Clinical application of a novel DNA fluorescence intercalator, pyrylium dye, for the quantification of PCR products
Yasushi IchikawaT. IshikawaNobuyoshi MomiyamaTakashi ChishimaSeiko HasegawaHiroyuki YamaokaNaoki YamamotoT. OKAMOTOMakiko KawaguchiHiroshi Shimada
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Polymerase chain reaction (PCR) is a powerful tool for clinical diagnosis in the field of oncology, infection, and allergy. However, a simple and sensitive method for quantifying PCR products has not been established. We therefore used a novel fluorescence DNA intercalator, pyrylium iodide (P2) to quantify PCR products. A 838-bp fragment of the human beta-actin gene and a 374-bp fragment of the gene for ornithine decarboxylase (ODC) and ODC primer dimers were generated by PCR and quantified using either P2 or another fluorescence DNA intercalator, YOYO-1. In addition, utilizing RT-PCR and the P2 method, the ODC mRNA expression from 10 colonic cancers was quantified. Serially diluted beta-actin and ODC PCR products could be quantified using P2 without having to separate them from primer dimers and other components of the reaction mixture. However, YOYO-1 could not be used to quantify the PCR products because of high background fluorescence from the primer dimers. In the clinical study, ODC mRNA expression as quantified by P2 was significantly higher in cancerous tissue (113.8 +/- 4.1; mean +/- SEM; plate reader units) than in mucosa of normal appearance (68.4 +/- 4.8). P2 is a promising tool for quantifying PCR products.Keywords:
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More than five different primer pairs have been used for the detection of Mycobacterium tuberculosis deoxyribonucleic acid (DNA) with the polymerase chain reaction (PCR).The sensitivity and specificity of PCR were evaluated using three different primer pairs in the detection of M. tuberculosis in paraffin-embedded tissues.Thirty-eight tissue specimens from 23 patients were studied. Eighteen samples were obtained from 10 tuberculosis patients, and 20 samples obtained from 13 patients with other diseases were used as negative controls. DNA extracted from paraffin-embedded tissues was used directly for PCR amplification using primers IS1 and IS2 to amplify a 123 base pair (bp) region of IS6110, sjMT3 and sjMTr2 to amplify a 281 bp region of protein antigen b, and INS1 and INS2 to amplify a 245 bp region of IS986. Each amplification was performed double-blinded and repeated three times including positive and negative control samples.IS1 and IS2 gave a positive result in each of the double samples obtained from eight tuberculosis patients and in the single samples obtained in the two others, sjMT3 and sjMTr2 detected 13 of the 18 tuberculosis samples, and INS1 and INS2 detected only three of the 18.These results highlight the importance of selecting appropriate primers to obtain high sensitivity in detecting M. tuberculosis in paraffin-embedded tissues by PCR.
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Two primer sets were chosen for the detection of Haemophilus influenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H. influenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H. influenzae tested. This primer set also reacted with the closely related species H. haemolyticus and H. aegyptius, and with two of nine H. parainfluenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H. influenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR; 40 were culture-positive for H. influenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H. influenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H. influenzae in CSF.
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INTRODUCTION The objective of a polymerase chain reaction (PCR) is to amplify a specific DNA segment without any nonspecific by-products. In principle, each physical and chemical component of PCR can be modified to produce a potential increase in yield, specificity, or sensitivity. Yet the most critical parameter for successful PCR is optimal primer design. A poorly designed primer can result in little or no product, due to nonspecific amplification and/or primer-dimer formation leading to reaction failure, even when all the other parameters are properly optimized. This article provides general guidelines for PCR primer design, tips for development of primer pairs for more complex applications, and advice on the development of probes for real-time PCR.
