In-house validation of two molecular tests. detecting. Globodera rostochiensis and G. pallida.

Transcription

1 In-house validation of two molecular tests detecting Globodera rostochiensis and G. pallida. 2009, Wageningen, Blgg bv. Validation performed by: Company Blgg bv. Address P.O. box 115, NL-6860 AC, Oosterbeek, The Netherlands Contact Dr. R. Landeweert / Ir. P. Veenhuizen / Ing. R. Staps Phone + 31 (0) Renske.landeweert@blgg.nl / Peter.veenhuizen@blgg.nl / Roel.staps@blgg.nl

2 Abstract In 2009, the molecular laboratory of Blgg performed an in-house validation of two molecular tests developed for detection of the potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida. Prior to test development, nematodes were collected from various habitats throughout The Netherlands or obtained from the Dutch Plant Protection Service. Nematodes were identified and sequenced. Subsequently, an alignment of 70 partial Heteroderidae LSU rdna sequences from 33 different species was used to identify species-specific sequence motives for primer development. Test validation included determination of the following performance characteristics: 1) specificity 2) selectivity and 3) robustness. Validation results show that species-specific detection with the described primer pairs is possible at the desired specificity and sensitivity. Based on the results of this in-house validation, Blgg concludes that molecular analyses is an effective method for detection of Globodera rostochiensis or Globodera pallida (cysts, egg or nematods) in a variety of samples. Blgg bv. Validation report for molecular species specific Globodera detection tests 2

3 Introduction The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida may cause major losses in potato crops. Soils that are found infested with PCN can only be used for the production of other crops or for the production of resistant potato varieties. In many countries, testing of fields for PCN is mandatory under governmental regulations before any seed potato may be planted. G. rostochiensis and G. pallida, members of the genus Globodera (Family Heteroderidae, Subfamily Heteroderinae) are morphologically closely related. The difference between the two species is confined to a minor morphological difference in the knobs at the base of the stylet and the stylet length. The most important cyst differences are in the perineal area, i.e. number of cuticular ridges between vulva-anus and Granek s ratio. In addition, there are three other Globodera species which could cause confusion during microscopic identification of potato cyst nematodes in Europe. G. achilleae and G. artemisiae are not parasitic on potato, while G. tabacum parasitizes tobacco, potato and solanaceous weeds in southern Europe. The problem with PCN detection and identification is that morphological methods are too cumbersome and error-prone and therefore, molecular methods have been proposed to distinguish between these two species. Molecular (DNA-based) identification is based on an large quantity of putative informative characters, and depending on the desired identification level order, family, genus, species or race appropriate stretches of DNA can be identified that are suitable for nematode identification at the desired taxonomic level. The PCN primers in the underlying report have been designed to hybridize to coding nucleic acid sequences (for PCN the rdna LSU sequences), since such sequences give rise to the presence of evolutionary conserved regions. Others (for instance Pylypenko et al. (Eur J Plant Pathol. (2005) 111:39-46) use the ITS (internal transcribed spacer a non-coding DNA fragment) sequence of ribosomal DNA to distinguish between Globodera rostochiensis and G. pallida by PCR. The problem with such a test is that it is based on a non-coding - hence moderate to highly variable - DNA region. Validation of such a test would require a worldwide survey to define the (substantial) within species variation. Moreover, the non-coding nature of these regions makes it is prone to change in time at a magnitude far higher than any of the coding ribosomal DNA regions. This may either result in false negative or falsepositive test results when the test is performed on soil samples in which the PCN population is uncharacterized. For instance, a test developed for potato cyst nematodes of the northern hemisphere, will not necessarily be suitable for detecting PCN in South America, where populations of nematodes have evolved entirely separately. Therefore, ITS based tests that are developed for the detection of cyst nematodes are of limited regional importance, and the use of ITS sequences implies that the test can only be used for a few years (until the moment that - in the absence of any selection pressure - a random mutation undermines the test). It would however be economically advantageous to provide a test that performs consistently and that provides valuable results in a wide variety of growing areas with equal confidence. In cooperation with the Laboratory of Nematology (Wageningen University, Wageningen, The Netherlands) Blgg bv. (Oosterbeek, The Netherlands) develops detection tests based on molecular barcoding for the detection of plant pathogenic nematodes in soil. Over the past years Blgg has shown that a detection system as outlined here can indeed be used for the high throughput detection of stem nematodes (Ditylenchus dipsaci) and root knot nematodes (Meloidogyne chitwoodi, M. fallax, M. minor, M. naasi and M. hapla) in nematode suspensions extracted from soil samples. Subsequently, detection tests were developed for the detection of PCN Globodera rostochiensis and G. pallida and cyst nematodes G. achilleae and G. artemisiae. Blgg bv. Validation report for molecular species specific Globodera detection tests 3

