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

The northern root-knot nematode (Meloidogyne hapla) is a damaging nematode that has caused serious economic losses worldwide. In the present study, a sensitive, simple and rapid method was developed for detection of M. hapla in infested plant roots by combining a Flinders Technology Associates (FTA) card with loop-mediated isothermal amplification (LAMP). The specific primers of LAMP were designed based on the distinction of internal transcribed spacer (ITS) sequences between M. hapla and other Meloidogyne spp. The LAMP assay can detect nematode genomic DNA at concentrations low to 1/200 000, which is 100 times more sensitive than conventional PCR. The LAMP was able to highly specifically distinguish M. hapla from other closely related nematode species. Furthermore, the advantages of the FTA-LAMP assay to detect M. hapla were demonstrated by assaying infected root galls that were artificially inoculated. In addition, M. hapla was successfully detected from six of forty-two field samples using FTA-LAMP technology. This study was the first to provide a simple diagnostic assay for M. hapla using the LAMP assay combined with FTA technology. In conclusion, the new FTA-LAMP assay has the potential for diagnosing infestation in the field and managing the pathogen M. hapla.

The loop-mediated isothermal amplification (LAMP) assay, originally developed by Notomi et al. 19 , is a simple and rapid method that allows DNA amplification under isothermal conditions. The technique can amplify DNA with high specificity and sensitivity under isothermal conditions within 1 hour based on the auto-cycling strand displacement DNA synthesis by Bst DNA polymerase 19 . LAMP utilizes four specific primers that were designed by six different regions of the target gene. The LAMP assay does not require much technical skill but a thiermal cycler. Additionally, the LAMP product can be seen with the naked eye by adding SYBR Green I, according to the colour change and the lateral flow dipstick (LFD). Because it is high efficient and does not require special laboratory facilities, this assay has recently shown promising in the specific and rapid detection of clinical 20,21 and agricultural pathogens [22][23][24] . However, in plant parasitic nematodes, only Bursaphelenchus xylophilus 25 , M. enterolobii 9 , M. incognita 26 Radopholus similis 27 and Tylenchulus semipenetrans 28 have been detected by LAMP. In this study, we established a sensitive and specific LAMP method for the direct detection of M. hapla from infected plant root galls based on the rDNA-ITS.

Results
Primer design and reaction optimization. The specific primers were designed for LAMP based on the sequence dissimilarity among M. hapla and other closely related Meloidogyne species at the primer positions, and the primer sets with high efficiency and low false positive rate were selected ( Fig. 1 and Supplemental Table 1). The LAMP reaction was optimized under the conditions of 5.0 mmol•L −1 Mg 2+ and 2.4 mmol•L −1 dNTPs, without betaine at 65 °C for 45 min.
Detection and confirmation of LAMP products. The LAMP products were detected by adding SYBR Green I fluorescence dye. After amplifications followed by adding SYBR Green I fluorescence dye, the tubes containing M. hapla samples produced positive reactions that the solution appeared green, while the solution remained orange in the negative reactions ( Fig. 2A). The LAMP products were separated using two percent agarose gel electrophoresis, and the bands presented ladder-shaped characteristic (Fig. 2B). To eliminate the false positive interference, the LAMP reaction products were evaluated by lateral flow strips (LFD): the sample with positive amplification showed both control and test lines, whereas the negative control only displayed the control line (Fig. 2C).
Specificity of LAMP assay. Specificity of the LAMP was evaluated using 9 Meloidogyne species and 3 other plant nematodes ( Table 1). The positive colour reactions were obtained with the DNA template from M. hapla, but were not observed in other nematode species (Fig. 3A). The LAMP amplifications were tested by LFD strips (Fig. 3B). The results indicated that the LAMP assay could distinguish M. hapla from closely related Meloidogyne species and other plant nematodes. The results were confirmed by an M. hapla-specific primer set, one band at 960 bp was detected in the four isolates of M. hapla (Fig. 3C).

Sensitivity comparison of LAMP with conventional PCR.
A series of 10-fold dilutions of M. hapla DNA extracted from a single adult female were used to determine the sensitivity of the LAMP assay. Positive results were observed when at least 1 μ l of the lysate was in the reaction mixture (that is, 5 × 10 −5 of an adult female in the reaction mixture) (Fig. 4A,B). In a comparative analysis of the LAMP and conventional PCR assays, the LAMP assay was 100-fold higher sensitivity than the conventional PCR, which had a detection limit of 5 × 10 −3 of single adult female lysates (Fig. 4C). No amplification was observed in the no-template control. LAMP analysis combined with FTA Technology. To prove the applicability of the FTA-LAMP assay for direct M. hapla detection from plant root galls, two different methods were compared using the DNA extracted from a single root gall. The FTA-based assay presented similar results to the conventional DNA isolation method (Fig. 5), indicating that the FTA technology has the potential to be used for detection of plant nematodes combine with the LAMP assay.  (Table 2). Furthermore, positive results were presented in 6 of the 42 field samples using FTA-LAMP with detection rates of 100% ( Fig. 6 and Supplemental Table 2). The results were confirmed by rDNA-ITS and morphological observation. In the negative samples, although there were other Meloidogyne species, no M. hapla specimens were detected.

