Abstract
Solid-phase isothermal recombinase polymerase amplification (RPA) offers many benefits over the standard RPA in homogeneous phase in terms of sensitivity, portability, and versatility. However, RPA devices reported to date are limited by the need for heating sources to reach sensitive detection. With the aim of overcoming such limitation, we propose here a label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle (AuNP)-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Plant disease (Citrus tristeza virus (CTV)) diagnostics was selected as a relevant target for demonstration of the proof-of-concept. RPA assay for amplification of the P20 gene (387-bp) characteristic of CTV was first designed/optimized and tested by standard gel electrophoresis analysis. The optimized RPA conditions were then transferred to the AuNP-modified electrode surface, previously modified with a thiolated forward primer. The in situ-amplified CTV target was investigated by EIS in a Fe(CN6)4–/Fe(CN6)3– red–ox system, being able to quantitatively detect 1000 fg μL–1 of nucleic acid. High selectivity against nonspecific gene sequences characteristic of potential interfering species such as Citrus psorosis virus (CPsV) and Citrus caxicia viroid (CCaV) was demonstrated. Good reproducibility (RSD of 8%) and long-term stability (up to 3 weeks) of the system were also obtained. Overall, with regard to sensitivity, cost, and portability, our approach exhibits better performance than RPA in homogeneous phase, also without the need of heating sources required in other solid-phase approaches.
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Claims 5
Claim · model-written summary
The in situ RPA/EIS approach quantitatively detected 1000 fg per microlitre of Citrus tristeza virus nucleic acid.
Verbatim
e0“The in situ-amplified CTV target was investigated by EIS in a Fe(CN6)4–/Fe(CN6)3– red–ox system, being able to quantitatively detect 1000 fg μL–1 of nucleic acid”
Claim · model-written summary
The assay showed high selectivity against nonspecific sequences of Citrus psorosis virus and Citrus caxicia viroid.
- Interactor
- Citrus psorosis virusCitrus psorosis viruse0other
- Second interactor
- Citrus caxicia viroide0other
- Outcome
- High selectivitye0
- Comparator
- nonspecific gene sequencese0
Verbatim
e0“High selectivity against nonspecific gene sequences characteristic of potential interfering species such as Citrus psorosis virus (CPsV) and Citrus caxicia viroid (CCaV) was demonstrated”
Claim · model-written summary
The system had good reproducibility (RSD of 8%).
Verbatim
e0“Good reproducibility (RSD of 8%)”
Claim · model-written summary
The system had long-term stability of up to 3 weeks.
Verbatim
e0“long-term stability (up to 3 weeks)”
Claim · model-written summary
The approach showed better sensitivity, cost and portability performance than RPA in homogeneous phase, without a heating source.
Verbatim
e0“our approach exhibits better performance than RPA in homogeneous phase”