Comparative analysis of different molecular and serological methods for detection of Xylella fastidiosa in blueberry

Abstract

Bacterial leaf scorch, caused by Xylella fastidiosa , is a major threat to blueberry production in the southeastern United States. Management of this devastating disease is challenging and often requires early detection of the pathogen to reduce major loss. There are several different molecular and serological detection methods available to identify the pathogen. Knowing the efficiency and suitability of these detection techniques for application in both field and laboratory conditions is important when selecting the appropriate detection tool. Here, we compared the efficiency and the functionality of four different molecular detection techniques (PCR, real-time PCR, LAMP and AmplifyRP® Acceler8™) and one serological detection technique (DAS-ELISA). The most sensitive method was found to be real-time PCR with the detection limit of 25 fg of DNA molecules per reaction (≈9 genome copies), followed by LAMP at 250 fg per reaction (≈90 copies), AmplifyRP® Acceler8™ at 1 pg per reaction (≈350 copies), conventional PCR with nearly 1.25 pg per reaction (≈ 440 copies) and DAS-ELISA with 1x10 5 cfu/mL of Xylella fastidiosa . Validation between assays with 10 experimental samples gave consistent results beyond the variation of the detection limit. Considering robustness, portability, and cost, LAMP and AmplifyRP® Acceler8™ were not only the fastest methods but also portable to the field and didn’t require any skilled labor to carry out. Among those two, AmplifyRP® Acceler8™ was faster but more expensive and less sensitive than LAMP. On the other hand, real-time PCR was the most sensitive assay and required comparatively lesser time than C-PCR and DAS-ELISA, which were the least sensitive assays in this study, but all three assays are not portable and needed skilled labor to proceed. These findings should enable growers, agents, and diagnosticians to make informed decisions regarding the selection of an appropriate diagnostic tool for X . fastidiosa on blueberry.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

“…The amplified products could be visualized using SYBR ® Green I nucleic acid gel staining, gel electrophoresis or real-time amplification by Genie ® III (Figures 2-4) as in previous reports [36,57]. Taken together, these results suggest that LAMP is a good substitute for conventional PCR and other PCR-based methods like qPCR due to its simplicity and rapidity without the need of sophisticated instruments for on-site detection of pathogens [52,53]. In conclusion, the LAMP assay developed in this study is an efficient, reliable, and sensitive method for rapid detection of CuLCrV.…”

“…Currently, the preferred method to differentiate the identity of the virus from other begomoviruses is PCR [3,5,6,12]. However, PCR is not sensitive enough compared to other molecular methods, is laborious and time-consuming, and requires a well-equipped laboratory, which makes this assay unavailable for detecting pathogens in field conditions [52,53]. The LAMP assay described here is a more rapid, accurate, sensitive, simple, and portable diagnosis method, which can be utilized in laboratory and field conditions for timely detection of this virus.…”

“…In addition, the sensitivity of the LAMP assay was 10 times higher than PCR ( Figure 5) and could detect the virus from symptomatic and asymptomatic plants with a greater efficiency compared to PCR (Table 1, Figure 6, Supplementary Figure S4). Although compared to LAMP, qPCR was more sensitive for detection of virus infection ( Figure 5, Table 1), it is not applicable for field detection of infection as the assay requires an expensive thermocycler and expert technicians [53]. Furthermore, the CuLCrV-LAMP assay was highly specific for detection of CuLCrV, as no amplification was observed from other closely related begmoviruses like SLCV (where the pairwise nucleotide identity is 81.8% and pairwise amino acid identity is 83.6% for DNA A sequences, Supplementary Table S2) in a mixed virus assay or mixed infected field samples or in healthy squash leaf samples (Figures 3, 4 and 7).…”

Comparative evaluation of lateral flow immunoassays, LAMP, and quantitative PCR for diagnosis of fire blight in apple orchards

“…The sensitivity of LAMP and qPCR were identified to be higher than conventional d i a g n o s t i c m e t h o d s f o r A l t e r n a r i a s o l a n i a n d Phytophthora infestans detection from potato, tomato, and other related host plants (Okiro et al 2019;Khan et al 2018;Lees et al 2019). The real-time PCR has previously also been identified as the most sensitive method, followed by LAMP assay, to detect the Xylella fastidiosa pathogen from blueberry (Waliullah et al 2019). However, both LAMP and qPCR-based diagnostic methods require technical expertise to perform and interpret the test results, and also require high initial investment costs (Notomi et al 2015;Tomlinson 2013;Pirc et al 2009;López et al 2009;Stöger et al 2006).…”

Rapid detection of Pecan Root-Knot Nematode,Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

“…Loop-mediated isothermal amplification (LAMP) is a novel technique that can overcome many of the limitations of traditional microscopy and molecular PCR based diagnostic assays [17–20]. LAMP has shown that the sensitivity can be 100 to 1,000 times higher than conventional methods and can easily detect below 1pg/µl or lower concentration [21]. This method costs less time per sample and is simpler to perform than other detection methods.…”