4 In 2009, the molecular laboratory of Blgg performed an in-house validation of two molecular tests developed for detection of Globodera rostochiensis and G. pallida. rdna sequence database The ribosomal DNA cistron is extensively used for the identification of organisms and for the establishment of phylogenetic relationships. It consists of conserved and more variable regions. This gene includes three coding regions: the Small Subunit gene (SSU, 1700 bp), the 5.8S gene (150 bp) and the Large Subunit gene (LSU, 3400 bp). In between there are two non-coding regions called internal transcribed spacer ITS-1 and ITS-2. For the identification of nematodes (in fact any organism) at any taxonomic level, a low intra-taxon variability should be combined with a relatively high inter-taxon variation. The Laboratory of Nematology, funded by the Dutch Technology Foundation STW, constructed a DNA sequence database of over 1600 nematode species collected in The Netherlands (Holterman et al., 2006). Nematodes were collected from various habitats throughout The Netherlands or obtained from the Dutch Plant Protection Service (Prof. dr. Gerrit Karssen). Nematodes were extracted from the soil using standard techniques and identified under a light microscope. Prior to DNA extraction, pictures were taken using a CCD camera (CoolSnap, RS Photometrics, Tucson AZ). LSU rdna sequences from collected species were supplemented with sequences taken from GenBank. For the systematics throughout this document we adhere to the classification of Siddiqi (2000). Clones of each species (voucher specimens) are kept at the Laboratory of Nematology (Wageningen University, Wageningen, The Netherlands). Information can be obtained from Dr. Hans Helder ( hans.helder@wur.nl). DNA extraction and rdna sequencing (as described in Holterman et al., 2008) Single nematodes were transferred to 0.2 ml PCR tube containing 25 l sterile water. An equal volume of lysis buffer containing 0.2 M NaCl, 0.2 M Tris-HCl (ph 8.0), 1% (v/v) - mercaptoethanol and 800 g/ml proteinase K was added. Lysis took place in a Thermomixer (Eppendorf, Hamburg, Germany) at 65 C and 750 r.p.m. for 2 hours followed by a 5 minute incubation at 100 C. Lysate was immediately used or stored at -20 C. SSU rdna was amplified as two partially overlapping fragments using three universal and one nematodespecific primer (1912R). For the first SSU fragment, either the primer 988F (5 - ctcaaagattaagccatgc-3 ) or the primer 1096F (5 -ggtaattctggagctaatac-3 ) was used in combination with the primer 1912R (5 -tttacggtcagaactaggg-3 ). The second SSU fragment was amplified with primers 1813F (5 -ctgcgtgagaggtgaaat-3 ) and 2646R (5 - gctaccttgttacgactttt-3 ). LSU rdna (5 end includingd D3 region ( 1,000 bp)) was amplified as two partially overlapping fragments using either primer 28 61for or 28 81for (28 61for, 5'-gtcgtgattacccgctgaactta-3'; 28 81for, 5'-ttaagcatatcatttagcggaggaa-3') in combination with either primer rev or rev ( rev, 5'- gttcgattagtctttcgcccct-3'; rev, 5'-tcggaaggaaccagctacta-3'). PCR was performed in a final volume of 25 l containing 3 l of 100x diluted crude DNA extract, 0.1 M of each PCR primer and a Ready-to-go PCR bead (GE Healthcare, Little Chalfont, UK). The following PCR program was used: 94 C for 5 min; 5 x (94 C, 30 s; 45 C, 30s; 72 C, 70s); 35 x (94 C, 30 s; 54 C, 30s; 72 C, 70s); 72 C for 5 min. Gel-purified (Marligen) amplification products (SSU and LSU rdna fragments) were cloned into a TOPO TA vector (Invitrogen) and sequenced using standard procedures. Blgg bv. Validation report for molecular species specific Globodera detection tests 4