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본 실험에서는 식품의약품안전평가원의 식품 중 사용원료 진위 판별 지침서에 명시 되어있는 소와 돼지에 대한 primer를 이용한 PCR 반응의 민감도와, 새롭게 선별한 primer의 PCR 민감도를 비교하였다. 새로운 primer의 타겟 부위는 cytochrome b이다. Primer의 적합성을 확인하기 위해 돼지고기와 소고기를 이용하여 DNA를 추출 후 PCR을 통하여 확인 하였다. 육포시료에서 식품의약품안전평가원의 식품 중 사용원료 진위 판별 지침서에 명시 되어있는 소에 대한 primer를 사용하였을 경우, PCR 산물을 얻을 수 있었음에 반해, 돼지 primer를 이용하였을 때는 PCR 산물을 얻을 수 없었다. 반면에 새로설정 한 primer에서는 전체 27개의 육포시료 중 13개에서 돼지 primer를 이용한 PCR 산물을 얻을 수 있었다. 이렇게 얻은 PCR 산물을 시퀀싱을 통하여 확인 해본 결과, 돼지 (Sus scrofa)임을 확인 할 수 있었다. 그러므로 본 실험에 사용 한 육포시료 중 일부는 돼지와 소고기의 혼합물임을 확인 할 수 있었다.We compared the sensitivity of polymerase chain reaction (PCR) using newly discovered bovine and porcine primers to previously reported primers of the Korea Food and Drug Administration (KFDA). The target site for the new primers was cytochrome b. The suitability of the primers was confirmed by conducting PCR on DNA extracted from beef and pork control. When the beef jerky samples were tested by PCR with the conventional primers of the KFDA, the DNA was successfully amplified from all samples with the bovine primer, whereas no PCR products were obtained with the porcine primer. By contrast, in the experiments with the new primers, we obtained PCR products from 13 of 27 beef jerky samples using the porcine primer. We have confirmed that the base sequences of these 13 samples match to the sequence of pigs (Sus scrofa) in the National Center for Biological Information (NCBI) database. Therefore, we determined that some of the beef jerky samples we received are mixture of beef and pork.
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Nested polymerase chain reaction (nested PCR) was used to separately amplify part of gag, pol, and env genes of human immunodeficiency virus type 1 (HIV-1) to evaluate that primer specific to either gag (SK380/390&SK38/39), pol (JA17/18&JA19/20), or env (JA9/10&JA11/12) genes is suitable for HIV-1 PCR based diagnosis in Thailand. The positive PCR results in 70 HIV-1 infected adults are 100, 97, 89 per cent and in 75 HIV-1 infected infants are 100, 94, 74 per cent by gag, pol, env primer, respectively. The specificity of all three primer sets is 100 per cent. The unamplified samples by pol and env primers were identified as HIV-1 subtype E by PELISA method. False negative in HIV-1 PCR based diagnosis caused by high genetic variation of HIV-1 can be overcome by using several primer sets as shown in this study.
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Editor,—Eales’ disease, first described by Henry Eales in 1880 is a primary retinal perivasculitis that predominantly affects the peripheral retina of young and otherwise healthy adults in the age group 15–40 years. Of the several aetiologies proposed, most favoured are tuberculosis and hypersensitivity to tuberculoprotein.1 Since polymerase chain reaction (PCR) using primers for the insertion sequence of IS6110 consisting of upstream primer: 5′ CCTGCGAGCGTAGGCGT CGG3′ and downstream primer: 5′CTC GTCCAGCGCCGCTTCGG 3′ …
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To investigate the generation of an abnormally long HIV-1 env PCR DNA product the latter was cloned and sequenced followed by sequence analysis of HIV-1 primer binding sites. We found that the formation of an abnormally long PCR product was due to HIV-1 env sequence alteration (a) in the reverse primer binding site resulting in faulty primer binding and (b) downstream from the forward primer sequence resulting in a new binding site with reverse complementary sequence with respect to the forward primer at the opposite end of the PCR product. Both changes led to amplification of a longer PCR product with forward primer alone. Our results indicate that the HIV-1 genetic diversity in the env gene can lead to amplification of a specific PCR product of unexpected size which can be disregarded in the absence of its cross-validation.
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A two-step polymerase chain reaction (PCR), with four double (nested) primer pairs, used for the detection of HIV-2 in clinical samples is described. With these four nested primer pairs we could detect HIV-2 DNA in 17 of 17 virus isolates and in blood mononuclear cell samples from 31 of 37 (83.7%) seropositive individuals after ethidium bromide staining of the amplified DNA. The nested primer PCR was also compared with a single primer pair-based PCR followed by hybridization. The sensitivities of the two methods were almost equal, but the nested primer PCR offered obvious technical advantages.
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