Recombinase polymerase amplification assay for the detection of piper yellow mottle virus infecting black pepper

Abstract

Recombinase polymerase amplification (RPA) is a quick, specific, sensitive molecular tool carried out at a constant temperature for pathogen detection. In the present study, RPA and reverse transcription (RT) RPA assays were optimized for the detection of piper yellow mottle virus (PYMoV) infecting black pepper. Out of the eight primer pairs targeted to amplify open reading frames (ORFs) 2 and 3 of the virus, the primer pair targeted to ORF2 gave specific amplification only with DNA isolated from infected plant but not with healthy plant. A magnesium acetate concentration of 18 mM, 40 min of incubation time and a temperature of 37-42 °C was found optimum for detection of the virus in RPA assay. Comparison of sensitivity of detection revealed that RPA could detect the virus up to 10^-5 dilution of the total DNA while PCR could detect the virus up to 10^-4 dilution indicating that RPA is 10 times more sensitive than PCR. RPA was further simplified using crude extract as template which could detect the virus up to 10^-3 dilution. RT-RPA was optimized for the detection of PYMoV using total RNA isolated from infected plants as the template. Both RT-RPA and RPA assays were validated using field samples of black pepper representing different varieties and geographical regions by using CTAB isolated DNA, crude DNA extract and cDNA. Our study showed that RPA and RT-RPA can be successfully adopted as a substitute to PCR for detection of PYMoV infecting black pepper.

Keywords: Diagnosis; Isothermal amplification; Polymerase chain reaction; Reverse transcription-RPA; Sensitivity.

Fig. 1

Optimization of recombinase polymerase amplification (RPA) assay for the detection of piper yellow mottle virus (PYMoV). a Identification of suitable primer pair. Lane M: 100 bp DNA ladder, Lane 1–8: Loaded with RPA product obtained with primers AIB 452–453, AIB 453–454, AIB 454–455, AIB 456–455, AIB 452–455, AIB 483–484, AIB 485–486, AIB 487–488. Details of the primers and expected product size are provided in supplementary Table 1. b determination of optimum concentration of magnesium acetate, c incubation time and d temperature. Lane HC: Healthy control

Fig. 2

a Detection of PYMoV through optimized RPA assay using CTAB isolated DNA as template. Lane M: 100 bp DNA ladder; Lane 1: DNA from infected plant; Lane 2: DNA from healthy plant; Lane 3: Water control. b Detection of PYMoV through optimized RPA assay using crude DNA extract. Lane 1: crude DNA extract from infected plant; Lane 2: 1:10 diluted DNA crude extract from infected plant; Lane 3: crude DNA extract from healthy plant; Lane 4: 1:10 diluted crude DNA extract from healthy plant; Lane 5: Water control. c Detection of PYMoV through reverse transcription (RT)-RPA assay using cDNA as template. Lane 1: cDNA from infected plant; Lane 2: cDNA from healthy plant; Lane 3: Water control

Fig. 3

Comparison of sensitivity of detection of PYMoV by RPA (a, c, e) and PCR (b, d, f) assays. Lane 2–10 loaded with RPA/PCR products obtained with template DNA dilutions from 10⁰–10⁻⁸ using template DNA isolated by CTAB (a, b), crude DNA extract (c, d) and cDNA (e, f). Lane M in a, c, d, e are loaded with 100 bp DNA ladder while lane M in b and f are loaded with 1 kb DNA ladder

Fig. 4

Validation of RPA (a, c, e) and PCR (b, d, f) assays for the detection of PYMoV using template DNA isolated by CTAB (a, b), crude extract (c, d) and cDNA (e, f) respectively. Lane M in a, c, d, e, f are loaded with 100 bp DNA ladder while lane M in b is loaded with kb DNA ladder; Lane N is DNA from known healthy plant while rest of the lanes are loaded with DNA/cDNA from test plants