5 Sequence alignment and phylogenetic analysis The sequences were aligned using the ClustalW algorithm as implemented in the program BioEdit (Hall, 1999). The alignment was then manually corrected using arthropod secondary structure information in accordance with Ben Ali et al. (1999) ( See appendix 1 and 2 for SSU and LSU phylogenetic trees. Primer design An alignment of 70 partial LSU rdna sequences from 33 different species belonging to the Heteroderidae family, was used to identify species-specific sequence motives (see appendix 2). To detect G. rostochiensis species-specific LSU rdna-based primers were designed (primer 269 and primer 270). To detect G. pallida, species-specific LSU rdna-based primers were designed (primer 185 and primer 117). Primer combinations were tested using cloned LSU rdna fragments. Bacterial clones harbouring a TOPO TA vector with a LSU rdna fragment of interest were grown in 2ml of LB medium (+100 µg/ml of ampicillin) at 37 C. Plasmid extraction was performed using the Wizard Plus Minipreps DNA Purification System (Promega, WI, USA). DNA concentrations were measured with a NanoDrop spectrophotometer (NanoDrop Technologies, DE, USA) and adjusted to10 ng/µl. For Q-PCR application 5 µl template was mixed with species-specific primers (final concentration for both primers 250 nm) and 10 µl Absolute QPCR SYBR green Fluorescein mix (Thermo scientific, UK) in a total reaction volume of 20 µl. Thermal cycling was performed on a Bio-Rad MyiQ thermal cycler (Bio-Rad, Hercules, CA, USA) and consisted of 95 o C for 15 min; followed by 40 cycles of 95 o C for 10 sec, 64 o C for 1 min and 72 o C for 20 sec. Intellectual property position relevant patents Patent application EPnummer EP Method for detecting cyst nematodes relates to the described molecular method of testing a sample for the presence of PCN species. Applicants are Wageningen University and Blgg bv. For the detection of PCN in soil or mixed cyst samples (in a complex DNA background) patent EP is essential as it includes the SSU rdna characteristics of virtual all Dutch plant parasitic and free-living (= bacterivorous / fungivorous) nematodes. This application is entitled Method for determining soil health ecologically and agriculturally. This patent is owned by Blgg bv. Blgg bv. Validation report for molecular species specific Globodera detection tests 5

6 Test specifications When processing soil, it is possible that more than one species of cyst nematodes is present. Intended use of the described molecular tests is species-specific detection of G. rostochiensis and G. pallida in a sample of up to 2 cysts. In addition, a sample as defined herein may also comprise eggs, juveniles or adult nematodes. Sensitivity of the tests should allow detection of one individual (egg, juvenile or adult nematode) of the target species in a background of non-target nematodes (eggs, juveniles or adult nematodes). Experimental validation An in-house validation of two molecular tests was performed by Blgg, in order to determine test performances. The following performance characteristics were determined: 1) specificity, 2) selectivity and 3) robustness. The organisms used for this validation were obtained from WUR and Prof. dr. Gerrit Karssen (Dutch Plant Protection Service). See table 1 for origin of the individual nematodes. Clones of each species (voucher specimens) are kept at the Laboratory of Nematology (Wageningen University, Wageningen, The Netherlands). Table 1: Identity and origin of the nematodes used for specificity testing Species Nematode no. Clone no. ID Phytosanitary Service Origin G. pallida Smilde (Netherlands) G. rostochiensis Munster (Germany) G. tabacum 1052 P83 C 6876 USA G. achilliae Slovenia G. artemisiae 1022 P86 E 1575 Sweden For detection of G. rostochiensis and G. pallida the following primer pairs are used: - Gros_forward (Blgg no.269) and Gros_reverse (Blgg no.270) - Gpal_forward (Blgg no.185) and Gpal_reverse (Blgg no.117) All primers are of Oligold ( quality (PAGE purified), obtained from Eurogentec Blgg bv. Validation report for molecular species specific Globodera detection tests 6

