QCM immunoassay for recombinant cysteine peptidase: A potential protein biomarker for diagnosis of citrus canker

Abstract

Citrus canker is one of the most important agricultural citrus diseases worldwide. It is caused by Xanthomonas citri subsp. citri (Xcc) bacterium that infects leaves and the fruits produce a cysteine peptidase (CPXaC), which makes it a potential target for the development of effective and rapid detection methods for citrus canker. We report here the studies on the development of piezoelectric immunoassay for CPXaC using a polyclonal antibody against CPXaC (anti-CPXaC). Three different strategies for covalent immobilization of anti-CPXaC on gold surfaces were evaluated by monitoring the frequency (Δf) and energy dissipation (ΔD) variation in real time when 64.5×10(-8) mol L(-1) CPXaC was added. Anti-CPXaC immobilized with 11-mercaptoundecanoic acid (MUA) showed the best relation between the frequency and dissipation factor variation, and strong values for the kinetic and equilibrium binding constant were obtained. The immunosensor showed a detection limit of 13.0 nmol L(-1) with excellent specificity, showing no response for different proteins that include another cysteine peptidase that is used as a target to detect Xylella fastidiosa bacterium, responsible for another important citrus disease. These results provide good perspectives for the use of CPXaC as a new biomarker for citrus canker.

Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence

“…This secretion strategy is known to also interfere with adherence to surfaces besides delivering virulence factors to sites within the plant that are distant from the producing bacterial cells [39,73]. Peptidases are important proteins in several physiological processes, such as the control of protein functionality throughout development and defense [74][75][76][77]. Strain 9a5c shows higher abundance of proteases, membrane-associated proteins, and toxins.…”

Peer Review #1 of "Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR (v0.1)"

“…Due to its high virulence and host range, the development of diagnostics is aimed towards the detection of XccA strains, the causative agent of Asian citrus canker. Currently, field detection is performed through rapid tests based on the immune-identification of its proteins as Xcc ImmunoStrip® Test, Agdia, Inc. (Catalog number: STX 92200), but there are several studies that have developed serological diagnoses for XccA [14,28,[43][44][45][46][47]. However, these tests have low sensitivity, and in some cases, false negative results have already been reported in phenotypically different strains of XccA [13,31].…”

A multiplexed immunoaggregation biomarker assay using a two-stage micro resistive pulse sensor

“…7 Many conventional immunosensors including surface plasmon resonance (SPR), [8][9][10] quartz crystal microbalance (QCM), 9,[11][12][13] and electrochemical sensors [14][15][16] have been demonstrated for the detection of single biomarker. 17 However, single biomarker detection is unable to provide sufficient information for disease diagnosis due to the complexity of human biology and heterogeneity of diseases.…”