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Multiplex qPCR of Ribonucleotide Reductase, 16S rRNA, Heat Shock Protein and Chaperonin genes for different Huanglongbing (HLB) detection purposes


<div>Huanglongbing (HLB) is a serious disease affecting citrus production worldwide. Among the three bacteria species associated with HLB, “<em>Candidatus</em>” Liberibacter asiaticus (<em>C</em>Las) is the only one currently present in the United States. Detection of infected trees with methods of optimal sensitivity and/or specificity may be tailored for different purposes of HLB research, quarantine and disease management. Five assays (four <em>C</em>Las specific and one COX control) were multiplexed in various combinations with probes of different dyes, and used to test well-characterized HLB <em>C</em>Las plant extract dilutions using ABI QuantStudio7. The <em>C</em>Las assays target individual 5-copy-per-genome Ribonucleotide Reductase/RNR, 3-copy 16S, single-copy Heat Shock Protein/HSP and Chaperonin/CPR genes. Sensitivity was slightly increased using an RNR-FAM/16S-FAM/COX-JOE assay that detects 8-copy targets per genome compared to RNR-FAM/COX-JOE (5 copies), producing 0.90±0.49 Ct lower (n=40) on higher titer dilutions, and showed 64% vs. 33% (n=95, P<0.0001) positive detection of very low titer dilutions where stochastic error becomes a factor. In additional testing, preliminary data of RNR-ABY/16S-FAM/COX-JOE and RNR-JUN/16S-FAM/CPR-VIC/HSP-ABY assays also showed promising sensitivity, and ability to detect up to three targets simultaneously in low titer samples. This study demonstrates the value of multiplexing previously validated <em>C</em>Las qPCR assays to increase either sensitivity or specificity.</div>

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