Application of duplex droplet digital PCR for detection of “Candidatus Liberibacter asiaticus” using 16S rRNA and ribonucleotide reductase genes

Authors: MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA); SELVARAJ, VIJAYANANDRAJ - Foreign Agricultural Service (FAS, USDA); HAJERI, S - Central California Tristeza Eradication Agency; Yokomi, Raymond - Ray

Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 3/27/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Huanglongbing (HLB) is the most destructive disease of citrus and has been detected in over 250 urban citrus trees in southern California. HLB is associated with the uncultivable bacterium “Candidatus Liberibacter asiaticus” (CLas) and is transmitted by the Asian citrus psyllid. Quarantines in California limit spread of CLas but methods are needed to improve sensitivity and early detection of HLB to aid eradication of infected trees. CLas detection is challenging due to low titer and uneven distribution in infected trees. The chromosome of CLas has three copies of the 16S rRNA gene and five copies of nrdB, encoding ß-subunit of ribonucleotide reductase (RNR). This study evaluated the applicability of duplex droplet digital PCR (ddPCR) detection of CLas at low titer using primers specific for the 16S and RNR genes. Standard curve analyses on the tenfold dilution series of plasmid, plant leaf tissue DNA and insect DNA showed that the duplex ddPCR exhibited good linearity and efficiency. The sensitivity for both the 16S and RNR plasmid was 2 copies/20µl reaction. However, low titer detection of CLas showed percent detection was higher with the RNR gene than the 16S rRNA gene with leaf tissue and insect DNA samples. The duplex ddPCR provided simultaneous comparison between both genes and proved valuable for CLas detection for samples with marginal Cq values in qPCR. This method may be adoptable in regulatory programs.