title: Improved real-time PCR detection of 'Candidatus Liberibacter asiaticus' from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes.
abstract: 'Candidatus Liberibacter asiaticus' (CLas) is a Gram-negative α-proteobacterium, and the prominent species of Liberibacter associated with a devastating worldwide citrus disease known as huanglongbing (HLB). This fastidious bacterium resides in phloem sieve cells of host plants and is vectored by the Asian citrus psyllid (Diaphorina citri). Due to its uneven distribution in planta and highly variable bacterial titers, detection of HLB bacteria can be challenging. Here we demonstrated a new utility of nearly identical tandem-repeats of two CLas prophage genes for real-time PCR by SYBR Green 1 (LJ900fr) and TaqMan(®) (LJ900fpr). When compared with conventional 16S rDNA-based real-time PCR, targeting the repeat sequence reduced the relative detectable threshold by approximately 9 and 3 real-time PCR cycles for LJ900fr and LJ900fpr, respectively. Additionally, both LJ900 methods detected CLas from otherwise non-detectable samples by other methods. CLas was also detected from globally derived samples including psyllids, various citrus varieties, periwinkle, dodder, and orange jasmine, suggesting the new detection method can be applicable worldwide. Additionally, we demonstrated the presence of the hyv(I)/hyv(II) repeat sequence within the 'Ca. Liberibacter americanus' strain. The method thereby provides sensitive HLB detection with broad application for scientific, regulatory, and citrus grower communities.
authors.affiliation: USDA-ARS, US Horticultural Research Laboratory, Fort Pierce, FL 34945, USA.
authors.forename: J Kent
authors.initials: JK
authors.lastname: Morgan
authors.forename: Lijuan
authors.initials: L
authors.lastname: Zhou
authors.forename: Wenbin
authors.initials: W
authors.lastname: Li
authors.forename: Robert G
authors.initials: RG
authors.lastname: Shatters
authors.forename: Manjunath
authors.initials: M
authors.lastname: Keremane
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Duan
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002957:Citrus / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry; D005814:Genes, Viral; D006430:Hemiptera / Q000382:microbiology*; D060885:Multiplex Polymerase Chain Reaction; D009930:Organic Chemicals / Q000737:chemistry; D010935:Plant Diseases / Q000382:microbiology; D039002:Prophages / Q000235:genetics; D011804:Quinolines; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012230:Rhizobiaceae / Q000235:genetics*; D020080:Tandem Repeat Sequences
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D015342:DNA Probes; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C098022:SYBR Green I