Dataset Information

A direct isothermal amplification system adapted for rapid SNP genotyping of multifarious sample types.

ABSTRACT: Genotyping of single nucleotide polymorphisms (SNPs) in point-of-care (POC) settings could be further improved through simplifying the treatment of samples. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on direct loop-mediated isothermal amplification (LAMP) without DNA extraction, known as Direct-LAMP. Samples from various sources (including whole blood, dried blood spot, buccal swab and saliva), treated with NaOH, can be used directly in amplification. The turnaround time was about 30?min from sample collection to provision of results. The accuracy was evaluated by assessing the polymorphisms of methylenetetrahydrofolate reductase (MTHFR) C677T and aldehyde dehydrogenase-2 (ALDH2) Glu504Lys, which are better known for their critical role in folate and ethanol metabolism, respectively. Completely consistent genotyping results reveal that Direct-LAMP is generally concordant with sequencing. This system can serve as a very promising platform in the fields of disease predisposition, drug metabolism and personalized medicine.

SUBMITTER: Liu X

PROVIDER: S-EPMC7126597 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A direct isothermal amplification system adapted for rapid SNP genotyping of multifarious sample types.

Liu Xiaonan X Zhang Chao C Zhao Mengye M Liu Kewu K Li Hang H Li Ningning N Gao Linlin L Yang Xuemin X Ma Ting T Zhu Juanli J Hui Wenli W Hua Kai K Cui Yali Y

Biosensors & bioelectronics 20180511

Genotyping of single nucleotide polymorphisms (SNPs) in point-of-care (POC) settings could be further improved through simplifying the treatment of samples. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on direct loop-mediated isothermal amplification (LAMP) without DNA extraction, known as Direct-LAMP. Samples from various sources (including whole blood, dried blood spot, buccal swab and saliva), treated with NaOH, can be used directly in amplification. ...[more]

PMID: 29803102

Similar Datasets

Project description:Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings. IMPORTANCE Current methods for diagnosing drug-resistant tuberculosis are time consuming, resulting in delays in patients receiving treatment and in transmission onwards. They also require a high level of laboratory infrastructure, which is often only available at centralized facilities, resulting in further delays to diagnosis and additional barriers to deployment in resource-limited settings. This article describes a new workflow that can diagnose drug-resistant TB in a shorter time, with less equipment, and for a lower price than current methods. The amount of TB DNA is first increased without the need for bulky and costly thermocycling equipment. The DNA is then read using a portable sequencer called a MinION, which indicates whether there are tell-tale changes in the DNA that indicate whether the TB strain is drug resistant. Our workflow could play an important role in the future in the fight against the public health challenge that is TB drug resistance.

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.

Dataset Information

A direct isothermal amplification system adapted for rapid SNP genotyping of multifarious sample types.

Publications

A direct isothermal amplification system adapted for rapid SNP genotyping of multifarious sample types.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets