Specific detection of topoisomerase i from the malaria causing P. falciparum parasite using isothermal Rolling Circle Amplification
Cinzia TesauroSissel JuulBarbara ArnòChristine Juul Fælled NielsenP FioraniRikke FrøhlichFélicie F. AndersenAlessandro DesideriMagnus StougaardEskild PetersenBirgitta R. Knudsen
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We present a Rolling-Circle-Enhance-Enzyme-Activity-Detection (REEAD) system with potential use for future point-of-care diagnosis of malaria. In the developed setup, specific detection of malaria parasites in crude blood samples is facilitated by the conversion of single Plasmodium falciparum topoisomerase I (pfTopI) mediated cleavage-ligation events, happening within nanometer dimensions, to micrometer-sized products readily detectable at the single molecule level in a fluorescence microscope. In principle, REEAD requires no special equipment and the readout is adaptable to simple colorimetric detection systems. Moreover, with regard to detection limit the presented setup is likely to outcompete standard gold immuno-based diagnostics. Hence, we believe the presented assay forms the basis for a new generation of easy-to-use diagnostic tools suitable for the malaria epidemic areas in developing countries.Keywords:
Rolling circle replication
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A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods. Key words: Toxoplasma gondii, loop-mediated isothermal amplification (LAMP), Q-PCR, pig
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Objective; To detect Salmonella choleraesuis rapidly, Loop-mediated isothermal amplification (LAMP) method was developed. Methods: According to fliC gene of S. choleraesuis flagellin protein, LAMP inner and outer primer sets were designed. Amplification reaction carried out within tens of minutes. Specificity of LAMP primers were validated by as-saying 10 different strains. Sensitivity of LAMP method was evaluated by serial gradient dilution of S. choleraesuis cultures. LAMP used for the detection of simulated meat samples. Results: S. choleraesuis LAMP method was rapid and specific. The detection limits of LAMP assay were 1.33×101 CFU/mL for pure cultures and 2.0×101 CFU/mL for simulated meat samples. Conclusion: LAMP method is a rapid and feasible method for the detection of S. choleraesuis.
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A loop-mediated isothermal amplification(LAMP) assay was developed for detection of Mycoplasma iowae(MI).According to the sequences specific to species protein1 of MI in GenBank,six primer pairs were designed,and the reaction conditions were optimized. In result,only LAMP reactions of Mycoplasma iowae were positive(visible green),while LAMP reactions of the other common pathogens were negative(visible red).The detection limit of this LAMP method was 10 fg for DNA,which was 100 times higher than routine PCR.The amplification could be finished within 1 h,and the presence of MI could be detected by naked eyes.These results show that this LAMP assay is a simple and specific method for rapid detection of MI for clinical samples.
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Loop‐mediated isothermal amplification ( LAMP ) is a promising nucleic acid‐based assay for quick, accurate and cost‐effective diagnosis of many infectious agents. The purpose of this study was to assess the diagnostic value of LAMP for rapid and accurate detection of Helicobacter pylori in biopsy specimens. Patients suffering from one or several gastroduodenal disorders were enrolled in the study. Specificity, sensitivity, and the positive and negative predictive values of LAMP were compared with the gold standard result, which was the assembled result of culture, rapid urease test and polymerase chain reaction. Sensitivity, specificity, and the positive and negative predictive values of LAMP in comparison with the gold standard result were 100%, 30.76%, and 87.67% and 100% respectively [%95 CI ]. As the diagnostic value of LAMP is favourable, the method is an optimum technique for diagnosis the presence of H. pylori in different clinical and environmental samples.
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A loop-mediated isothermal amplification(LAMP)assay was developed for detection of Salmonllain fecal samples of experimental monkeys.According to the specific sequences(fimY)of Salmonllain GenBank,one set of primers was selected and the reaction condition was optimized.The results showed that the detection limit of LAMP method was 1.35×101 and 1.35×103 CFU/mL in Salmonellapure culture and clinical samples,respectively,which was the same as routine PCR.The amplification could complete in one hour,and the result could be distinguished by naked eyes.There was non-specific amplification of other pathogens.These results suggested that this LAMP assay was a simple and specific method for rapid detection of Salmonllain fecal samples.
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Rolling circle amplification (RCA) and loop mediated isothermal amplification (LAMP) were combined to establish the rolling circle and loop mediated isothermal amplification (RC-LAMP) method for miRNA detection. With the participation of Bst 2.0 DNA Polymerase, the method enabled RCA and LAMP amplification to occur simultaneously without thermal cycling. The limit of detection of RC-LAMP was 500 amol/L, which is comparable to previously reported amplification strategies. Moreover, its upper limit of quantitation was higher and showed a stronger resistance to matrix interference. Therefore, it is possible to detect low concentrations of miRNA in samples by increasing the total RNA added. Owing to its facile detection mode and simple operation, this method has great potential in clinical sample detection.
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Plasmodium infection causes devastating disease and high mortality in young children. Immunity develops progressively as children acquire protection against severe disease, although reinfections and recrudescences still occur throughout life in areas of endemicity, partly due to parasite immunoevasion via switching of variant proteins such as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the infected erythrocyte surface.
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The assay was aimed to establish a rapid,sensitive and specific method of loop-mediated isothermal amplification(LAMP)for the detection of Salmonella.According to the highly conserved STN gene of Salmonellareported in GenBank, we designed a set of primers,and then followed by a series of LAMP optimization of reaction conditions,specificity test,sensitivity test,stability test and enzyme test.The sensitivity of LAMP and conventional PCR were compared.The results showed that the optimal reaction temperature of LAMP was 65 ℃,when only amplification of Salmonella,the minimum detectable concentration was 101 CFU/mL,which was more than conventional PCR detection by one order of magnitude.The results showed that LAMP method had the qualities of specificity,high sensitivity,short time-consuming and convenience for the detection of Salmonella,which was expected to develop into an effective method.
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Loop-mediated isothermal amplification(LAMP) is a sensitive,specific,convenient and rapid DNA amplification technique that has developed rapidly over the past few years.Unlike conventional PCR,LAMP is an isothermal amplification assay that does not require an expensive thermocycler.Moreover,the LAMP reaction produces a large amount of magnesium pyrophosphate,a white precipitate by-product,that makes results easier to judge with the naked eye.If dye is added before or after reaction,the results are readily visible so further electrophoretic analysis is not needed.Therefore,LAMP is an efficient nucleic acid amplification assay with a high level of sensitivity and specificity;the technique is convenient and is suitable for rapid detection of parasitic diseases during field surveys or in poorly equipped laboratories.Numerous reports have described using LAMP to detect parasitic diseases.This paper reviews recent advances in the use of LAMP techniques to rapidly detect parasitic diseases.
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