Discussion
M. hapla has a wide range of hosts, and can be distributed in various climate areas 29 . However, a rapid and precise diagnosis method is urgently required. Molecular techniques can help species identification, but all current techniques have limitations. Traditional M. hapla detection assays based on PCR methods [11][12][13]15,30 are time-consuming and require sophisticated equipments as well as expertise. LAMP is a novel, simple, rapid and precise amplification method that can be adapted for diagnosing plant-parasitic nematodes, as previously reported 9,[25][26][27][28] . In this study, we designed a set of five specific LAMP primers to detect M. hapla. The reaction can be completed in a water bath, and the amplified products can be detected visually by the naked eye rather than expensive instruments within 1 hour. Niu et al. 26  M. incognita-specific LAMP set (Mi-LAMP), however, the M. hapla-specific LAMP assay was not recorded. In our study, the specific LAMP assay for detection of M. hapla was developed.
Several specific primers of M. hapla have been developed based on the ribosomal intergenic spacer (rDNA) 10,15,16 and RAPD patterns 12 . Zijlstra et al. 14 cloned and sequenced the ITS regions and found sufficient variability in those regions to separate M. hapla from M. chitwoodi. Then, the PCR primers were designed to separate M. chitwoodi, M. fallax, M. hapla, and M. incognita based on the ITS sequences 11 . In this study, we designed a set of five specific LAMP primers for M. hapla based on the ITS regions. LAMP based on these five primers is more specific compared to the conventional PCR pair-primers. Furthermore, the LFD strips were a great improvement to specificity and effectively avoided presenting false positives in the reactions. Additionally, the positive results were only present in four populations of M. hapla but did not appear in the other eight closely related Meloidogyne species, the test showed high specificity, but we tested a limited number of nematodes populations and did not assess other related species such as M. chitwoodi and M. fallax. Even the primers used in this study were selected in the ITS regions where there exist differences between M. hapla and other Meloidogyne species ( Fig. 1) and had high specificity to avoid the risk of misdetection. More isolates of M. hapla and other related species should be examined using the LAMP method in the future.
Previous results found that the sensitivity of the LAMP assay was higher than conventional PCR 9,26-28 . In the present study, the detection limit was low to 1/200 000 nematode DNA, whereas the conventional PCR approach usually requires a single nematode, Williamson et al. 11 designed the RAPD primers and Zijlstra et al. 12   the sensitivity was 1/2000 nematode DNA (Fig. 4). Therefore, the detection limit of the LAMP was 100 times higher than that of PCR-based detection methods. The sensitivity of the LAMP was equal to B. xylophilus 25 , M. enterolobii 9 , M. incognita 26 , R. similis 27 and T. semipenetrans 28 .
In a previous study, isolating nematodes from a Baermann funnel or direct picking nematodes from plant root galls required more time and specialized technique. The direct detection of plant nematodes using DNA from infected plant tissues has been published 27,31-34 , although those methods for preparing nucleic acids require toxic reagents and expansive instruments. In this study, direct extraction of DNA from plant root galls greatly improved the efficiency when using the FTA technology. This assay rarely reported in molecular plant nematology was not time-consuming and is using a nontoxic reagent. Marek et al. 35 developed an FTA-based technology for the collection, long-term archiving and molecular analysis of three species of nematodes, including Ditylenchus dipsaci, Heterodera schachtii and M. hapla that involved PCR amplification. Our study demonstrated that the LAMP assay combined with FTA technology was fully applicable to plant nematode detection as well as the established conventional methods (Fig. 5). To the best of our knowledge, this is the first evidence of detecting plant nematodes using the LAMP assay combined with FTA technology, and the total detection time was shortened to one hour.
To evaluate the practical application of the FTA-LAMP for analyzing infected root galls, this method was tested on a collection from both artificially inoculated samples and field root galls. For artificially inoculated samples, 19 of the 20 (95%) M. hapla-induced root galls were successfully amplified. In contrast, only 16 of the 20 (80%) samples were successfully detected by PCR assay. Subsequently, the validity of the FTA-LAMP assay for M. hapla detection was also performed in 42 field samples (Fig. 6). All of the root galls infested with M. hapla were successfully detected by the FTA-LAMP assay with a detection rate of 100%, which indicated the high potential of this method. Combined with similar observations from previous reports 9, 25 , these results showed that this assay has great stability and sensitivity, and can overcome interference from various types of PCR inhibitors, such as humic acid, proteins and non-target DNA. Furthermore, the FTA-captured nematode DNA could be stored at room temperature for many years 35 , which means that the field samples could be collected and that DNA purification could be accomplished using the FTA protocol; then, those samples could be tested with the LAMP assay, which it is simple, rapid and reliable.
In conclusion, the present study developed a FTA-LAMP assay for M. halpa detection. This novel FTA-LAMP assay could easily be used as a more sensitive, specific, and practical method for directly detecting M. halpa in infected plant tissues compared to previous methods. It will be potentially useful for monitoring and managing of M. halpa in the field.

