The PCR primers were validated and designed to amplify DNA fragments no longer than 700 bp to ensure that all multiplex PCR systems are well suited for tested insects with samples collected from only legs/antenna/whole body. After testing individual primer pairs in singleplex reactions to ensure proper amplification, optimal annealing temperatures were determined using PCRs, and primer concentrations were adjusted to balance the successful amplification of all fragments using standardized DNA templates, as described by Yashiro et al. [2 (link)]
TOYOBO KOD-FX neo (Toyobo Life Science) was used for general PCR to confirm the species through sequencing and primer checking. Appropriate primers were used with the PCR amplification protocol that included 2 min denaturation at 94 °C, followed by 40 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 15 s, and extension at 68 °C for 15 s. SYBR green was used to visualize the amplified DNA fragments after they were separated on 1.5% agarose gel electrophoresis (Life Technologies, Grand Island, NY, USA). All experiments were conducted with at least three biological replicates.
The multiplex primer mixture comprised BPH_lamp3_F33/B33, SBPH_lamp1_F311/B34, and WBPH_lamp1_F31/B312 in a ratio of 1:1:1. A multiplex reaction (20 μL volume) was conducted with 3 μL of the respective primer mixture (10 pmol/μL), 1 μL gDNA (10 ng/μL), 10 μL 2X buffer with 15 mM MgCl2, 1.6 μL dNTP (each dNTP), including 0.15 KOD-FX neo (1 unit/μL), and PCR grade water 5.25 μL. The multiplex PCR reaction was conducted for 2 min at 94 °C, followed by denaturation at 94 °C for 20 s, annealing at 53 °C for 20 s, extension at 68 °C for 20 s, and final extension at 68 °C for 2 min, and holding at 8 °C for an unlimited period. Amplification was carried out using an Applied Biosystems ProFlex PCR system (Thermo Fisher Scientific, Waltham, MA, USA).
The LAMP assay was carried out according to the manufacturer’s instructions using a WarmStart® LAMP Kit (New England Biolabs, Ipswich, MA, USA). The LAMP conditions were selected based on our previous studies [30 (link),40 (link)]. The LAMP assay was run for 60 min at 61 °C, 63 °C, and 65 °C with four primers (F3, B3, FIP, and BIP) to optimize the reaction temperature using an Applied Biosystems ProFlex PCR system (Thermo Fisher Scientific). The efficiency of loop primer(s) was tested/evaluated in the presence of additional loop primer(s) at 61 °C for 30 min. The detection limit of gDNA was also tested using six primers at 61 °C for 30 min.
TOYOBO KOD-FX neo (Toyobo Life Science) was used for general PCR to confirm the species through sequencing and primer checking. Appropriate primers were used with the PCR amplification protocol that included 2 min denaturation at 94 °C, followed by 40 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 15 s, and extension at 68 °C for 15 s. SYBR green was used to visualize the amplified DNA fragments after they were separated on 1.5% agarose gel electrophoresis (Life Technologies, Grand Island, NY, USA). All experiments were conducted with at least three biological replicates.
The multiplex primer mixture comprised BPH_lamp3_F33/B33, SBPH_lamp1_F311/B34, and WBPH_lamp1_F31/B312 in a ratio of 1:1:1. A multiplex reaction (20 μL volume) was conducted with 3 μL of the respective primer mixture (10 pmol/μL), 1 μL gDNA (10 ng/μL), 10 μL 2X buffer with 15 mM MgCl2, 1.6 μL dNTP (each dNTP), including 0.15 KOD-FX neo (1 unit/μL), and PCR grade water 5.25 μL. The multiplex PCR reaction was conducted for 2 min at 94 °C, followed by denaturation at 94 °C for 20 s, annealing at 53 °C for 20 s, extension at 68 °C for 20 s, and final extension at 68 °C for 2 min, and holding at 8 °C for an unlimited period. Amplification was carried out using an Applied Biosystems ProFlex PCR system (Thermo Fisher Scientific, Waltham, MA, USA).
The LAMP assay was carried out according to the manufacturer’s instructions using a WarmStart® LAMP Kit (New England Biolabs, Ipswich, MA, USA). The LAMP conditions were selected based on our previous studies [30 (link),40 (link)]. The LAMP assay was run for 60 min at 61 °C, 63 °C, and 65 °C with four primers (F3, B3, FIP, and BIP) to optimize the reaction temperature using an Applied Biosystems ProFlex PCR system (Thermo Fisher Scientific). The efficiency of loop primer(s) was tested/evaluated in the presence of additional loop primer(s) at 61 °C for 30 min. The detection limit of gDNA was also tested using six primers at 61 °C for 30 min.
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