Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for rapid and sensitive detection of bean common mosaic virus

Abstract

Bean common mosaic virus (BCMV) is one of the most widespread and damaging viruses of cultivated legumes in the world. In addition to serious yield reduction and germplasm decline, BCMV infection also makes legumes more vulnerable to other pathogens. Early diagnosis of the virus is particularly important in limiting its spread. Recombinase polymerase amplification (RPA) is a novel isothermic amplification technology. The whole reaction can be done outside the laboratory environment after the nucleic acid sample is obtained. In this study, we established a rapid and sensitive RPA combined with the lateral flow dipstick (LFD) assay for detection of BCMV, based on the conserved BCMV coat protein (CP) gene sequence. Specific primers and a probe were designed, which amplify ~ 150 bp CP fragments from BCMV-infected samples under a constant temperature of 37 °C for 20 min. The end-labeled amplification products were detected by high-affinity LFD within 5 min. Sensitivity of this RPA-LFD assay was 1000 times greater than that of the conventional polymerase chain reaction (PCR) assay. Furthermore, when the primers/probe were used against related potyviruses including soybean mosaic virus (SMV), bean yellow mosaic virus (BYMV), and turnip mosaic virus (TuMV), the three potyviruses were not detected, indicating that the assay was BCMV species-specific. The RPA-LFD assay was also successfully applied for the detection of seed-borne BCMV in beans. The RPA-LFD assay has great potential application in the rapid diagnosis of BCMV in the field.

Recombinase polymerase amplification combined with lateral flow dipstick assay for rapid visual detection of A.simplex (s. s.) and A.pegreffii in sea foods

“…This difference may have resulted from the primer specificity under room temperature being lower than that of PCR, resulting in non-specific amplification and primer dimer. This disadvantage can be made up by optimizing the conditions for RPA to minimize false-positive results, as suggested in other reports [ 41 , 43 ]. Furthermore, the quality of DNA extracted methods may also affect the results of the RPA-LFD assay, and the high quality and concentration of pure DNA can improve its sensitivity and efficiency.…”

“…The RPA reaction system is typically sufficient for 10–20 min detection at 37 °C. RPA has been widely used for detecting bacteria [ 36 , 37 ], fungi [ 38 , 39 ], viruses [ 40 , 41 ], and parasites [ 42 , 43 ], with the aid of fluorescently labeled probes or the lateral flow dipstick (LFD) for visual analysis of the amplified products. Additionally, the RPA reaction system has advantages in identifying specific genes, e.g., those linked with drug-resistant genes, transfected genes, and novel coronaviruses-specific genes [ 7 , 35 , 44 ].…”

Genome-wide analysis of key gene families in RNA silencing and their responses to biotic and drought stresses in adzuki bean

“…Powdery mildew is a common fungal disease threatening the cultivation of adzuki bean in China especially under high humidity or high temperature [ 9 ]. Bean common mosaic virus (BCMV) is one of the most damaging viruses infecting legume plants with worldwide distribution and has been reported in adzuki bean in China with serious symptoms [ 10 , 11 ]. For abiotic stress, drought is one of the most devastating environmental stresses that restricts the root and shoot growth and the final productivity [ 12 ].…”