7 1. Specificity Specificity tests were performed using plasmid DNA containing LSU rdna from the target organism (G. rostochiensis or G. pallida) and the closest related non-target organisms (being G. achilleae, G. artemisiae and G. tabacum) (For results see table 2). Plasmid DNA amplifies well, making it relatively easy to detect false positives. Clone DNA concentrations were measured with a NanoDrop spectrophotometer (NanoDrop Technologies, DE, USA) and adjusted to10 ng/µl. For Q-PCR application 5 µl of 10 5 diluted template (0.5 pg) was mixed with species-specific primers (final concentrations for both primers 250 nm) and 10 µl Absolute QPCR SYBR green Fluorescein mix (Thermo scientific, UK) in a total reaction volume of 20 µl. Thermal cycling was performed on a Bio-Rad MyiQ thermal cycler (Bio-Rad, Hercules, CA, USA) and consisted of 95 o C for 15 min; followed by 40 cycles of 95 o C for 10 sec, 64 o C for 1 min and 72 o C for 20 sec. Results: Table 2: Ct values of Globodera samples (in duplicates) tested with two different primer combinations. 0.5 pg plasmid DNA was mixed with species-specific primers. Ct values were measured after the threshold line was set manually at 100. No. Sample Primer pair Ct value Melting curve * 1 Clone 926 G.pallida Gpal 185/ OK 2 Clone 926 G.pallida Gpal 185/ OK 3 Clone 938 G.rostochiensis Gpal 185/117 N/D - 4 Clone 938 G.rostochiensis Gpal 185/117 N/D - 5 Clone P83 G.tabacum Gpal 185/ OK 6 Clone P83 G.tabacum Gpal 185/117 N/D - 7 Clone 1210 G.achilliae Gpal 185/117 N/D OK 8 Clone 1210 G.achilliae Gpal 185/ OK 9 Clone P86 G.artemisiae Gpal 185/117 N/D - 10 Clone P86 G.artemisiae Gpal 185/117 N/D - 11 Clone 926 G.pallida Gros 269/270 N/D - 12 Clone 926 G.pallida Gros 269/270 N/D - 13 Clone 938 G.rostochiensis Gros 269/ OK 14 Clone 938 G.rostochiensis Gros 269/ OK 15 Clone P83 G.tabacum Gros 269/ OK 16 Clone P83 G.tabacum Gros 269/ OK 17 Clone 1210 G.achilliae Gros 269/270 N/D - 18 Clone 1210 G.achilliae Gros 269/270 N/D - 19 Clone P86 G.artemisiae Gros 269/270 N/D - 20 Clone P86 G.artemisiae Gros 269/270 N/D - * OK: melting temperature PCR product corresponds to melting temperature positive control target DNA N/D: not detected Blgg bv. Validation report for molecular species specific Globodera detection tests 7

8 Figure 2: Amplification chart of sample 1 10 as given in table 2. Green: 0.5 pg G.pallida plasmid DNA containing LSU rdna. Figure 3: Amplification chart of sample as given in table 2. Red: 0.5 pg G.rostochiensis plasmid DNA containing LSU rdna Blgg bv. Validation report for molecular species specific Globodera detection tests 8

9 Table 3: Ct values after amplification of whole cyst DNA. Healthy cysts were crushed and DNA was extracted as described in Holterman et al., Ct values were measured after the threshold line was set manually at 100. No G. pal G. ros G.tab Dil. Primer pair Ct value Melting curve * 1 1 cyst x Gpal 185/ OK 2-1 cyst - 100x Gpal 185/117 N/D cyst 100x Gpal 185/117 N/D cyst x Gros 269/270 N/D cyst - 100x Gros 269/ OK cyst 100x Gros 269/ OK * OK: melting temperature PCR product corresponds to melting temperature positive control target DNA N/D: not detected Figure 4: Amplification chart of sample 1 6 as given in table 3. Green: 1 cyst G.pallida (primer pair G.pallida), red: 1 cyst G.rostochiensis (primer pair G.rostochiensis), blue: 1 cyst G.tabacum (primer pair G.rostochiensis). Blgg bv. Validation report for molecular species specific Globodera detection tests 9

10 Conclusions specificity testing: Primer pairs Gpal_forward (Blgg no.185) and Gpal_reverse (Blgg no.117) and Gros_forward (Blgg no.269) and Gros_reverse (Blgg no.270) give the correct amplification products and detect G. pallida and G. rostochiensis respectively. Primer pair Gros_forward (Blgg no.269) and Gros_reverse (Blgg no.270) also amplifies G.tabacum, however, specificity tests performed using plasmid DNA containing LSU rdna indicate an 1000-fold difference in signal ( Ct 10). Similar results were obtained when using genomic DNA from cysts. Analyses of a (partly) degraded cyst mixed with healthy G. tabacum cysts however have to be approached with caution because of potential cross-reactivity with primer pair Gros_forward (Blgg no.269) and Gros_reverse (Blgg no.270) (see tabel 3). Blgg bv. Validation report for molecular species specific Globodera detection tests 10

