Dataset Information

Novel miniaturized fluorescence loop-mediated isothermal amplification detection system for rapid on-site virus detection.

ABSTRACT: New pathogen outbreaks have progressed rapidly and are highly infectious in recent years, increasing the urgency of rapid and accurate detection of pathogenic microorganisms. Based on the point-of-care testing (POCT) requirements, in this study, a real-time fluorescent loop-mediated isothermal amplification (LAMP) detection system was developed and applied to pathogen detection. The system is compact and portable, with good uniformity and reproducibility, and it can detect pathogens rapidly and effectively. For norovirus detection, the linear range was 10⁰-10⁶ copies/μL. The system can achieve the theoretical sensitivity of LAMP detection, conclusions could be obtained within 35 min, and quantitative detection was possible. The test results of 45 clinical samples were consistent with quantitative PCR (qPCR) and clinical results, and the accuracy could reach 100%. This system has the characteristics of portability, speed, and POCT accuracy, and the cost is much lower than that of commercial qPCR. Therefore, it is suitable for remote areas or places with relatively poor conditions and environments requiring on-site conditions. It can also be widely used to detect various epidemics and unexpected diseases.

SUBMITTER: Wu Y

PROVIDER: S-EPMC9448916 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Novel miniaturized fluorescence loop-mediated isothermal amplification detection system for rapid on-site virus detection.

Wu Yanqi Y Bai Liping L Ye Chengfu C Yuhong Guan Kunming Yan Chen Hui H Jiang Zhihong Z

Frontiers in bioengineering and biotechnology 20220824

New pathogen outbreaks have progressed rapidly and are highly infectious in recent years, increasing the urgency of rapid and accurate detection of pathogenic microorganisms. Based on the point-of-care testing (POCT) requirements, in this study, a real-time fluorescent loop-mediated isothermal amplification (LAMP) detection system was developed and applied to pathogen detection. The system is compact and portable, with good uniformity and reproducibility, and it can detect pathogens rapidly and ...[more]

PMID: 36091427

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Project description:Background:Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods:A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results:Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion:In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

Project description:BackgroundDetection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.Methodology and significant findingsSpecies-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.ConclusionThe RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.

Dataset Information

Novel miniaturized fluorescence loop-mediated isothermal amplification detection system for rapid on-site virus detection.

Publications

Novel miniaturized fluorescence loop-mediated isothermal amplification detection system for rapid on-site virus detection.

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