The study began with symptomatic bell pepper plants grown under protected cultivation at the Centre for Protected Cultivation Technology of the Indian Agricultural Research Institute in New Delhi. Affected plants showed upward and downward leaf curling, mosaic patterns, thickened veins, chlorosis and stunted growth. Leaf-dip transmission electron microscopy revealed geminate particles about 31.13 nanometres long in symptomatic tissue, a morphology consistent with begomoviruses, while asymptomatic controls showed no comparable particles. Rolling circle amplification, used in the study to enrich circular viral DNA rather than to identify viruses diagnostically, produced products that were digested with five restriction enzymes. BamHI, KpnI and SalI linearized an approximately 2.7-kilobase component, while HindIII and EcoRI released fragments of about 2.7 and 1.3 kilobases, consistent with a begomovirus DNA-A genome and an associated betasatellite.

Sequence analysis of those components found that the DNA-A shared 100% nucleotide identity with a pepper leaf curl virus isolate generated from the same symptomatic sample, and the betasatellite shared 99.8% identity with tomato leaf curl betasatellite isolate B1. On the basis of the current International Committee on Taxonomy of Viruses criterion of about 91% genome-wide identity, the authors placed the DNA-A within the pepper leaf curl virus species. They nevertheless designed the diagnostic assay to detect the broader begomovirus complex associated with chilli leaf curl disease, rather than distinguish individual species. Two primer sets were selected from conserved sequences of the AV1 gene, which encodes the coat protein. The larger pair, ChiLCD-F1 and ChiLCD-R1, generated a 234-base-pair product and was carried forward for subsequent tests.

Both primer sets amplified the expected products in symptomatic samples by conventional PCR and RPA, while asymptomatic controls produced no bands. In silico searches found broad matches among representative begomoviruses infecting peppers, tomatoes, cotton, papaya and other crops. The researchers also compared four crude-extraction buffers: sodium hydroxide–EDTA, General Extraction Buffer 3, phosphate buffer and AP1 lysis buffer. Only the sodium hydroxide–EDTA preparation, mixed at a 1:1 ratio and adjusted to pH 8.0, consistently produced a clear 234-base-pair product in RPA. The other buffers gave variable amplification, leading the team to use 1 microlitre of the sodium hydroxide–EDTA crude extract directly in subsequent reactions without a purification step.

Temperature and duration tests were conducted with purified DNA and crude sap from one infected plant source. Expected amplification occurred from 30 to 40 °C, with the strongest signal at 35 °C; at 45 °C, purified DNA failed to amplify and crude sap produced only a faint band. At 35 °C, product was detectable after five minutes, became stronger at 10 minutes and was strongest around 20 to 25 minutes. Signals were also seen at 15 minutes, but no clear increase occurred after 25 minutes through 40 minutes, while negative controls remained free of amplification. These optimization experiments relied on a single biological source, so the selected conditions have not yet been shown to capture the full variation associated with different plants, cultivars, extract preparations or reagent batches.

The specificity panel included cucumber mosaic virus, pepper mild mottle virus and chilli veinal mottle virus, three non-begomoviral viruses associated with Capsicum or related solanaceous crops. No RPA product was detected from those templates, healthy bell pepper DNA or sap, or non-template controls, whereas the positive control repeatedly yielded the expected fragment. The same primers amplified cotton leaf curl virus, okra enation leaf curl virus, mungbean yellow mosaic India virus and tomato leaf curl New Delhi virus–bitter gourd strain from purified DNA by PCR and from crude sap by RPA. These results support cross-species detection of the four tested begomoviruses, but the researchers did not evaluate mixed infections, a broad set of additional begomovirus isolates or every non-target virus likely to occur in Capsicum.

In dilution experiments, RPA and PCR both amplified purified pepper DNA through a 10−6 dilution under the conditions tested. RPA also detected the target through a 10−6 dilution of infected-leaf crude sap, while conventional PCR did not amplify when crude sap was used directly. The authors stress that this endpoint is not a formal limit of detection: the series came from a single infected plant, replicate detection frequencies were not established across independent experiments, and no probability-based value such as LOD95 could be calculated. A separate SYBR Green qPCR standard curve was generated with cloned plasmid DNA rather than plant-derived dilutions, so it was not used to assign copy numbers to the infected-leaf series. The comparison instead shows a comparable dilution endpoint for purified DNA and better performance of RPA than PCR in the tested crude-sap matrix.

