Rapid Newcastle Disease Virus Detection Based on Loop-Mediated Isothermal Amplification and Optomagnetic Readout

Abstract

Rapid and sensitive diagnostic methods based on isothermal amplification are ideal substitutes for PCR in out-of-lab settings. However, there are bottlenecks in terms of establishing low-cost and user-friendly readout methods for isothermal amplification schemes. Combining the high amplification efficiency of loop-mediated isothermal amplification (LAMP) with an optomagnetic nanoparticle-based readout system, we demonstrate ultrasensitive and rapid detection of Newcastle disease virus RNA. Biotinylated amplicons of LAMP and reverse transcription LAMP (RT-LAMP) bind to streptavidin-coated magnetic nanoparticles (MNPs) resulting in a dramatical increase in the hydrodynamic size of the MNPs. This increase was measured by an optomagnetic readout system and provided quantitative information on the amount of LAMP target sequence. Our assay resulted in a limit of detection of 10 aM of target sequence with a total assay time of 30 min. The assay has also been tested on clinical samples (vaccine and tissue specimens) with a performance comparable to realtime RT-PCR. By changing the LAMP primers, this strategy can serve as a general method for the detection of other DNA/RNA targets with high specificity and sensitivity.

Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry

“…2A, 1 pM target sequence), which is much lower than observed in previous work (approximately 36 Hz, 1 pM target sequence) (Tian et al, 2016b). The frequency peak obtained by the optomagnetic setup is not exactly equal but very close to the B f (Donolato et al, 2015;Tian et al, 2016a).…”

“…The assay has a dynamic detection range of 1 aM-10 fM targets in 20% serum sample. Although we only detected synthetic Zika virus DNA, we have demonstrated in a previous paper that the enzyme (Bst 3.0 polymerase) could amplify both RNA and DNA sequences under identical conditions, and performs well when detecting clinical RNA samples (vaccine and tissue specimens) (Tian et al, 2016b). This method is approximately two orders of magnitude more sensitive and faster than the reported RT-PCR based ZIKV serum detection (Faye et al, 2008;Faye et al, 2013), and is thus a promising method for ZIKV diagnosis and control.…”

“…In our previous work (Tian et al, 2016b), we reported a LAMP based optomagnetic biosensor, which could detect 10 aM synthetic Newcastle disease virus DNA in 30 min. It has been demonstrated that Bst 3.0 polymerase could amplify both RNA and DNA sequences under identical conditions, and the LAMP-optomagnetic biosensor was verified by performing tests on clinical RNA samples (vaccine and tissue specimens) (Tian et al, 2016b). The LAMPoptomagnetic assay was performed in a three-step procedure including amplification, labeling and read-out.…”

Rotating magnetic particles for lab-on-chip applications – a comprehensive review

“…Another way to measure the concentration of target molecules is by a cluster assay. Here MPs are provided with capture molecules and form clusters in the presence of target molecules [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] (Fig. 1C).…”

“…One year later the same group published a paper showing that these rotating magnetic particle chains can be used to capture biological molecules. 9 This principle has been extensively studied in the years thereafter 11 and was used in many bio-sensing applications 35,37,38,42,43 (Fig. 1B and C) to significantly shorten assay times, typically from 60-120 min to 15-30 min.…”