 세균성갈색무늬병 병원균 Pseudomonas tolaasii 검출)

  • Jeong, Kyu-Sik;Kim, Woo-Jae;Chang, Who-Bong;Cha, Jae-Soon
    • Journal of Mushroom
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    • v.1 no.1
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    • pp.28-33
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    • 2003
  • Pseudomonas tolaasii causing brown blotch disease was detected by PCR from water samples collected from the oyster mushroom cultivation farms to find the contamination level of the pathogen in water. Sixteen water samples (28.1%) contain less than 1,000 cfu, 31 samples (54.4%) contain 1,001-10,000 cfu, 6 samples (10.5%) contain 10,001-100,000 cfu, and 4 samples (7%) contain of bacteria per milliliter. P. tolaasii-specific DNA band was amplified in 3 samples (5.3%) by nested-PCR and in 20 samples (35.1%) by immunocapture (IC)-nested PCR respectively. These results suggest that IC-nested-PCR was much more sensitive than nested-PCR in detection of P. tolaasii and a quite few waters using for oyster mushroom cultivation were contaminated with P. tolaasii.

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Detection of Plasmodium vivax by Nested PCR and Real-Time PCR

  • Genc, Ahmet;Eroglu, Fadime;Koltas, Ismail Soner
    • The Korean Journal of Parasitology
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    • v.48 no.2
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    • pp.99-103
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    • 2010
  • Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.

Specific Detection of Root Rot Pathogen, Cylindrocarpon destructans, Using Nested PCR from Ginseng Seedlings (Nested PCR 기법을 이용한 인삼 뿌리썩음병원균의 특이적 검출)

  • Jang, Chang-Soon;Lee, Jung-Ju;Kim, Sun-Ick;Song, Jeong-Young;Yoo, Sung-Joon;Kim, Hong-Gi
    • Research in Plant Disease
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    • v.11 no.1
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    • pp.48-55
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    • 2005
  • Cylindrocarpon destructans is a soil-borne plant pathogenic fungus causing root rot on ginseng and trees. Rapid and exact detection of this pathogen was practiced on ginseng seedlings by nested PCR using speciesspecific primer set. The second round of PCR amplification by Dest 1 and Dest 4 primer set formed 400 bp of species-specific fragment of C. destructans from the product of first round of amplification by ITS 1 and ITS 4 primer set. In the PCR sensitivity test based on DNA density, nested PCR detected to the limit of one fg and it meant the nested PCR could detect up to a few spores of C. destructans. Also, nested PCR made it possible to detect the pathogen from ginseng seedlings infected by replantation on artificial infested soil. Our nested PCR results using species-specific primer set could be utilized for diagnosis of root rot disease in ginseng cultivation.

국내 돼지 설사 유발 칼리시 바이러스 감염증의 발생현황

  • 김현진;조경오;조호성;강성귀;박남용
    • Proceedings of the Korean Society of Veterinary Pathology Conference
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    • pp.139-139
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    • 2002
  • 돼지 설사유발 칼리시 바이러스(Porcine enteric calicivirus: PECV)도 자돈에서 설사를 일으키는 바이러스로 이미 알려졌다. RT-PCR과 nested PCR을 이용하여 국내 양돈장에서 PECV의 발생을 조사하고자 본 연구를 시도하였다. 설사 분변은 경기, 충남, 전북, 전남과 제주지역에 분포한 31개의 농장 102마리의 자돈에서 채취하여 의뢰된 것을 조사하였다. RT-PCR 과 nested PCR 을 위하여 RNA dependent RNA Polymerase (RDRP) 부위와 capsid 부위에서 각각 2 쌍의 primer를 작성하였다. RDRP 부위에서 RT-PCR을 시행했던 바, 3마리 (2.9%) 에서, nested PCR에서는 18마리 (17.6%)에서 양성반응이 나왔으며 capsid 부위에서 RT-PCR 결과 5마리 (4.9%), nested PCR에서는 18마리(17.6%)가 양성반응으로 확인되었다. 본 연구를 통하여 PECV가 국내에서 돼지 설사를 일으키는 주요 원인체 중 하나라는 것이 밝혀졌으며, nested PCR 기법이 돼지 설사분변에서 PECV를 검출하는 좋은 진단방법이었다.

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