Rapid Detection of the Bursaphelenchus Xylophilus by Denaturation Bubble-mediated Strand Exchange Amplification

Abstract

that the pathogenic species B. xylophilus and non-pathogenic related species B. mucronatus have very similar morphologies, 9 the operation staff has required specialized knowledge about the nematode morphology, and it is extremely difficult to distinguish the pathogenic species from non-pathogenic related species. In recent years, polymerase chain reaction (PCR)-based detection methods for nematodes have been developed, such as conventional 10 or real-time PCR. 11 Real-time PCR can offer an accurate diagnosis of B. xylophilus. However, complicated operation and relatively expensive equipment required during the PCR reaction limit it within a laboratory area. 12,13 Thus, a simple and rapid detection method is a very important research goal for the on-site detection of B. xylophilus.Herein, we proposed a simple and robust detection strategy of B. xylophilus using the strand exchange amplification (SEA) technique. 14 The SEA technique is identified as isothermal PCR, which only requires a pair of specific primers, and then utilizes successive natural strand "breathing" to unwound double-stranded DNA, rather than heat denaturation. 15 Thus, our strategy was expected to provide a novel nucleic acid amplification method for detecting pinewood nematode, which can be further applied to other target specific detection and electrochemical signal amplification strategy research. [16][17][18] 2019 Bursaphelenchus xylophilus (B. xylophilus) is one of the most important causal agents of infectious diseases in forest pathology. Obviously, the rapid detection of B. xylophilus is an urgent need for its prevention and cure. We have developed a detection method of B. xylophilus by strand exchange amplification (SEA). This method could detect 10 5 copies of genomic DNA of B. xylophilus, and it was sufficiently sensitive to detect a single nematode as short as 40 min. Moreover, because the amplification result could be visualized by the naked eyes, the only equipment required throughout the process was a simple isothermal block. Therefore, our method would be a potential for developing on-site detection of B. xylophilus to prevent and control its spread.

Rapid and sensitive detection of Staphylococcus aureus via an all-in-one staggered strand exchange amplification platform

“…Primer design and establishing an optimal SSEA assay As previous studies have shown that the design of SEA primers was as simple as PCR, a variety of nucleic acid hybridization parameters were calculated to assess the primer quality. 24,25 To improve the amplification specificity and sensitivity, two sets of forward and reverse primers were designed and deployed simultaneously in the SSEA method (Table S1 †). The basic principle of SSEA reaction is based on the generation of denaturation bubbles and guanidine hydrochloride-mediated promotion of primers and template sequence binding.…”

Rapid On-Site Detection of the Bursaphelenchus xylophilus Using Recombinase Polymerase Amplification Combined With Lateral Flow Dipstick That Eliminates Interference From Primer-Dependent Artifacts

“…Alternatively, isothermal DNA amplification techniques that do not require the use of thermal cycling apparatus have been applied to detect B. xylophilus. These include loop-mediated isothermal amplification (LAMP) (Kikuchi et al, 2009;Meng et al, 2018;Ahuja and Somvanshi, 2021), denaturation bubblemediated strand exchange amplification (SEA) (Liu C. et al, 2019), and recombinase polymerase amplification (RPA) (Cha et al, 2019(Cha et al, , 2020Fang et al, 2021). RPA, an isothermal nucleic acid amplification method that has attracted much attention because it is sensitive, rapid, works at isothermal temperature (Piepenburg et al, 2006;Glökler et al, 2021).…”

Global Occurrence of Pine Wilt Disease: Biological Interactions and Integrated Management

“…Alternatively, isothermal DNA amplification techniques that do not require the use of thermal cycling apparatus have been applied to detect B. xylophilus. These include loop-mediated isothermal amplification (LAMP) Ahuja and Somvanshi, 2021), denaturation bubble-mediated strand exchange amplification (SEA) (Liu C. et al, 2019), and recombinase polymerase amplification (RPA) (Cha et al, 2019(Cha et al, , 2020Fang et al, 2021). RPA, an isothermal nucleic acid amplification method that has attracted much attention because it is sensitive, rapid, works at isothermal temperature (Piepenburg et al, 2006;Glökler et al, 2021).…”