|MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA)|
|SELVARAJ, VIJAY - Foreign Agricultural Service (FAS, USDA)|
|HAJERI, SUBHAS - Central California Tristeza Eradication Agency|
|Yokomi, Raymond - Ray|
Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2017
Publication Date: 9/11/2017
Citation: Maheshwari, Y., Selvaraj, V., Hajeri, S., Yokomi, R.K. 2017. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR. PLoS ONE. 12(9):e0184751. https://doi.org/10.1371/journal.pone.0184751.
Interpretive Summary: Citrus stubborn disease (CSD) is caused by the bacterium Spiroplasma citri and is transmitted plant to plant by leafhopper vectors. CSD induces leaf chlorosis, stunting, off-shaped fruit, premature fruit drop, and yield reduction. This research was conducted to develop a precise method to measure the population of S. citri to determine limits of accurate detection. Two diagnostic procedures were compared: real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR). qPCR is the current technology for S. citri detection. ddPCR is a unique approach to measure the absolute copy number of nucleic acid targets without the need for external standards. Two S. citri gene targets were examined; spiralin, a single copy gene and a S. citri multicopy prophase gene, ORF1. Results indicated that ddPCR was more robust and accurate than qPCR and that detectable levels of pathogen were higher in fruit columella than in leaf petioles. Detection was more reliable and sensitive with less variation using the ORF1 vs. spiralin primers due to a higher copy number of targets in the sample.
Technical Abstract: Droplet digital Polymerase chain reaction (ddPCR) is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. It is a promising DNA quantification technology for medical diagnostics but there are only a few reports of its use for plant pathogen detection. This study evaluated the applicability of ddPCR as a quantitative detection tool for Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiralin housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissue. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better that qPCR to detect S. citri infection in fruit columella due to high pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets.