Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited.
This study explores the development of Loop-mediated isothermal amplification (LAMP) for detection of rose rosette virus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense, single-stranded RNA virus which is a member of the genus Emaravirus (Family Fimoviridae) and the causal agent of the rose rosette disease (RRD). Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear like herbicide damage. Moreover, it may take incubation time for symptoms to appear after virus infection. Two sets of RRV gene sequences RNA3 and RNA4 were analyzed and two sets of four LAMP primers were designed. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. RT-LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. RT-qLAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) was 1pg/μL and 1 fg/μL using Bst 2.0 LAMP and GspSSD LD quantitative LAMP, respectively. In visual colorimetric pre- and post-reactions, the LoD was 10 pg/μL and 0.1 pg/μL using hydroxy naphthol blue (HNB, 120 μM) and SYBR green I (1:10 dilution), respectively. No cross-reactivity was detected in the RT-LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses and one virus taxonomically related to RRV. Four different dyes were tested, and visible colorimetric reactions were obtained with RT-LAMP Bst 2.0 combined with SYBR I or HNB. RT-qLAMP with GspSSD2.0 offers LoD equal to RT-PCR and it is faster since it works with RNA directly.
This study explores the development of Loop-mediated isothermal amplification of DNA (LAMP) for detection of rose rosette emaravirus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense single-stranded RNA Emaravirus and causal agent of the rose rosette disease (RRD). Transmission of RRV is by Phyllocoptes fructiphilus, an eriophyid mite . Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear similar to herbicide damage. Two sets of RRV gene sequences composed of twenty-two accessions of RRV-P3 (RNA 3) and another twenty-four from RRV-P4 (RNA 4) were analyzed and two sets of four LAMP primers were designed for broad-range detection of RRV isolates. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. LAMP reactions were optimized for Bst 2.0 DNA polymerase using the outer RRV-F3/RRV-B3 primers, and internal RRV-FIP/RRV-BIP primers. LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. LAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) using Bst 2.0 LAMP was 1pg/μL and 1 fg/μL with GspSSD LD quantitative LAMP. The LoD of pre-reaction hydroxy naphthol blue (HNB, 120 μM) for colorimetric (visual) reactions was 10 pg/μL and 0.1 pg/μL using SYBR green I (1:10 dilution) in colorimetric post-reactions. No cross-reactivity was detected in LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses (INSV, ArMV, MSpV, TSWV, ApMV, PNRSV, ToRSV, and TMV), and one virus taxonomically related to RRV (HPWMoV). RNA from healthy rose tissues and non-template controls (water) were included in all LAMP assays.
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