11 2. Selectivity The selectivity of the species-specific primer pairs was tested by adding plasmid or genomic DNA from the close related non-target organism to G. rostochiensis or G. pallida (being G.pallida or G. rostochiensis respectively) to the PCR reaction in two different concentrations (for results see table 4 and 5). For Q-PCR application 5 µl of 10 5 diluted template (0.5 pg) was mixed with species-specific primers (final concentrations for both primers 250 nm) and 10 µl Absolute QPCR SYBR green Fluorescein mix (Thermo scientific, UK) in a total reaction volume of 20 µl. Thermal cycling was performed on a Bio-Rad MyiQ thermal cycler (Bio-Rad, Hercules, CA, USA) and consisted of 95 o C for 15 min; followed by 40 cycles of 95 o C for 10 sec, 64 o C for 1 min and 72 o C for 20 sec. Blgg bv. Validation report for molecular species specific Globodera detection tests 11

12 Results: Table 4: Ct values after amplification of target DNA (plasmid DNA in 10 7 and 10 8 dilution) in the presence of non-target DNA (plasmid DNA in 10 5 dilution). Ct values were measured after the threshold line was set manually at 100. No. Sample Dil. target Dil. Non target Primer pair Ct value Melting curve * 1 Clone 926 G.pallida Gpal 185/ OK 2 Clone 926 G.pallida Gpal 185/ OK 3 Clone 926 G.pallida Gpal 185/ OK 4 Clone 926 G.pallida Gpal 185/ OK 5 Clone 926 G.pallida in clone 938 G.rostochiensis Gpal 185/ OK 6 Clone 926 G.pallida in clone 938 G.rostochiensis Gpal 185/ OK 7 Clone 926 G.pallida in clone 938 G.rostochiensis Gpal 185/ OK 8 Clone 926 G.pallida in clone 938 G.rostochiensis Gpal 185/ OK 9 Clone 938 G.rostochiensis Gros 269/ OK 10 Clone 938 G.rostochiensis Gros 269/ OK 11 Clone 938 G.rostochiensis Gros 269/ OK 12 Clone 938 G.rostochiensis Gros 269/ OK 13 Clone 938 G.rostochiensis in clone 926 G.pallida Gros 269/ OK 14 Clone 938 G.rostochiensis in clone 926 G.pallida Gros 269/ OK 15 Clone 938 G.rostochiensis in clone 926 G.pallida Gros 269/ OK 16 Clone 938 G.rostochiensis in clone 926 G.pallida Gros 269/ OK 17 Water - - Gpal 185/117 N/D - 18 Water - - Gros 269/270 N/D - * OK: melting temperature PCR product corresponds to melting temperature positive control target DNA N/D: not detected Figure 5: Amplification chart of sample 1 8 as given in table 4. Primer pair Gpal_forward (Blgg no. 185) and Gpal_reverse (Blgg no. 117) Blgg bv. Validation report for molecular species specific Globodera detection tests 12

13 Figure 6: Amplification chart of sample 9 16 as given in table 4. Primer pair Gros_forward (Blgg no. 269) and Gros_reverse (Blgg no. 270) Table 5: Ct values after amplification of PCN cyst/nematode DNA in the presence of non-target DNA. Ct values were measured after the threshold line was set manually at 100. No. G. pallida G. rostochiensis Dil. Primer pair Ct value Melting curve * 1 1 nematode - 100x Gpal 185/117 29,64 OK 2 1 nematode - 100x Gpal 185/117 28,65 OK 3-1 nematode 100x Gpal 185/117 N/A nematode 100x Gpal 185/117 N/A nematode 1 cyst 100x Gpal 185/117 28,16 OK 6 1 cyst 1 nematode 100x Gpal 185/117 22,01 OK 7 1 nematode - 100x Gros 269/270 N/A nematode - 100x Gros 269/270 N/A nematode 100x Gros 269/270 32,55 OK 10-1 nematode 100x Gros 269/270 30,93 OK 11 1 nematode 1 cyst 100x Gros 269/270 23,10 OK 12 1 cyst 1 nematode 100x Gros 269/270 30,60 OK * OK: melting temperature PCR product corresponds to melting temperature positive control target DNA N/D: not detected Figure 7: Amplification chart of sample 1 12 as given in table 5. Green: primer pair Gpal_forward (Blgg no. 185) and Gpal_reverse (Blgg no. 117) Red: primer pair Gros_forward (Blgg no. 269) and Gros_reverse (Blgg no. 270) Blgg bv. Validation report for molecular species specific Globodera detection tests 13

