Detection of Plant Nematodes by Recombinase Polymerase Amplification

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Recombinase polymerase amplification (RPA) technology mainly relies on recombinant enzymes that bind single-stranded nucleic acid, single-strand binding protein, and strand displacement DNA polymerase. The recombinase binds to the amplification primer to form a recombinase primer complex. After that, the recombinase is hydrolyzed from the complex, the 3' end primer is exposed. It binds to DNA polymerase, and the DNA begins to replicate and extend, forming two new complementary double-stranded DNA, thus achieving exponential amplification of the target region on the template.

Lifeasible develops an advanced platform that provides services to customers worldwide covering the detection of plant nematodes by recombinase polymerase amplification. We customize featured services according to customer needs with decades of experience in plants. In addition, we deliver satisfactory and reliable results and reports on time to our customers worldwide.

Detection of Plant Nematodes by RPA

RPA does not require thermal denaturation and temperature control equipment. Under the condition of 23 - 45°C, the amplification process can be completed within 20 min to achieve the goal of rapid detection.
Lifeasible provides detection of plant nematodes with RPA, which enables the field detection of plant nematodes.
In addition, we offer RPA in combination with detection methods such as real-time fluorescence detection, side chromatography, and gel electrophoresis, bridge-linked flocculation analysis, colorimetric reactions, electrochemical transduction, electrochemical biosensors, silicon micro ring resonator photon detection, and surface-enhanced Raman scattering for rapid detection.

Fig.1 Schematic of the RPA assay for plant nematodes in the field.

Workflows of RPA Detection

Steps	Operation Methods
DNA Extraction	Plants' nematodes are isolated from culture by the Baermann funnel technique and washed three times with phosphate-buffered saline-Twain. After incubation with 100 µl of extraction buffer, the lysate is extracted once with PCI (phenol / chloroform / isoamyl alcohol), followed by ethanol precipitation, and finally dissolved in 50 µl Tris-EDTA buffer.
Composition and Procedure of the RPA Assay	Reagents for the RPA reaction are pure DNA from each nematode as the reaction template, primers, nuclease-free water untreated with diethyl pyrrolcarbonate (DEPC), rehydration buffer, and magnesium acetate. The master mixture is incubated for 15 min at the optimal temperature (37°C) using the portable optical isothermal device. After incubation, SYBR green I dye mixture and RPA reaction product are mixed, and the absorbance levels are confirmed under ultraviolet light and by the portable optical isothermal device.
The Specificity Test of the RPA Assay	A specificity test of the RPA assay is conducted to confirm the primers that could amplify the specific fragment from plant nematodes but not from the others.
The limit of the Detection Test of the RPA Assay	Tenfold serial dilutions of DNA from nematodes are used to determine the detection limit of RPA.

Lifeasible offers professional services covering the detection of plant nematodes to meet your research needs. With years of experience in plants, our advanced platforms can help our clients solve various difficulties and conduct research. If you are interested in our services or have any questions, please feel free to contact us or make an online inquiry.

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