|Yan, Z - Animal And Plant Health Inspection Service (APHIS)|
|Rascoe, J - Animal And Plant Health Inspection Service (APHIS)|
|Constanzo, S - Animal And Plant Health Inspection Service (APHIS)|
|Stulberg, M - Animal And Plant Health Inspection Service (APHIS)|
|Liu, Z - Animal And Plant Health Inspection Service (APHIS)|
|Nakhla, M - Animal And Plant Health Inspection Service (APHIS)|
Submitted to: International Research Conference on Huanglongbing
Publication Type: Abstract Only
Publication Acceptance Date: 2/1/2017
Publication Date: 3/14/2017
Citation: Yan, Z., Rascoe, J., Constanzo, S., Stulberg, M., Liu, Z., Chen, J., Nakhla, M. 2017. Adoption and validation of Ribonucleotide Reductase (RNR)-based real-time assays for detection of HLB ‘Candidatus Liberibacter asiaticus’ (CLas). International Research Conference on Huanglongbing.
Technical Abstract: Huanglongbing (HLB), aka Citrus Greening, is a well-known destructive disease that threatens the multi-billion dollar citrus industry in the United States and citrus production in other countries around the world. The presumptive causal agent of HLB, ‘Candidatus Liberibacter asiaticus' (CLas), is of Asian origin and the only species associated with HLB currently found in the U.S. Sensitive methods for detection are needed to help prevent spread of the HLB pathogen. The ribonucleotide reductase (RNR) gene sequence is conserved and present in five-copies per CLas genome, presumptively making it a more sensitive target for detection than the 3-copy 16S sequence used in the HLBaspr assay. RNR-based real-time PCR assay was adopted and validated for detection of CLas. Testing a total of 223 diverse citrus samples, the RNR assay showed improvement in analytical sensitivity and specificity over the 16S HLBaspr method. Results for both the 56 CLas positive isolates and 167 CLas negative samples were 100% match with determinations made by APHIS HLB confirmatory methods (i.e., CLas 16S sequencing, Heat Shock Protein (HSP), and Chaperonin (CPR) based real-time PCRs). Serial dilutions of 4 CLas positive plant DNA samples from CA, TX, FL, and India were tested to compare efficiency and limits of detection. RNR Ct values dropped 1 to 2.5 in a side-by-side comparison with the 16S HLBaspr assay. The data suggest that the RNR based real-time PCR could be a more sensitive and specific method for detection of CLas than the 16S HLBaspr assay and warrants further studies. Converting this assay into isothermal technology for potential detection of field samples also is being further investigated.