Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

Peng, Huan; Long, Haoyu; Huang, Wenkun; Liu, Jing; Cui, Jiangkuan; Kong, Lingan; Hu, Xiaoge; Gu, Jianfeng; Peng, Deliang

doi:10.1038/srep44853

Cited by 36 publications

(28 citation statements)

References 30 publications

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“…Traditionally M. partityla was diagnosed based on morphological observation, and PCR‐based molecular methods which is a time‐consuming process and requires highly traned personnel and laboratory instruments [26, 27]. The LAMP assay is an effective tool for plant-parasitic nematode identification because of its capability of DNA amplification at isothermal conditions with high sensitivity and efficiency [18, 22, 24]. In this report, we present a novel method to detect the pecan RKN, M. partityla .…”

Section: Discussionmentioning

confidence: 99%

“…[22, 24, 25]. Some of these LAMP sets are very specific to a certain RKN species, and in some cases, a single LAMP primer set can also be used for detecting double or multiple RKNs simultaneously [24, 25]. In 2019, Zhang et al developed a LAMP primer set which can detect two potato nematodes, M. chitwoodi and M. fallax .…”

Section: Discussionmentioning

confidence: 99%

“…It can be carried out rapidly (often in 30 min) with minimal equipment (a water bath or isothermal heat block) which is highly applicable for the onsite diagnosis of pathogens [22, 23]. Recently LAMP technique has been used for identifying different species of RKNs including M. enterolobii , M. incognita , M. arenaria , M. javanica , M. hapla , M. chitwoodi and M. fallax [22, 24, 25]. However, so far there is no report for using LAMP technique for the identification of M. partityla .…”

Section: Introductionmentioning

confidence: 99%

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

Waliullah

Bell

Stackhouse

et al. 2020

Preprint

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2Meloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in 3 Georgia. This species is known to cause a reduction in root growth and a decline in yields from 4 mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode 5 disease and reduce losses in pecan production. In this study, a loop-mediated isothermal 6 amplification (LAMP) method was developed for simple, rapid and on-site detection of M. 7 partityla in infested plant roots and validated to detect the nematode in laboratory and field 8 conditions. Specific primers were designed based on the sequence distinction of internal 9 transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other 10 Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla 11 genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from 12 other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We 13 also conducted a traditional morphology-based diagnostic assay and conventional polymerase 14 chain reaction (PCR) assay to determine which of these techniques was less time consuming, more 15 sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, 16 amplifying the target nematode species in less than 60 min at 65°C, with results 100 times more 17 sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly 18 time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These 19 features greatly simplified the operating procedure and made the assay a powerful tool for rapid, 20 on-site detection of pecan RKN, M. partityla. The LAMP assay will facilitate accurate pecan 21 nematode diagnosis in the field and contribute to the management of the pathogen. 3 1 diagnosis 3 Introduction 4 Pecan (Carya illinoinensis) is an important nut crop in North America. A variety of diseases caused 5 by bacteria, fungi, virus, and nematode can attack these trees, and if not properly managed, they 6 can cause economic damage to pecans. Root-knot nematodes (RKNs) of the genus Meloidogyne 7 are economically important plant-parasitic nematodes, which cause significant damage to pecan 8 production. Three species of RKNs, Meloidogyne incognita, M. arenaria and M. partityla have, 9 have been reported as pathogenic to pecan [1-3]. Among these species M. partityla is the dominant 10 RKN parasitizing pecan which has a greater incidence in southern United States [2-6]. M. partityla 11 was originally found infecting pecan in South Africa, and was likely introduced into the United 12 States during importation of infected pecan seedlings [7]. Due to the complexity and lengthy 13 process of diagnosis and species identification, it is difficult to conduct extensive surveys, which 14are required for determining the pecan RKN distribution. Therefore, a quick diagnosis approach 15 will be helpful to know the latest status of the pecan RKNs in s...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Waliullah

Bell

Stackhouse

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Several authors also developed a TaqMan real-time PCR assay with species-specific primers for the detection of M. hapla from root galls and soil samples [ 8 , 9 , 10 , 11 , 12 ]. Recently, Peng et al [ 13 ] developed loop-mediated isothermal amplification methods (LAMP) combined with a Flinders Technology Associates card for the identification of M. hapla .…”

