Dataset Information

Detection of epidermal growth factor receptor T790M mutation by allele-specific loop mediated isothermal amplification.

ABSTRACT:

Introduction

Targeted therapy using specific inhibitors against tyrosine kinases (TKs) is a paradigm in non-small-cell lung cancer management. However, the success of TK inhibitor (TKI) therapy depends on certain activating or acquired mutations, which render sensitivity or resistance to TKIs in the patients. The acquisition of epidermal growth factor receptor (EGFR) T790M point mutation is the most common mechanism of resistance to TKI in non-small cell lung cancer. A number of molecular strategies are now available for molecular testing of non-small cell lung cancers. However, almost all of them are cost-intensive and laborious and require high-end advanced equipment. Thus, assays that are rapid, simple, and cost-effective, yet sensitive, are most ideal in clinical settings for screening such therapeutically relevant mutations.

Materials and methods

Allele-specific loop-mediated isothermal amplification assay (AS-LAMP), which is a variant of the original LAMP assay, is a promising diagnostic technique for screening single-nucleotide polymorphisms. Using commercially available plasmid constructs as template DNA, AS-LAMP assay for EGFR T790M mutation was optimized with six different sets of reaction mixture containing varying concentrations of buffer and primers. The results of AS-LAMP assay were further validated by ultrasensitive droplet digital polymerase chain reaction.

Results

Only one of the six sets of reaction mixture could accurately distinguish between wild type and mutated DNA, indicating that the primers and buffer are the two most critical components that determine the accuracy of AS-LAMP. The optimized AS-LAMP assay was further used to screen germ line and somatic T790M mutations in non-small cell lung cancer using blood and tissue samples collected from patients.

Conclusion

Development of an accurate and rapid diagnostic assay that can detect resistant mutations without the need for sequencing is highly useful for clinicians in deciding on the eligibility of patients for TKI therapy. Considering its several inherent advantages, AS-LAMP assay could become an effective molecular tool for screening baseline or acquired EGFR T790M mutations in non-small cell lung cancer patients.

SUBMITTER: Arjuna S

PROVIDER: S-EPMC7363156 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Detection of epidermal growth factor receptor T790M mutation by allele-specific loop mediated isothermal amplification.

Arjuna Srividya S Chakraborty Gunimala G Venkataram Rajesh R Dechamma Pandyanda Nanjappa PN Chakraborty Anirban A

Journal of carcinogenesis 20200611

<h4>Introduction</h4>Targeted therapy using specific inhibitors against tyrosine kinases (TKs) is a paradigm in non-small-cell lung cancer management. However, the success of TK inhibitor (TKI) therapy depends on certain activating or acquired mutations, which render sensitivity or resistance to TKIs in the patients. The acquisition of epidermal growth factor receptor (EGFR) T790M point mutation is the most common mechanism of resistance to TKI in non-small cell lung cancer. A number of molecula ...[more]

PMID: 32684851

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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.

Dataset Information

Detection of epidermal growth factor receptor T790M mutation by allele-specific loop mediated isothermal amplification.

Introduction

Materials and methods

Results

Conclusion

Publications

Detection of epidermal growth factor receptor T790M mutation by allele-specific loop mediated isothermal amplification.

Similar Datasets

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