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

Zhou, Qinzheng; Liu, Ya; Wang, Zheng; Wang, Huimin; Zhang, Xingyao; Lü, Quan

doi:10.3389/fpls.2022.856109

Cited by 10 publications

(15 citation statements)

References 47 publications

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“…The cost of detection is a major problem that has restricted the popularization and application of detection methods. For example, the fluorescence quantitative PCR instrument is expensive and recombinant polymerase amplification-lateral flow dipsticks are approximately 50 CHY each ( Zhou et al., 2022 ). The DETECTR detection method ( Chen et al., 2018 ) was shown to accurately detect the risk of human papillomavirus (HPV) infection, HPV16 (100%) and HPV18 (92%), and DETECTR tests cost less than one U.S. dollar each.…”

Section: Discussionmentioning

confidence: 99%

“…Isothermal amplification approaches, such as loop-mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA), have been successfully applied to detect B. xylophilus . These approaches have eliminated the need for thermocycling instruments and can be used for real-time detection, thereby supporting field and point-of-care testing ( Zhou et al., 2022 ; Meng et al., 2022 ). However, these technologies generally are unable to distinguish nucleotide sequences that differ in only one, or even several, bases; they need to be optimized, and may result in false positives because of non-specific amplification, cross-contamination, and/or primer dimerization ( Wang et al., 2018 ; Joung et al., 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

Wang

Cao

et al. 2022

Front. Plant Sci.

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Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

Wang

Cao

et al. 2022

Front. Plant Sci.

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“…RPA combined with lateral flow (LF) test strips possesses the advantages of rapidity and portability in the POCT and has been widely studied. Zhou et al [ 55 ] developed an on-site detection method RPA-LFD for Bursaphelenchus xylophilus based on RPA in combination with LF test strips (Fig. 5 ).…”

Section: Main Textmentioning

confidence: 99%

Microfluidic chip and isothermal amplification technologies for the detection of pathogenic nucleic acid

Gao

Guo²,

Yang³

et al. 2022

J Biol Eng

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The frequency of outbreaks of newly emerging infectious diseases has increased in recent years. The coronavirus disease 2019 (COVID-19) outbreak in late 2019 has caused a global pandemic, seriously endangering human health and social stability. Rapid detection of infectious disease pathogens is a key prerequisite for the early screening of cases and the reduction in transmission risk. Fluorescence quantitative polymerase chain reaction (qPCR) is currently the most commonly used pathogen detection method, but this method has high requirements in terms of operating staff, instrumentation, venues, and so forth. As a result, its application in the settings such as poorly conditioned communities and grassroots has been limited, and the detection needs of the first-line field cannot be met. The development of point-of-care testing (POCT) technology is of great practical significance for preventing and controlling infectious diseases. Isothermal amplification technology has advantages such as mild reaction conditions and low instrument dependence. It has a promising prospect in the development of POCT, combined with the advantages of high integration and portability of microfluidic chip technology. This study summarized the principles of several representative isothermal amplification techniques, as well as their advantages and disadvantages. Particularly, it reviewed the research progress on microfluidic chip–based recombinase polymerase isothermal amplification technology and highlighted future prospects.

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“…The development of isothermal ampli cation technology (IAT) makes on-site assays possible, including the most commonly used loop-mediated isothermal ampli cation (LAMP) and recombinase polymerase ampli cation (RPA), which are promising methods that do not require complicated and expensive equipment (Li and ). The end-point readout of RPA combined with the lateral ow dipstick (LFD) allows for the direct interpretation of results by the naked eye within a few minutes (Wu et al 2020;Zhou et al 2022). In brief, it is a rapid detection technology based on an antigen-antibody immune reaction with gold nanoparticles (AuNPs) (Fig.…”

Section: Introductionmentioning

confidence: 99%

Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali, a serious wood-boring beetle of the western Tianshan Mountains in China

Sun

Zhao

et al. 2023

Preprint

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Agrilus mali is a major wood-boring pest of Northeast Asia. Recently, it destroyed Malus sieversii and caused the decline of the wild fruit forests of the Tianshan Mountains in the Xinjiang Uygur Autonomous Region, China. The pest beetle is often not identified due to the absence of affective rapid discrimination methods beyond adult morphological examination. This study reports a simple, rapid, specific and sensitive method based on recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD), which is able to quickly define the beetle even when it is young. RPA primers were designed based on the internal transcribed spacer 1 region (ITS1). The introduction of the probe in the reaction eliminated the false-positive signals. The probe (Probe1-b) was specificity and sensitivity with as little as 10-3 ng of purified DNA needed to detect A. mali. Moreover, the RPA reaction was carried out using crude extracted DNA, which was only extracted by immersion in sterile distilled water. The LFD is used to visualize the detection results. The results show that the whole process of the RPA-LFD assay can be completed at 38 °C in approximately 30 min, including 10 min for A. mali gDNA extraction and 15 min for RPA-LFD detection, and the amplification can be carried out in an individual’s hand; thus, this method has very good application prospects in the field of quarantine and pest management.

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

Cited by 10 publications

References 47 publications

Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12

Microfluidic chip and isothermal amplification technologies for the detection of pathogenic nucleic acid

Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali, a serious wood-boring beetle of the western Tianshan Mountains in China

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