14 Conclusions selectivity testing: Primer pairs Gpal_forward (Blgg no. 185) and Gpal_reverse (Blgg no. 117) and Gros_forward (Blgg no. 269) and Gros_reverse (Blgg no. 270) can detect a single nematode in the presence of one close related non-target cyst giving the correct amplification product. Presence of a 1000 fold excess of non-target plasmid DNA compared to the amount of target plasmid DNA (table 3) does not influence the detected Ct values (allowing a variance of 1 Ct) when amplifying G. pallida or G. rostochiensis DNA. Samples no15 and 16 give somewhat higher variance, this is probably due to the very high dilution of the target DNA. Blgg bv. Validation report for molecular species specific Globodera detection tests 14

15 3. Robustness The annealing temperature of all primers was designed to be 64 ºC and primer performance was validated at an annealing temperature of 64 ºC. The temperature variance of the Bio-Rad thermal cyclers as given by Bio-Rad is + or 0.5 ºC. Possible temperature variance would most likely affect annealing of the primer pairs and therefore the two primer pairs were subjected to a range of annealing temperatures ( ºC) in order to determine their performance at temperatures other than 64 ºC. Tests were performed with target and nontarget plasmid DNA. For Q-PCR application 5 µl of 10 5 diluted template (0.5 pg) was mixed with species-specific primers (end concentrations for both primers 200 nm) and 10 µl Absolute QPCR SYBR green Fluorescein mix (Thermo scientific, UK) in a total reaction volume of 20 µl. Thermal cycling was performed on a Bio-Rad MyiQ thermal cycler (Bio-Rad, Hercules, CA, USA) and consisted of 95 o C for 15 min; followed by 40 cycles of 95 o C for 10 sec, o C for 1 min and 72 o C for 20 sec. Blgg bv. Validation report for molecular species specific Globodera detection tests 15

16 Table 6: Ct values of primer pair Gpal_forward (Blgg no. 185) and Gpal_reverse (Blgg no. 117) at different annealing temperatures (ranging from ºC). Ct values were measured after the threshold line was set manually at 100. No. Sample Primer pair Ta ( C) Ct value Melting curve * 1 Clone 926 G.pallida Gpal 185/ OK 2 Clone 926 G.pallida Gpal 185/ OK 3 Clone 926 G.pallida Gpal 185/ OK 4 Clone 926 G.pallida Gpal 185/ OK 5 Clone 926 G.pallida Gpal 185/ OK 6 Clone 926 G.pallida Gpal 185/ OK 7 Clone 926 G.pallida Gpal 185/ OK 8 Clone 926 G.pallida Gpal 185/ OK 9 Clone 938 G.rostochiensis Gpal 185/ N/D - 10 Clone 938 G.rostochiensis Gpal 185/ N/D - 11 Clone 938 G.rostochiensis Gpal 185/ N/D - 12 Clone 938 G.rostochiensis Gpal 185/ N/D - 13 Clone 938 G.rostochiensis Gpal 185/ N/D - 14 Clone 938 G.rostochiensis Gpal 185/ N/D - 15 Clone 938 G.rostochiensis Gpal 185/ N/D - 16 Clone 938 G.rostochiensis Gpal 185/ N/D - 17 Clone P83 G.tabacum Gpal 185/ N/D - 18 Clone P83 G.tabacum Gpal 185/ N/D - 19 Clone P83 G.tabacum Gpal 185/ N/D - 20 Clone P83 G.tabacum Gpal 185/ N/D - 21 Clone P83 G.tabacum Gpal 185/ N/D - 22 Clone P83 G.tabacum Gpal 185/ N/D - 23 Clone P83 G.tabacum Gpal 185/ N/D - 24 Clone P83 G.tabacum Gpal 185/ N/D - 25 Clone 1210 G.achilliae Gpal 185/ N/D - 26 Clone 1210 G.achilliae Gpal 185/ N/D - 27 Clone 1210 G.achilliae Gpal 185/ N/D - 28 Clone 1210 G.achilliae Gpal 185/ N/D - 29 Clone 1210 G.achilliae Gpal 185/ N/D - 30 Clone 1210 G.achilliae Gpal 185/ N/D - 31 Clone 1210 G.achilliae Gpal 185/ N/D - 32 Clone 1210 G.achilliae Gpal 185/ N/D - 33 Clone P86 G.artemisiae Gpal 185/ N/D - 34 Clone P86 G.artemisiae Gpal 185/ N/D - 35 Clone P86 G.artemisiae Gpal 185/ N/D - 36 Clone P86 G.artemisiae Gpal 185/ N/D - 37 Clone P86 G.artemisiae Gpal 185/ N/D - 38 Clone P86 G.artemisiae Gpal 185/ N/D - 39 Clone P86 G.artemisiae Gpal 185/ N/D - 40 Clone P86 G.artemisiae Gpal 185/ N/D - * OK: melting temperature PCR product corresponds to melting temperature positive control target DNA N/D: not detected Figure 8: Amplification chart of sample 1 40 as given in table 6. Green: G.pallida. Blgg bv. Validation report for molecular species specific Globodera detection tests 16

