Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus"

Authors: SELVARAJ, VIJAY ANANDRAJ - Foreign Agricultural Service (FAS, USDA); MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA); HAJERI, SUBHAS - Central California Tristeza Eradication Agency; Chen, Jianchi; McCollum, Thomas; Yokomi, Raymond - Ray

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/11/2018
Publication Date: 5/17/2018
Citation: Selvaraj, V., Maheshwari, Y., Hajeri, S., Chen, J., Mccollum, T.G., Yokomi, R.K. 2018. Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus". PLoS One. 13(5):e0197184. https://doi.org/10.1371/journal.pone.0197184.
DOI: https://doi.org/10.1371/journal.pone.0197184

Interpretive Summary: Huanglongbing (HLB) (aka citrus greening) is a devastating citrus disease associated with "Candidatus Liberibacter asiaticus" (CLas), an unculturable bacterium transmitted by the Asian citrus psyllid (ACP). Quarantines in California require that HLB-affected trees be removed immediately upon detection to eliminate pathogen reservoirs and minimize further spread by ACP. The current regulatory detection protocol for CLas is quantitative real time PCR (qPCR) targeting 16S rRNA (16S). A critical need exists to improve early detection of CLas to assist in timely HLB eradication. To this end, a duplex assay was developed that targeted the multi-copy 16S and ribonucleotide reductase (RNR) genes in a droplet digital polymerase chain reaction (ddPCR) format. This assay resulted in simultaneous absolute quantification (copy number) of both CLas targets in the same reaction mixture. Because the CLas RNR is more conserved than the 16S region, the duplex format improved diagnostic performance and precision by checking for non-specific target amplification in the same sample. In addition, sample partitioning in ddPCR resulted in a greater tolerance to PCR inhibitors. These results showed that detection of CLas was more precise by ddPCR due to direct pathogen quantification without need for standards; whereas, qPCR provided an indirect relative measure of the pathogen titer requiring adding target standard dilutions to interpolate pathogen titer. Moreover, using the duplex assay, low titer detection of CLas- infected citrus leaf and insect tissues was shown to be significantly greater than in the duplex assay rather than the singleplex assay. Given the need to produce rapid unambiguous results for CLas, the duplex ddPCR for simultaneous detection of the 16S and RNR targets in the same sample showed great advantages which are critical for regulatory samples.

Technical Abstract: Huanglongbing (HLB) (aka citrus greening) is a devastating citrus disease associated with “Candidatus Liberibacter asiaticus” (CLas). Currently, diagnosis of CLas in regulatory samples is based on a real-time quantitative polymerase chain reaction (qPCR) assay using 16S rRNA gene specific primers/probe. qPCR detection of CLas is challenging due to low pathogen titer and uneven distribution in infected plants exacerbated by sampling issues and inhibitors. To improve diagnosis of CLas, duplex assays were developed using multicopy 16S and ribonucleotide reductase (RNR) genes for droplet digital polymerase chain reaction (ddPCR). The ddPCR assay was evaluated in comparison with qPCR for CLas detection. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf DNA, and Asian citrus psyllid (ACP) DNA showed that both ddPCR and qPCR exhibited excellent linearity and efficiency in the duplex assays. Validation experiments with low titer samples showed that the detection rate was higher when both 16S and RNR primers were used in a duplex format than if either target was used in a singleplex ddPCR or qPCR format. However, receiver operating characteristic analysis indicated that area under the curve for the RNR target reaction was significantly broader compared to 16S target at low pathogen titer. Moreover, absolute quantification of CLas at variable titers showed that the duplex ddPCR assay was more reproducible and repeatable for both primers and showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts than that for duplex qPCR.