Materials and Methods
Biological materials. Nine Meloidogyne species and three other plant nematode species used in this study are listed in Table 1. M. mali and M. camelliae were intercepted by China entry-exit Inspection and Quarantine in imported plant material, M. graminicola were collected from Myanmar and China. All Meloidogyne species except M. graminicola were purified from single egg-mass and reared on the susceptible tomato cv. Jiafen No. 9. The M. graminicola was cultured on the Rice cv. Nipponbare. Non-Meloidogyne genus including Pratylenchus coffeae, D. destructor and H. glycines were collected from China and used to verify the specificity test. All populations had been identified and previously diagnosed by morphological characteristics, rDNA-internal transcribed spacer (ITS) and species-specific primers 11,12 . Detailed protocols were described as below.
DNA extraction. The genomic DNA of nematode was extracted by two different methods, and a portion of the samples was completed as described in Ou et al. 36 . Another group of samples and root galls was isolated by an FTA card (Whatman, GE Healthcare, USA) using a process that as previously described 35 , the root gall was placed  on the FTA card and crushed with a pestle, a 2 mm diameter piece of the FTA card containing biological material was manually isolated and transferred into a tube, the disk was washed with FTA purification solution (Whatman, GE Healthcare, USA) and Tris-EDTA (TE) buffer (10 mM Tris and 0.5 mM EDTA [pH 8.0]) and air dried, the disk was directly used as a template for LAMP amplification.
PCR amplification. Primers for rDNA-ITS amplification were rDNA1 and rDNA2 37 , a M. hapla specific SCAR primer 11,12 were used to evaluate the specificity of the LAMP assays (Supplementary Table 1   Analysis of LAMP products. The LAMP amplification results were detected with three methods: adding the fluorescent dye SYBR green I (Invitrogen, 1:1000 TE buffer) to the reaction mixture and visually inspecting the results with the naked eye or under UV light; a Lateral-flow dipstick (LFD) assay that as visually observed with naked eyes, and 2% agarose gel electrophoresis.

Lateral-flow dipstick (LFD) assay.
In the LAMP-LFD assay, the 5′ biotin-labeled inner primer FIP was used. A DNA probe labelled by FITC at the 5′ end was designed from the sequence between the F3 and B3 regions, and the other primers were designed using the same procedure as previously described (Supplementary Table 1). After the LAMP reaction, 4 μ l of the FITC-labeled probe (20 pmol/μ L) were added into the LAMP reaction solution and incubated at 63 °C for 5 min to hybridize. Then, 8 μ l hybridized product was transferred into 100 μ l assay buffer in the reaction well. The LFD Strip (Milenia Biotec, Germany) was dipped into the mixer for approximately 5 min to detect the amplicon-probe hybrid; the positive result showed a test line and a control line, whereas the negative control only had a control line.

Specificity and sensitivity comparison of LAMP to conventional PCR.
To detect specificity of the LAMP assay, genomic DNA isolated from several Meloidogyne spp. and other plant nematode species was compared (Table 2). Meanwhile, a set of M. hapla-specific PCR primers Mh-0F and Mh-1F were used to verify the accuracy of the LAMP assay 12 . Specificity tests were repeated three times.
To determine the LAMP sensitivity, a series of 10-fold dilution of single female M. hapla genomic DNA (10 0 , 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 and 10 −6 ) was prepared and used for both the LAMP and traditional PCR assays. SYBR Green I dye and electrophoresis were used to detect the LAMP products. Sensitivity tests were repeated three times.  39 . The rice Oryza sative L cv. Nipponbare was also planted in sand soil and inoculated with M. graminicola as described by Haegeman et al. 40 . Thirty days post inoculation (dpi), the single gall was collected and used to extract genomic DNA with FTA technology as described above. The healthy roots were used as negative control. Every sample was repeated twenty times.

Field evaluation of FTA-LAMP.
To determine the practical application of the FTA-LAMP process in the field, 42 field samples, including root galls and soil in different regions across China, were collected and tested (Supplementary Table 2). DNA extraction from a single root gall by an FTA card and the LAMP reaction were performed as described above. The results were confirmed by both an rDNA-ITS assay as described by Vrain et al. 37 and the morphological identification 41 . As a comparison, the purified DNA from M. hapla and sterilized water were used as positive and negative controls, respectively. Three independent tests were performed for each sample.