17 Table 7: Ct values of primer pair Gros_forward (Blgg no. 269) and Gros_reverse (Blgg no. 270) at different annealing temperatures (ranging from ºC). Ct values were measured after the threshold line was set manually at 100. No. Sample Primer pair Ta ( C) Ct value Melting curve * 1 Clone 926 G.pallida Gros 269/ N/D - 2 Clone 926 G.pallida Gros 269/ N/D - 3 Clone 926 G.pallida Gros 269/ N/D - 4 Clone 926 G.pallida Gros 269/ N/D - 5 Clone 926 G.pallida Gros 269/ N/D - 6 Clone 926 G.pallida Gros 269/ N/D - 7 Clone 926 G.pallida Gros 269/ N/D - 8 Clone 926 G.pallida Gros 269/ N/D - 9 Clone 938 G.rostochiensis Gros 269/ OK 10 Clone 938 G.rostochiensis Gros 269/ OK 11 Clone 938 G.rostochiensis Gros 269/ OK 12 Clone 938 G.rostochiensis Gros 269/ OK 13 Clone 938 G.rostochiensis Gros 269/ OK 14 Clone 938 G.rostochiensis Gros 269/ OK 15 Clone 938 G.rostochiensis Gros 269/ OK 16 Clone 938 G.rostochiensis Gros 269/ OK 17 Clone P83 G.tabacum Gros 269/ OK 18 Clone P83 G.tabacum Gros 269/ OK 19 Clone P83 G.tabacum Gros 269/ OK 20 Clone P83 G.tabacum Gros 269/ OK 21 Clone P83 G.tabacum Gros 269/ OK 22 Clone P83 G.tabacum Gros 269/ OK 23 Clone P83 G.tabacum Gros 269/ OK 24 Clone P83 G.tabacum Gros 269/ OK 25 Clone 1210 G.achilliae Gros 269/ N/D - 26 Clone 1210 G.achilliae Gros 269/ N/D - 27 Clone 1210 G.achilliae Gros 269/ N/D - 28 Clone 1210 G.achilliae Gros 269/ N/D - 29 Clone 1210 G.achilliae Gros 269/ N/D - 30 Clone 1210 G.achilliae Gros 269/ N/D - 31 Clone 1210 G.achilliae Gros 269/ N/D - 32 Clone 1210 G.achilliae Gros 269/ N/D - 33 Clone P86 G.artemisiae Gros 269/ N/D - 34 Clone P86 G.artemisiae Gros 269/ N/D - 35 Clone P86 G.artemisiae Gros 269/ N/D - 36 Clone P86 G.artemisiae Gros 269/ N/D - 37 Clone P86 G.artemisiae Gros 269/ N/D - 38 Clone P86 G.artemisiae Gros 269/ N/D - 39 Clone P86 G.artemisiae Gros 269/ N/D - 40 Clone P86 G.artemisiae Gros 269/ N/D - * OK: melting temperature PCR product corresponds to melting temperature positive control target DNA N/D: not detected Figure 9: Amplification chart of sample 1 40 as given in table 7. Red: G.rostochiensis, blue: G.tabacum. Blgg bv. Validation report for molecular species specific Globodera detection tests 17