For field validation, the researchers collected 36 independent leaf samples from three Indian Capsicum-growing regions: the CPCT site in New Delhi, Pataudi in Haryana and Dehradun in Uttarakhand. The collection covered multiple commercially cultivated varieties and included 29 symptomatic and seven asymptomatic samples. Ten symptomatic samples from New Delhi were positive by both crude-sap RPA and PCR using extracted DNA. The other 19 symptomatic samples, all from Pataudi and Dehradun, and all seven asymptomatic samples were negative in both assays. Cohen’s kappa was 1.0, McNemar’s exact test gave p = 1.0, and PCR-based calculations yielded 100% sensitivity and 100% specificity, with 95% confidence intervals of 69.2 to 100% and 86.8 to 100%, respectively. The wide intervals and modest sample size show why the findings should not be treated as definitive estimates of performance across all field populations.

Hydroxynaphthol blue was then added to a 25-microlitre RPA format to replace agarose-gel analysis with direct visual inspection. Among 15 field-collected samples from the New Delhi protected-cultivation site, ten positive reactions changed from violet to sky blue and five remained violet, fully matching gel-based RPA outcomes. Healthy and buffer controls stayed violet, while the positive control became blue. The colorimetric method was performed under controlled laboratory conditions with field-collected leaves rather than during an operational field trial. It still required extract preparation, centrifugation, pipetting and controlled incubation, and the study did not assess extract stability, operator performance, blinded interpretation, throughput, contamination controls, reagent transport or total sample-to-result time.

A SYBR Green qPCR assay targeting the same AV1 fragment served as a laboratory reference and quantitative complement. A plasmid-derived standard curve spanning 100 to 0.000001 nanograms per microlitre showed a linear relationship with R² of 0.9882, a slope of −3.2893 and an estimated amplification efficiency of about 101.37%. Single sharp melt peaks supported a specific amplification product. When applied to purified DNA from 15 New Delhi samples, qPCR produced Ct values from 15.09 to 30.31 and estimated target copy numbers of approximately 1.45 × 10⁴ to 6.14 × 10⁸. Late signals in samples negative by RPA and PCR were not treated as confirmatory infections because SYBR Green can report non-specific amplification. The study did not establish an independent qPCR detection or quantification limit, evaluate matrix inhibition or recovery, verify plasmid concentration by an independent method, or relate viral copy number to systematically scored disease severity.

Estimated reagent costs further favored the simplified format: 263.40 Indian rupees per sample for colorimetric RPA, compared with 363 rupees for RPA, 370.61 rupees for qPCR and 450.14 rupees for PCR. These calculations excluded equipment, personnel, infrastructure and other operating expenses, so they do not establish the assay’s total cost-effectiveness. The work demonstrates a technically promising combination of direct crude-sap preparation, rapid isothermal amplification and naked-eye readout, with performance fully concordant with PCR in the tested sample set. Its practical value now depends on broader validation across independent operators, locations, cultivars, viral isolates and environmental conditions, along with an internal amplification control, reproducibility studies, mixed-infection testing and direct evaluation under field-deployment conditions.

Subject of Research: Development of a crude-sap colorimetric RPA assay for detecting begomoviruses associated with chilli leaf curl disease in Capsicum.

Article Title: Colorimetric RPA and SYBR green qPCR enable rapid detection of begomoviruses in Capsicum using crude plant sap

Article References: Kumar, R., Kumari, S., Rawat, A., Diksha, D., Srivastava, N., Singhal, P., Bhardwaj, P., Bharti, H., Baranwal, V. K., Sharma, S. K., & Gupta, N. (2026). Colorimetric RPA and SYBR green qPCR enable rapid detection of begomoviruses in Capsicum using crude plant sap. Current Research in Biotechnology, Article 100430. https://doi.org/10.1016/j.crbiot.2026.100430

Image Credits: AI Generated

DOI: 10.1016/j.crbiot.2026.100430

Keywords: begomovirus detection, Capsicum annuum, chilli leaf curl disease, recombinase polymerase amplification, crude plant sap, colorimetric diagnostics, hydroxynaphthol blue, SYBR Green qPCR, plant virus surveillance, Colorimetric, SYBR, green