Section: Introductionmentioning

confidence: 99%

Sensitive, Accurate and Rapid Detection of the Northern Root-Knot Nematode, Meloidogyne hapla, Using Recombinase Polymerase Amplification Assays

Subbotin

Burbridge

2021

Plants

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Rapid and reliable diagnostics of root-knot nematodes are critical for selections of effective control against these agricultural pests. In this study, recombinase polymerase amplification (RPA) assays were developed targeting the IGS rRNA gene of the northern root-knot nematode, Meloidogyne hapla. The RPA assays using TwistAmp® Basic, TwistAmp® exo and TwistAmp® nfo kits (TwistDx, Cambridge, UK) allowed for the detection of M. hapla from crude extracts of females, eggs and juveniles without a DNA extraction step. The results of the RPA assays using real-time fluorescence detection (real-time RPA) in series of crude nematode extracts showed reliable detection after 13 min with a sensitivity of 1/100 of a second-stage juvenile and up to 1/1000 of a female in reaction tubes. The results of the RPA assays using lateral flow dipsticks (LF-RPA) showed reliable detection within 30 min with a sensitivity of 1/10 of a second-stage juvenile and 1/1000 of a female in reaction tubes. The RPA assay developed here is a successful tool for quick, accurate and sensitive diagnostics of M. hapla. The application of the LF-RPA assay has great potential for diagnosing infestation of this species in the lab, field or in areas with a minimal laboratory infrastructure.

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“…These characteristics fit in well with the epidemiological investigation and on-site detection especially when experimental settings are limited (Niu et al, 2018; Hirayama et al, 2006). Until now, LAMP assay has been regarded as an excellent diagnosis approach in detecting parasites, including Schistosoma japonicum (Xu et al, 2010), Meloidogyne (Peng et al, 2017), Trichinella spiralis (Li et al, 2012), Plasmodium falciparum (Kersting et al, 2014), Giardia (Crannell et al, 2015), Leishmania donovani (Mondal et al, 2016) and so on. The application of LAMP assay in diagnosing parasitic diseases have also been reported in recent years, such as toxoplasmosis (Sotiriadou & Karanis, 2008), angiostrongylosis (Liu et al, 2011), and cryptosporidiosis (Crannell et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Sensitive and rapid detection of Ortleppascaris sinensis (Nematoda: Ascaridoidea) by loop-mediated isothermal amplification

Zhao

et al. 2019

PeerJ

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Ortleppascaris sinensis is the dominant nematode species infecting the gastrointestinal tract of the captive Chinese alligator, a critically endangered species. Gastrointestinal nematode infection may cause a loss of appetite, growth, a development disorder, and even mortality in alligators, especially young ones. This research first establishment a loop-mediated isothermal amplification (LAMP) assay in rapidly identifying O. sinensis, upon the basis of the complete internal transcribed spacers (ITS) gene. Eight sets of primers were designed for recognition of the unique conserved ITS gene sequences, and one set was selected to be the most suitable primer for rapid detection. The specific as well as the sensitive features of the most appropriate primer in LAMP reactions for O. sinensis, and feces specimens of Chinese alligators suffering from O. sinensis were determined. Turbidity monitoring and Te Visual Reagent methods were used for determining negative and positive consequences. According to this study, amplification and visualization of the target DNA could be realized through two detection approaches during 50 min at 65 °C isothermal temperature. The sensitivity of LAMP was a detecting limitation of 3.46 pg/µl DNA. No cross-reactions were found between O. sinensis and any other of the nine heterologous nematode parasites, which shows the outstanding specific features of the primers. The LAMP assay could also perform a detection of target DNA of O. sinensis in the feces samples of Chinese alligators. This LAMP assay is useful for directly detecting O. sinensis in the Chinese alligator breeding centers, particularly due to its rapidity, simplicity and low cost.

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Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

Cited by 36 publications

References 30 publications

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

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

Sensitive, Accurate and Rapid Detection of the Northern Root-Knot Nematode, Meloidogyne hapla, Using Recombinase Polymerase Amplification Assays

Sensitive and rapid detection of Ortleppascaris sinensis (Nematoda: Ascaridoidea) by loop-mediated isothermal amplification

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