Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil

Abstract

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10−2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10−1 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

Development and Validation of a Loop-Mediated Isothermal Amplification Diagnostic Method to Detect the Quarantine Potato Pale Cyst Nematode, Globodera pallida

“…Many other LAMP assays have been developed to detect PPN, such as Aphelenchoides besseyi [42], A. ritzemabosi [43], Anguina wevelli [44] and A. agrostis [45], Radopholus similis, directly from infected plant tissues [46], Ditylenchus destructor from complex plant/nematode DNA mixtures [47] and Tylenchulus semipenetrans in soil samples [48,49].…”

Detection of Globodera pallida directly from soil sample using mt-COI region based LAMP assay

“…Above all LAMP technique is friendly to people and the environment, the detection process does not require the use of Ethidium bromide and other toxic agents. Many researchers developed LAMP assays for the detection of many plant parasitic nematodes such as tropical root-knot nematodes (Meloidogyne incognita and M. enterolobii) (Niu et al, 2011;Niu et al 2012), burrowing nematode (Radopholus similis) (Peng et al 2012), pine wood or pine wilt nematode (Bursaphelenchus xylophilu) (Kikuchi et al, 2009;Kang et al 2015;Meng et al 2018), citrus root nematode (Tylenchulus semipenetrans) (Lin et al 2016;Song et al 2017), red ring nematode B. cocophilus (Ide et al 2017), temperate root-knot nematode M. hapla (Peng et al 2017), apple root-knot nematode M. mali (Zhou et al 2017), grass nematode (Anguina wevelli) (Yu et al 2018), M. chitwoodi, andM. fallax (Zhang andGleason 2019).…”

“…In view of these advantages, this technology has been used commercially in a variety of pathogens detection kits (Mori and Notomi 2009). In the eld of plant nematology, LAMP assays have been developed for the detection of parasitic worms of pine wood Bursaphelenchus xylophilus (Kikuchi et al 2009;Meng et al 2018), coconut red ring worm B. cocophilus (Ide et al 2017), burrowing worm Radopholus similis (Peng et al 2012), citrus root worm Tylenchulus semipenetrans (Song et al 2017), grass pararsitic worm Anguina wevelli (Yu et al 2018) and tropical root-knot worm Meloidogyne incognita, M. enterolobii (Niu et al 2011;Niu et al 2012) as well as the temperate root-knot worm M. hapla (Peng et al 2017), and apple root-knot worm M. mali (Zhou et al 2017). Quick and speci c detection of G. pallida in the soil is imperative for development of management strategy against the particular species.…”

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection and Analysis of the Root-Knot Nematode Meloidogyne hapla in Soil

“…DNA used for testing the specificity of the HSP primers and generating standard curves for real-time LAMP (rt-LAMP) was prepared according to the reported method [30]. Meloidogyne hapla juveniles were collected in 0.2 mL sterile PCR tubes containing 5 µL of MQ water, 4 µL of 10× LAMP buffer (NEB, Ipswich, MA, USA), and 1 µL of proteinase K (1 mg/mL) (Thermo Scientific, Vilnius, Lithuania).…”