18 Conclusions robustness testing: The robustness is tested with relatively high concentrations of (non) target DNA (5 µl of 10 pg/µl template). Primer pairs Gpal_forward (Blgg no. 185) and Gpal_reverse (Blgg no. 117) give the correct amplification products with G.pallida plasmid DNA. at annealing temperatures ranging from 63.0 to 65.5 ºC. Gros_forward (Blgg no. 269) and Gros_reverse (Blgg no. 270) give the correct amplification products (with high Ct values) with G.rostochiensis and G.tabacum plasmid DNA. However, the difference with the expected Ct value is over 14 cycles. The temperature variance of the Bio-Rad thermal cyclers (as given by Bio-Rad: + or 0.5 ºC) falls within the temperature variance of the primer pairs (at Ta = 64 º C) and will not affect the detection results. Blgg bv. Validation report for molecular species specific Globodera detection tests 18

19 Final conclusions in-house validation The intended use of the described molecular tests is species-specific detection of G. rostochiensis or G. pallida in a sample of multiple cysts, comprising various cyst nematode species, without isolating the Globodera cysts or individuals first. Validation results show that species-specific detection with the described primer pairs is possible at the desired specificity and sensitivity. There is a clear distinction in Q-PCR signal between target DNA and close related non-target DNA. It is possible to detect a single nematode in the presence of a close related non-target cyst. Document approval Name/function Date Signature Dr. R. Landeweert Sr. Scientist Blgg May 1, 2009 Blgg bv. Validation report for molecular species specific Globodera detection tests 19

20 References Ben Ali A, Wuyts J, De Wachter R, Meyer A, Van de Peer Y (1999) Construction of a variability map for eukaryotic large subunit ribosomal RNA. Nucleic Acids Research, 27, Hall, TA (1999) BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, Holterman, M., G. Karssen, S. van den Elsen, H. van Megen, J. Bakker, and J. Helder (2009) Small Subunit rdna-based Phylogeny of the Tylenchida Sheds Light on Relationships Among Some High-Impact Plant-Parasitic Nematodes & the Evolution of Plant Feeding. Phytopathology (in press) (accepted on 22 October 2008) Holterman, M., K. Rybarczyk, S. Van den Elsen, H. Van Megen, P. Mooyman, R. P. Santiago, T. Bongers, J. Bakker, and J. Helder (2008) A ribosomal DNA-based framework for the detection and quantification of stress-sensitive nematode families in terrestrial habitats. Mol. Ecol. Resour. 8: Holterman, M; van der Wurff, A; van den Elsen, S; van Megen, H; Bongers, T; Holovachov, O; Bakker, J; Helder, J (2006) Phylum-wide analysis of SSU rdna reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biology and Evolution. 23 (9): Karssen, G., T. Vanhoenselaar, B. Verkerkbakker, and R. Janssen (1995) Species Identification of Cyst and Root-Knot Nematodes from Potato by Electrophoresis of Individual Females. Electrophoresis 16: Ludwig, W., Strunk O, Westram R, et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Research 32, Pylypenko, L.A., Uehara, T., Phillips, M.S., Sigareva, D.D. and Blok V.C. (2005) Identification of Globodera rostochiensis and G. pallida in the Ukraine by PCR. European Journal of et Plant Pathology 111, Siddiqi, M. R. (2000) Tylenchida: parasites of plants and insects. Wallingford, UK: CABI publishing. Blgg bv. Validation report for molecular species specific Globodera detection tests 20

21 Appendix 1: LSU rdna-based Heteroderidae tree Bayesian inference-based LSU rdna tree of Heteroderidae. Numbers near nodes indicate posterior probabilities (p.p.). Nodes with p.p. values above 0.95 are generally considered as robust. Holterman et al. (2009) Phytopathology (in press) Wageningen University, Laboratory of Nematology, dr. Hans Helder Plant Protection Service of The Netherlands, prof. dr. Gerrit Karssen Blgg bv. Validation report for molecular species specific Globodera detection tests 21

22 Appendix 2: LSU rdna-based Heteroderidae tree Bayesian inference-based LSU rdna tree of Heteroderidae. Numbers near nodes indicate posterior probabilities (p.p.). Nodes with p.p. values above 0.95 are generally considered as robust. Unpublished results Wageningen University, Laboratory of Nematology, dr. Hans Helder Plant Protection Service of The Netherlands, prof. dr. Gerrit Karssen Blgg bv. Validation report for molecular species specific Globodera detection tests 22