Fig 1.
Schematic illustration of HLB-RPA-LFA principle for the detectionof ‘Ca. L. asiaticus’.
(A) Amplification of FAM-biotin-linking ‘Ca. L. asiaticus’ 16S rRNA amplicons. The RPA driven primers (DKG-F/ Biotin labelled-DKG-R) first generate templates for the annealing of the nfo probe. In the resulting double strand context, nfo endonuclease present in the TwistAmpnfo kit recognizes the double-strand hybridization complex and cuts the THF residue. Polymerisation of ‘Ca. L. asiaticus’ above mentioned template using RPA enzymes. The DNA amplicons produced effectively co-join the two antigenic residues (FAM and biotin) in one DNA molecule. (B) Detection of FAM-Biotin linking ‘Ca. L. asiaticus’ RPA product by LFA using PCRD nucleic acid detector. The appearance of two red bands indicates the positive RPA assay for ‘Ca. L. asiaticus’ whereas appearance of a single red line indicates the negative results.
Table 1.
Conventional PCR primers used in present study.
Table 2.
RPA primers and probe used in present study.
Fig 2.
Agarose gel electrophoresis of PCR products.
Amplified products using OI1/OI2c ‘Ca. L. asiaticus’ specific primers were separated by 1% agarose gels. Observed amplification product of 1160 bp product is shown.C1 to C6 represent the ‘Ca. L. asiaticus’ isolates maintained in the screenhouse. H: Healthy control; -Ve: negative control and L: 1 kb marker.
Fig 3.
Agarose gel electrophoresis of PCR products.
Amplified products using A2/J5 ‘Ca. L. asiaticus’ specific primers were separated by 1% agarose gels. Observed amplification product of 703bp product is shown. C1 to C6 represent the ‘Ca. L. asiaticus’ isolates maintained in the screenhouse. H: Healthy control; -Ve: negative control and L: 100bp marker.
Fig 4.
Agarose gel electrophoresis (1.5%) of PCR products amplified using DKG-F/DKG-R ‘Ca. L. asiaticus’ specific primers.
Observed amplification of 170 bp product is shown.C1 to C6 represent the ‘Ca. L. asiaticus’ isolates maintained in the screenhouse. H: Healthy control, -Ve: negative control and L: 100 bp marker.
Fig 5.
Agarose gel electrophoresis of recombinase polymerase amplification products amplified using DKG-F/DKG-R ‘Ca. L. asiaticus’ specific primers.
Observed amplification of 170 bp product is shown.C1 to C6 represent the ‘Ca. L. asiaticus’ isolates maintained in the screenhouse, H: Healthy control, -Ve: negative control and L: 100bp marker.
Fig 6.
HLB-RPA-LFA; Huanglongbing-recombinase polymerase amplification-lateral flow assay with DNA as a template, showing three reaction lines: C is control; 2 is for detection of FAM/Biotin-labelled HLB amplicons.
(1 is not used in present assay). C1 to C6 represents the ‘Ca. L. asiaticus’ isolates maintained in the screenhouse, H: Healthy control and -Ve: negative control.
Fig 7.
HLB-RPA-LFA, Determination of optimum reaction time.
The best visibility of the test line was observed in the ranges20, 25, 30 and 35 min.
Fig 8.
HLB-RPA-LFA, Determination of optimum reaction temperature.
The best visibility of the test line was observed in the ranges 35°C, 37°C, 38°C and 40°C.
Fig 9.
Detection limit of HLB-RPA-LFA.
The amplified RPA products were detected on PCRD strip. The order of samples is lanes 1–7, amplified RPA products with concentrations 10 ng/μl, 1ng/μl, 0.1 ng/μl, 10 pg/μl, 1 pg/μl, 0.1 pg/μl, 0.01 pg/μl template DNA of HLB infected plant. Lane 8, NTC (non template control).
Fig 10.
Detection limit of conventional PCR using OI1/OI2c ‘Ca. L. asiaticus’ specific primers.
The amplified PCR products are resolved on 1% agarose gel. The order of samples is lane L, 1kb DNA marker; lanes 1–7, amplified PCR products with concentrations 10 ng/μl, 1 ng/μl, 0.1 ng/μl, 10 pg/μl, 1 pg/μl, 0.1 pg/μl, 0.01 pg/μl template DNA of HLB infected plant. Lane 8, NTC (non template control).
Fig 11.
Validation of HLB-RPA-LFA assay by using AmplifyRP Acceler8 Las detection kit showing two reaction lines: C is control and T is the test line.
C1 to C6 represents the ‘Ca. L. asiaticus’ isolates maintained in the screenhouse, H: Healthy control and -Ve: negative control.
Fig 12.
Validation of HLB-RPA-LFA assay by using TaqMan-qPCR with HLBas-F/R-HLBp primer probe pair.
Amplification plot for sample C1 to C6 represent the ‘Ca. L. asiaticus’ isolates showing average Ct value, 23.29, 18.78, 22.23, 22.76, 21.50 and 20.62 respectively whereas undetermined Ct observed for non template control and H: Healthy control.
Table 3.
Specificity of HLB-RPA-LFA, HLB-RPA, PCR with RPA primers and conventional PCR for the detection of ‘Ca. L. asiaticus’.