Taqman qpcr mix

Leaves for DNA extraction and analysis were rinsed three times with sterile water. Midribs were separated from the leaf samples and cut into 1.0 to 2.0 mm pieces. DNA was extracted from 0.1 g (fresh weight) of leaf midrib tissue using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. qPCR was performed with primers and probe (HLBas, HLBr and HLBp) specific for Las [26 (link)] using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) in a 20 μl reaction volume consisting of the following reagents: 300 nM (each) target primer (HLBas and HLBr), 150 nM target probe (HLBp), and 1× TaqMan qPCR Mix (Applied Biosystems). The amplification protocol was 95°C for 20 s followed by 40 cycles at 95°C for 3 s and 60°C for 30 s. All reactions were performed in triplicate and each run contained one negative (DNA from healthy plant) and one positive (DNA from Las-infected plant) control. Data were analyzed using the ABI 7500 Fast Real-Time PCR System with SDS software.

Each leaf was rinsed three times with sterile water. Midribs were separated from the leaf samples and cut into pieces 1.0 to 2.0 mm in size. DNA was extracted from 0.1 g of the midrib tissue (fresh weight) using a Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. qPCR was performed with primers and probes (HLBas, HLBr, and HLBp) specific for the detection of Las using an ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA) [26 (link)]. A PCR reaction at a volume of 20 μl was composed of the following reagents: 300 nM (each) target primer (HLBas and HLBr), 150 nM target probe (HLBp), and 1 × TaqMan qPCR Mix (Applied Biosystems). The cycling conditions were 95°C for 20 s followed by 40 cycles at 95°C for 3 s and 60°C for 30 s. All reactions were performed in triplicate and each run contained one negative (DNA from healthy plant) and one positive (DNA from Las-infected plant) control.

Genomic DNA extraction and qPCR analysis of plant samples were performed according to Zhang et al. (2011) (link). Plant samples were rinsed three times with sterile water. DNA was extracted from 0.1g of plant samples (fresh weight) using Qiagen’s DNeasy Plant Mini Kit (Qiagen, Valencia, CA). qPCR was performed with primers and probes (HLBas, HLBr, and HLBp) for ‘Ca. L. asiaticus’ using the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA) in a 20 µl reaction volume consisting of the following reagents: 300nM (each) target primers (HLBas and HLBr), 150nM target probe (HLBp), and 1× TaqMan qPCR Mix (Applied Biosystems). All reactions were performed in triplicate and each run contained negative (DNA from healthy plants) and positive (DNA from HLB-affected plants) controls. Data were analysed using the ABI 7500 Fast Real-Time PCR System with SDS software. The cycle threshold (Ct) values were converted to estimated bacterial titres using the grand universal regression equation Y=13.82–0.2866 X, where Y values are the estimated log concentrations of templates and X values are the qPCR Ct values. Plants were considered to be PCR negative for Las when the Ct values were >36.0, which is equivalent to an estimated bacterial titre of <1 60 cells g^–1 of plant tissue.