One-step colorimetric genotyping of single nucleotide polymorphism using probe-enhanced loop-mediated isothermal amplification (PE-LAMP)

Abstract

Single nucleotide polymorphism (SNP) is the most abundant molecular marker associated with many physiologic and pathologic phenotypes. An isothermal, accurate and cost-effective SNP detection could make a great difference in point-of-care testing (POCT) or on-site diagnosis. However, there are two challenges, the expensive instrument and labor-intensive process, faced by the development of on-site SNP detection. We reported a novel SNP typing method based on the probe-enhanced loop-mediated isothermal amplification (PE-LAMP), which combines the oligonucleotide probe with a conventional LAMP to realize the SNP discrimination by analyzing the great discrepancy in amplification efficiency. Methods : We firstly constructed the genotyping method by combining the hybridization of the specific probe with the powerful amplification of LAMP. Then we validated the method by genotyping the SNP rs3741219 and we sought to realize one-step visualized typing. Finally, we applied the method to pharmacogenomic testing by genotyping CYP2C19 *2 and MDR1 C3435T. Results : The PE-LAMP was successfully constructed to detect SNP and the sensitivity of our method is as low as 1000 copies of target DNA, which is sufficient to routine diagnosis. The high specificity in detecting mutant in the presence of excess wild-type allele could be achieved. It has shown good performance in helping predict the individual response of antiplatelet drug Clopidogrel through typing simply treated saliva samples. Conclusions : The proposed method is one-step, colorimetric, specific and sensitive enough to detect crudely treated samples, showing great potential in the pharmacogenomic study and POCT use.

A Fast, Naked-Eye Assay for Varietal Traceability in the Durum Wheat Production Chain

“…In several cases, however, these approaches lead to high background and low discrimination efficiency due to the poor inhibition of the non-specific amplification. Herein, we focused on the optimization of a recently published methodology proposed for the detection of salivary SNPs [ 26 ] based on the use of loop primers as allele-discriminating primers. In this strategy, two reactions are performed in parallel, one with the loop primer hybridizing with the wild-type base and one with the loop primer perfectly matched to the polymorphic one.…”

“…Since loop primers are designed short, one mismatch significantly destabilizes the annealing, and the obtained effect is comparable to that of a reaction performed without loop primers. For instance, in the case of a wild-type target gene, the reaction performed with the polymorphic loop primer resulted in a strongly decreased amplification rate, creating a significant time gap between the two reactions [ 26 ].…”

Point-of-care cervical cancer screening using deep learning-based microholography

“…Expanding on this inaugural DNA microholography platform, we intend to enhance the assay for expanded testing in resource-poor geographies. These will include i) integrating an isothermal amplification method such as recombinase polymerase amplification (Figure S7) or loop-mediated isothermal amplification 23,24; ii) extending the assay for other high-risk HPV strains ( e.g. , HPV 31, 58) 25,26; and iii) employing reagent lyophilization steps for long-term storage.…”

Ultra-Rapid Somatic Variant Detection via Real-Time Threshold Sequencing

“…Once dumbbells are formed, extension can initiate from 3' looped ends or inner-primer annealing sites on the loops themselves resulting in rapid concatemeric extension and copying of the target region (Figure 5a). LAMP amplification has been used in a variety of use-cases where rapid time-to-result is important including pathogen detection 26,30 and targeted-allele genotyping 29,37 . However, the complex machinery of LAMP can easily malfunction and lead to spurious amplification.…”