Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification

Abstract

Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR.

Emerging Loop-Mediated Isothermal Amplification-Based Microchip and Microdevice Technologies for Nucleic Acid Detection

“…In other work, a multiplex e ndonuclease restriction real-time LAMP assay was developed to detect multiple genome targets in one LAMP sample. 84 FIP and BIP primers, which contain 5′-end short sequences, were recognized by restriction endonuclease enzyme and new FIP and BIP were modified at the 5′ end with fluorophore as well as a dark quencher. Later, restriction endonuclease digested the newly synthesized double-stranded terminal sequences, releasing the quenching and gaining the florescent signal.…”

Development of Multiple Cross Displacement Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Shigella spp.

“…(McKillip and Drake, 2004; Warren et al, 2006; Mandal et al, 2011; Villalobo and Torres, 1998). These methodologies require a sophisticated thermal cycling apparatus to denature target templates and still analysis of the amplified products with either agarose gel electrophoresis or probe hybridization techniques, which significantly hampered its application in the laboratories with limited resources settings (Wang et al, 2015a,b, 2016a). Although, newer approaches, including chemical and biological sensors, have been reported to be very rapid, sensitive and specific for detecting PCR amplicons of different target, thermal cycling of PCR-based methods during the amplification stage imposed instrumental constraints, limiting these assays to a low-resource setting (Chua et al, 2011; Liao et al, 2016).…”

Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus

“…Multiplex detection is the development trend of pathogen detection technology due to the properties of cost savings and high efficiency. In order to achieve multiplex LAMP detection, methods based on end point analysis2425 as well as real-time detection262728 have been employed to differentiate multiple target sequences, while these strategies all required complicated and specialized instruments. To enable multiplex pathogen detection without the need of specialized instruments, Dou et al .…”

“…However, the differentiation of the ladder-like LAMP amplicons derived from multiple targets is still challengeable to date, although previous studies have described several methods for multiplex LAMP detection. These multiplex LAMP methods either used end point analysis, through gel electrophoresis24 or pyrosequencing25, or used real-time detection, through annealing curve analysis26, DARQ27 or MERT-LAMP technique28. However, these techniques all require complicated and specialized instrumentations, which diminish the point-of-care testing capability of multiplex LAMP.…”