• Title/Summary/Keyword: First PCR

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DNA Microarray Probe Preparation by Gel Isolation Nested PCR

  • Wang, Hong-Min;Ma, Wen-li;Huang, Hai;Xiao, Wei-Wei;Wang, Yan;Zheng, Wen-Ling
    • BMB Reports
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    • v.37 no.3
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    • pp.356-361
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    • 2004
  • To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

Microarrays for the Detection of HBV and HDV

  • Sun, Zhaohui;Zheng, Wenling;Zhang, Bao;Shi, Rong;Ma, Wenli
    • BMB Reports
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    • v.37 no.5
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    • pp.546-551
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    • 2004
  • The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

Development of RT-PCR Kit for Diagnosis of Pathogenic Agent of Ginseng Root Rot in the Ginseng Field (인삼포장에서 뿌리섞음병원균의 진단을 위한 RT-PCR KIT의 개발)

  • 도은수
    • Korean Journal of Plant Resources
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    • v.16 no.1
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    • pp.40-48
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    • 2003
  • Cylindrocarpon destructans is the major pathogen inducing the root rot disease in ginseng. Up to now, there is no reliable and convenient method to analyze the spore density or population of this pathogen in ginseng-growing soil or any contaminated farmlands. Therefore, it will be very valuable to develop a new and reliable method in detecting the spore of this pathogen. In this study, a molecular biological technique using two step nested PCR method, was developed. Two universal ITS primers, ITS5F and ITS4R were used in the first round of PCR to amplify a fragment of ITS region from the genomic DNA of C. destructans. The specific prmers Nest 1 and Nest 2 were designed and used in the second round of PCR to amplify a inner fragment from the first round PCR product of C. destructans. C. destructans spore, only soil samples from the diseased ginseng farm produced the positive bands, suggesting its usefulness in detecting the C. destructans spores in soil samples. Thus it is recommended to first extract the whole genomic DNA from soil samples and use it for the PCR reaction, thereby eliminating the inhibitory activity of soil components.

A Method for Evaluation of the Quality of DNA Microarray Spots

  • Zhang, Bao;Ma, Wen-Li;Hu, Zi-You;Shi, Rong;Song, Yan-Bin;Zheng, Wen-Ling
    • BMB Reports
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    • v.35 no.5
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    • pp.532-535
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    • 2002
  • To establish a method to evaluate the quality of the printed microarray and DNA fragments' immobilization. The target gene fragments that were made with the restriction display PCR (RD-PCR) technique were printed on a superamine modified glass slide, then immobilized with UV cross-linking and heat. This chip was hybridized with universal primers that were labeled with cy3-dUTP, as well as cDNA that was labeled with cy3-dCTP, as the conventional protocol. Most of the target gene fragments on the chip showed positive signals, but the negative control showed no signal, and vice versa. We established a method that enables an effective evaluation of the quality of the microarrays.

Possibility of Using DNA Chip Technology for Diagnosis of Human Papillomavirus

  • Liu, Cui-Hua;Ma, Wen-Li;Shi, Rong;Ou, Yang-Qian;Zhang, Bao;Zheng, Wen-Ling
    • BMB Reports
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    • v.36 no.4
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    • pp.349-353
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    • 2003
  • To explore the application of DNA chip technology for the detection and typing of Human Papillomavirus (HPV), the HPV6, 11, 16 and 18 gene fragments were isolated and printed onto aminosilane-coated glass slides by a PixSys 5500 microarrayer as probes to prepare the HPV gene chips. HPV samples, after being labeled with fluorescent dye by restriction display PCR (RD-PCR) technology, were hybridized with the microarray, which was followed by scanning and analysis. The experimental condition for preparing the HPV gene chips was investigated, and the possibility of HPV genotyping using gene chips was discussed. The technique that was established in this study for preparing HPV gene chips is practical. The results of the present study demonstrated the versatility and inspiring prospect of using this technology to detect and genotype HPV.

Application of Multiplex Nested Methylated Specific PCR in Early Diagnosis of Epithelial Ovarian Cancer

  • Wang, Bi;Yu, Lei;Yang, Guo-Zhen;Luo, Xin;Huang, Lin
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.7
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    • pp.3003-3007
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    • 2015
  • Objective: To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR) in the early diagnosis of epithelial ovarian carcinoma (EOC). Materials and Methods: Serum and fresh tissue samples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylation levels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared with those for carcinoma antigen 125 (CA125). Results: The serum free deoxyribose nucleic acid (DNA) methylation spectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accurate reflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOC group were evidently higher than those in benign lesion and control groups (p<0.05). Patients with early EOC had markedly lower serum CA125 than those with advanced EOC (p<0.05), but there was no significant difference in free DNA methylation (p>0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplex nested methylated specific PCR were significantly higher for detection of all patients and those with early EOC than those for CA125 (p<0.05). In the detection of patients with advanced EOC, the PPV of CA125 detection was obviously lower than that of multiplex nested methylated specific PCR (p>0.05), but there was no significant difference in sensitivity (p>0.05). Conclusions: Serum free DNA methylation can be used as a biological marker for EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it can accurately determine tumor methylation conditions.

