Designing of Las specific primers and experimental validation of the specificity and sensitivity of qRT-PCR assay to detect Las Based on the genome sequence of Las strain psy62, we designed 34 qRT-PCR primer pairs that specifically target the 34 unique sequences identified in our bioinformatic analyses (Additional file 4: Table S1). We designed the melting temperature (Tm) of each of these primers to range from 59°C to 65°C with an optimum of 62°C. The GC content of the primers ranged from 35% to 65% with an optimum of 50%. The PCR amplicon sizes for each primer set are between 84 to 185 bp (Additional file 4: Table S1). In addition to the novel RG7204 cost primers designed in this work, we also used a set
of control primers that have been previously used in a qRT-PCR based detection of Las. These known primers include 16S rDNA pairs specific to the three different Candidatus
Liberibacter species (HLBasf/r: Las, HLBamf/r: Lam and HLBaf/r: Laf) [23], β-operon (CQULA04f/r: β-operon) [26], www.selleckchem.com/products/ABT-263.html intragenic repeats regions of the prophage sequence (LJ900f/r: Prophage) [25], and the primer pair specific to the plant cytochrome oxidase (COXf/r: COX) gene [23] as a positive endogenous control. We performed qRT-PCR assays to test the specificity of the designed primers using total DNA extracted from Las-infected citrus plants as a template. To further validate the specificity of these primers, we also included total DNA from the phylogenetically closely related species Lam and Laf in our test. Additionally, DNA extracted from healthy citrus plant was used as a negative control, whereas water served as a no template control. The results of qRT-PCR assays are listed in Table 1. Table 1 Specificity and sensitivity of the novel primers in the detection of Las as shown by qRT-PCR assay Primer pairs Target gene Las CT value of the qRT-PCR# Negative control Other controls CT value R 2 value† Slope†
Laf Lam Healthy plant tissue Water C1 C2 C3 C4 C5 C6 P1 CLIBASIA_05555 20.54 0.9944 -0.2883 UD UD UD UD UD UD UD UD UD UD P2 CLIBASIA_04315 19.99 0.9867 -0.2849 UD UD UD UD UD UD UD UD UD UD P3 CLIBASIA_05575 20.15 0.9991 -0.2847 UD UD UD UD UD UD UD UD UD UD P4 CLIBASIA_05465 19.52 0.9618 -0.2897 UD UD UD UD UD UD UD UD UD UD P5 CLIBASIA_01460 19.48 0.9995 -0.2969 UD UD UD UD UD UD UD Molecular motor UD UD UD P6 CLIBASIA_05145 22.29 0.9971 -0.3057 UD UD UD UD UD UD UD UD UD UD P7 CLIBASIA_05545 20.11 0.9972 -0.3407 UD UD UD UD UD UD UD UD UD UD P8 CLIBASIA_05560 19.92 0.9982 -0.3132 UD UD UD UD UD UD UD UD UD UD P9 CLIBASIA_02025 20.12 0.9875 -0.2743 UD UD UD UD UD UD UD UD UD UD P10 CLIBASIA_05605 20.18 0.9945 -0.2781 UD UD UD UD UD UD UD UD UD UD P11 CLIBASIA_03090 23.61 0.9997 -0.2867 UD UD UD UD UD UD UD UD UD UD P12 CLIBASIA_03875 27.47 0.9992 -0.2563 UD UD UD UD UD UD UD UD UD UD P13 CLIBASIA_02305 UD NT NT UD UD UD UD UD UD UD UD UD UD P14 CLIBASIA_05495 21.25 0.9974 -0.