T/JSF 009-2024 Molecular Detection Technique of Bursaphelenchus xylophilus by RPA

Scope

Scope: This document describes the molecular detection of pine wood nematode (RPA), the detection objects, detection process, result determination, and methods of recording and archiving. This document is applicable to plant quarantine for import and export, domestic plant quarantine, and investigations of forestry pests, as well as the molecular detection and identification of pine wood nematode in host plants and products. Main technical content: The main technical objective is to achieve rapid amplification of pine wood nematode DNA under constant temperature conditions. The specific techniques are as follows: (1) Sample preparation: Use the Baermann separation method or a rapid separator to separate the nematodes, or directly drill wood chips with a power drill; (2) DNA extraction from samples: Use self-developed extraction reagents to lyse the samples and release nucleic acids; (3) Add uniquely designed specific molecular probes, primers, and sample nucleic acids; (4) Under the presence of ATP, the recombinase combines with the primers to form a protein-nucleic acid complex and searches for the paired pine wood nematode DNA template; (5) The recombinase leaves the primers and synthesizes new DNA strands under the action of DNA polymerase. At the same time, SSB binds to the displaced single-stranded DNA; (6) The newly synthesized DNA strands can be used as templates for the next round of amplification, thus achieving exponential amplification; (7) Real-time signal acquisition is performed using a matching nucleic acid fluorescence detector, and the fluorescence signal-time curve is plotted. The Ct value is calculated, and the results are automatically determined based on the size of the sample Ct value and the shape of the curve. Innovation points: (1) Currently, the average detection time for pine wood nematode on the market is 40 minutes. This technology greatly shortens the detection time to 15 minutes; (2) The entire amplification

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