Evaluation of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR for the Diagnosis of Invasive Pulmonary Aspergillosis in a Rat Model

  • Lin, Jian-Cong;Xing, Yan-Li;Xu, Wen-Ming;Li, Ming;Bo, Pang;Niu, Yuan-Yuan;Zhang, Chang-Ran
    • Journal of Microbiology and Biotechnology
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    • v.24 no.8
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    • pp.1044-1050
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    • 2014
  • Since there is no consensus about the most reliable assays to detect invasive aspergillosis from samples obtained by minimally invasive or noninvasive methods, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis. Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, $200{\pm}20g$) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. The A. fumigatus DNA detection sequence was detected in the following number of samples from 12 immunosuppressed, infected rats examined on the scheduled days: day 1 (0/12), day 3 (0/12), day 5 (6/12), and day 7 (8/12) post-infection. The sensitivity and specificity of the qRT-PCR assay was 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct (cycle threshold) cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal Ct cut-off value was 1.40 (AUC, 0.919). The GM assay was more sensitive than the qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.

High-accuracy quantitative principle of a new compact digital PCR equipment: Lab On An Array

  • Lee, Haeun;Lee, Cherl-Joon;Kim, Dong Hee;Cho, Chun-Sung;Shin, Wonseok;Han, Kyudong
    • Genomics & Informatics
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    • v.19 no.3
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    • pp.34.1-34.6
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    • 2021
  • Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

Studies on the mycoplasmal pneumonia in slaughter pigs. 2. Isolation of mycoplasmas from lung tissues and identification of isolates by nested-PCR technique (도축돈의 마이코플라즈마성 폐렴에 관한 연구 2. 폐조직에서의 균분리와 nested-PCR방법에 의한 동정)

  • Lim, Young-Taek;Seok, Ho-Bong
    • Korean Journal of Veterinary Research
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    • v.42 no.2
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    • pp.225-229
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    • 2002
  • We report that mycoplasma organisms from lung tissues of slaughter pigs were identified to genes fragments with references use of nested-PCR technique(nPCR). Seven strains of mycoplasma species were isolated from 70 lung tissues. The organisms were detected by in vitro amplification of 16S rRNA and 23S rRNA genes. Nucleotide sequences of the spacer between 16S and 23S in the ribosomal RNA operons of mycoplasma were identified by the analysis of products from the nested PCR. Four common PCR primers, MhF1, MhF2 MhR1 and MhR2, were designed by analysis between these sequences by first amplified with F1, R1 and second with F2, R2, respectively. Specific amplification of the spacer region for reference strains of M. hyopneumoniae, M. hyorhinis, M. flocculare were confirmed by first round of PCR in which the traduced fragments of 690bp, 460bp, 630bp. But amplications of second round was changed to 240bp, 210bp, 230bp, respectively. Three different strains (M. hyopneumoniae:4, M. hyorhinis:2, M. flocculare:1) were detected by the nested-PCR technique. The results suggest that the detection of swine mycoplasma by n-PCR can be analyzed the nucleotide sequences between rRNA operons and homology study.

A Study on Simultanious Detection of Bovine Rotavirus, Coronavirus and Virai Diarrhea virus by Multiplex RT-PCR (Multiplex RT-PCR 기법을 이용한 소의 로타바이러스, 코로나바이러스 및 설사병바이러스의 동시진단)

  • Nho, W.G.;Lee, J.H.
    • Journal of Practical Agriculture & Fisheries Research
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    • v.5 no.1
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    • pp.57-63
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    • 2003
  • The bovine rotavirus(BRV), bovine coronavirus(BCV) and bovine viral diarrhea virus(BVDV) are main viruses of bovine viral diarrhea disease. These viruses could be rapidly amplified by the reverse transcriptase polymerase chain reaction(RT-PCR). This study was conducted to develop rapid and accurate diagnostic methods of these viral diseases by multiplex RT-PCR. Specific primers were designed based on the sequences reported by Chang KO et. al. (1997) and Schroeder BA, et. al. (1990), RNA were prepared from the cultured viruses, first-stranded DNAs were synthesised by reverse transcriptase. PCR were conducted to amplify specific regions of the viruses by multiplex. Three bands such as 1,062bp for BRV, 458bp for BCV, and 300bp for BVDV were successfully produced by multiplex RT-PCR. In conclusion, this result suggested that these viruses could be diagnosed rapidly and accurately by multiplex RT-PCR.