Effectiveness of Screening of Meat Samples for Enterohemorrhagic Escherichia coli Serovar O157 and O26 by LAMP Assay
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We investigated the effectiveness of a loop-mediated isothermal amplification (LAMP) assay to screen meat samples to detect enterohemorrhagic Escherichia coli serovar O157 and O26. Ninety beef rumen samples were cultured in mEC broth at 37°C and noboviocin-containing mEC broth (N-mEC) at 42°C, and then assayed by LAMP reactions. Twenty-eight mEC culture samples were positive by LAMP assay but no samples were positive for EHEC O157 or O26 by the immuno-magnetic separation (IMS) method. Among N-mEC culture samples, 17 samples were positive by LAMP assay and EHEC O157 was isolated from one sample by IMS method, but no sample was positive for EHEC O26 by IMS method. The rate of concordance for negative results between LAMP assay and IMS method (true negative fraction) was 0.8202 for EHEC O157 and 0.8111 for EHEC O26 by N-mEC culturing. By mEC culturing, true negative fraction was 0.6889 for both EHEC O157 and O26. The effectiveness of LAMP assay for screening was also confirmed using retailed meat samples. Four out of 100 retailed meat samples had positive LAMP reactions, and no EHEC O157 or O26 was isolated from the positive samples. For retailed meat samples, true negative fraction was 0.98 for EHEC O157 or O26. These results suggested that LAMP assay was effective for screening meat samples for EHEC O157 and O26.Loop-mediated isothermal amplification method (LAMP) is a novel nucleic acid amplification technology. The LAMP method amplifies DNA with rapidity, high specificity and sensitivity under isothermal conditions. This thesis took toxic Alexandrium as the major research object. DNA template was extracted by boiling method and specific primer was designed for LAMP assays. Toxic Alexandrium were quickly detected by LAMP technology. LAMP and PCR were successfully compared in the sensitivity of detecting Alexandrium minutum. Without gel electrophoresis, the LAMP amplification was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The result indicated that toxic Alexandrium was detected by LAMP technology within 1 h under isothermal condition at 65 ℃. The lower detection limit of the LAMP reaction was 200 cell/ml, while that of the PCR was 1000 cell/ml. The LAMP assay is more simple, rapid and sensitive than the PCR. Furthermore, as LAMP technology does not require sophisticated instrumentation, it is very suitable for diagnosis in the fields.
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Loop-mediated isothermal amplification(LAMP) was a novel method for nucleic acid amplification with the advantages of high specificity,high sensitivity,simple operation,and fitness for detection of varieties environments,and had been widely used in many fields as a molecular biological detection technology.This paper briefly introduced the principle of LAMP,reviewed its application in detecting plant pathogens,discussed the problems occurring in its application and provided relavent solutions according to these problems,and finally outlooked its application prospect.
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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), a rapid nucleic acid amplification method, was developed for the clinical diagnosis of toxoplasmosis. Three LAMP assays based on the SAG1, SAG2, and B1 genes of Toxoplasma gondii were developed. The sensitivities and specificities of the LAMP assays were evaluated by comparison with the results of conventional nested PCR. The LAMP assays were highly sensitive and had a detection limit of 0.1 tachyzoite, and no cross-reactivity with the DNA of other parasites was observed. Blood was collected from 105 individuals to test the LAMP assays: 40 patients with active toxoplasmosis, 40 negative controls, and 25 patients with other parasitic infections. The SAG2-based LAMP (SAG2-LAMP) had a greater sensitivity (87.5%) than the SAG1-LAMP (80%), B1-LAMP (80%), and nested PCR (62.5%). All the LAMP assays and nested PCR were 100% specific. This is the first report of a study which applied the LAMP method to diagnose toxoplasmosis from human blood samples. Due to its simplicity, sensitivity, and specificity, LAMP is suggested as an appropriate method for routine diagnosis of active toxoplasmosis in humans.
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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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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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