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14Russian Journal of Nematology, 2023, 31 (2), 115 - 120
On the reliability of recombinase polymerase amplification - lateral flow assay using ITS rRNA gene primers and probe as a new detection method of the golden potato cyst nematode, Globodera rostochiensis
Sergei A. Subbotin
Plant Pest Diagnostic Centre, California Department of Food and Agriculture, 3294 Meadowview Road, 95832-1448, Sacramento, CA, USA e-mail: [email protected]
Accepted for publication 01 August 2023
Summary. Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technique, which has been adopted as a novel molecular technology for simple, rapid, reliable, and low-resource diagnostics of nematodes and other damaging organisms. Recently, RPA integrated with lateral flow assay (LFA) using the ITS rRNA gene primers and probe was proposed by Wang et al. (2022) as a new detection method of Globodera rostochiensis. The reliability of any diagnostic method should be validated on a broad range of populations of different species from different geographical locations as well as on related taxa to avoid erroneous identification with false positive results. A total of 24 isolates of 13 cyst nematode species were used to validate the analytical specificity of the method mentioned above. The results of these tests showed that the ITS rRNA gene primers and probe did not give specific RPA-LFA detection for the golden potato cyst nematode and gave positive reactions not only with G. rostochiensis, but also with other Globodera parasitising solanaceous plants, G. artemisiae, and representatives of the genus Punctodera parasitising grasses.
Key words: cross-reactions, Globodera ellingtonae, Globodera pallida, molecular diagnostics, specific primers, specificity assay.
The worldwide distributed golden potato cyst nematode, Globodera rostochiensis (Wollenweber, 1923) Skarbilovich, 1959, is probably the most economically important nematode pest of potato in temperate regions and in some warm areas of the world where short-cycle potato crops are produced in winter months (Inserra et al., 1996). There are estimates indicating that the potato cyst nematodes are responsible for the loss of 9% of this crop worldwide. The golden cyst nematode is recognised as a significant global threat to food security and it is the subject of quarantine regulations in most countries where it occurs (Turner & Subbotin, 2013). The finding of the golden cyst nematode in samples have serious implications for market access and international trade of seed potato. Accurate identification is critical for the implementation of phytosanitary and control measures against this pest.
Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technique, which has been adopted as a novel molecular technology for simple, robust, rapid, reliable and low-resource diagnostics of nematodes and other organisms. RPA represents a versatile alternative to PCR (Tan et al., 2022). RPA assays show high sensitivity and specificity for detecting various pests including nematodes (Subbotin & Burbridge, 2021). Recently, a new detection method of G. rostochiensis using RPA integrated with lateral flow assays (LFA) was published by Wang et al. (2022). The method allowed the rapid diagnosis and detection of this nematode from crude extracts of cysts and juveniles within 30 min. Moreover, the RPA-LFA method can also directly diagnose and detect G. rostochiensis from infested field soil. The authors stated that they designed species-specific
© Russian Society of Nematologists, 2023; doi: 10.24412/0869-6918-2023-2-115-120
Table 1. Cyst-forming nematode isolates tested in RPA-LFA assay.
Species Sample code Origin RPA experiment
Globodera rostochiensis CD2176 Russia, Kaliningrad region, Pravdinskii district +
G. rostochiensis CD2598a Bolivia, Cochabamba, Tapacari, Chuna Chununi +
G. rostochiensis CD2197 German, Hannover +
G. rostochiensis CD2200a Germany, Harmerz +
G. rostochiensis CD2617a Bolivia, La Paz, Los Andes, Lacaya +
G. pallida CD2740a The Netherlands +
G. pallida CD2553b Peru, Cusco, Quispicanchis +
G. pallida CD2555a Peru, Cruz Pampa +
G. pallida CD2554b Peru, Cusco, Marangani, Canchis +
G. tabacum CD2689a, b USA, Virginia, Paulette +
G. tabacum CD2684a USA, Virginia, Baines +
G. mexicana CD3563a Mexico, State of Mexico, Juchitepec County +
G. mexicana CD3565b Mexico, State of Tlaxcala, Santiago Cuaula +
G. ellingtonae CD3815a Bolivia, Tarija, El Puente, Chilcayo Sud +
G. ellingtonae CD3887a Bolivia, Tarija, El Puente, Chorcoya Mendez +
G. artemisiae CD2178a Russia, Primorskii Krai +
Punctodera punctata CD2715a USA, Michigan +
P. stonei CD3088a Russia, Jaroslavl region, Myshkin +
P. stonei CD2994a Canada, Ontario, Ottawa Arboretum +
P. chalcoensis CD2813 Mexico +
Cactodera rosae CD3562 Mexico, Tlaxcala State, Huamantla, Francisco Villa Tecoac -
C. solani CD3586a Mexico, Tlaxcala State, Huamantla, Francisco Villa Tecoac -
Heterodera avenae CD2006b Turkey, Mardin, Midyat, Idil Road -
H. humuli CD3144b Russia, Moscow -
Note: + - test line; - no test line.
primers and probe, which amplified the internal transcribed spacer of nuclear ribosomal rRNA gene (ITS rRNA) of target nematode (Wang et al., 2022). To determine the specificity in RPA-LFA assay, the authors used the single cyst crude extracts from 14 cyst nematode populations, including nine G. rostochiensis samples from different geographical origins, one sample of the pale cyst nematode, Globodera pallida Stone, 1973, one sample of Globodera artemisiae (Eroshenko & Kazachenko, 1972) Behrens, 1975, and three other cyst nematodes species. The authors stated that the RPA-LFA result showed high level specificity to
G. rostochiensis and did not detect other potato or other cyst nematode species. Only the strip of G. rostochiensis showed both test line and control line, but the other cyst nematode species and negative control showed no test line (Wang et al., 2022).
For validation of any diagnostic methods, the analytical specificity should be assessed by testing a broad range of species of the same taxonomical group, including those from different geographical locations as well as related taxa to avoid erroneous identification with false positive results. The goal of this study was to evaluate primers and probe proposed by Wang et al. (2022)
for the G. rostochiensis using RPA-LFA detection assay with some representatives of Globodera and related genera.
MATERIAL AND METHODS
Nematode samples. DNA was obtained from 24 isolates of 13 cyst-forming nematode species. The nematodes samples included: five isolates of G. rostochiensis from Germany, Russia and Bolivia, four isolates of G. pallida from Peru and The Netherlands, two isolates of Globodera tabacum (Lownsbery & Lownsbery, 1954) Skarbilovich, 1959 from USA, two isolates of Globodera mexicana Subbotin, Mundo-Ocampo & Baldwin, 2010 from Mexico, two isolates of Globodera ellingtonae Handoo, Carta, Skantar & Chitwood, 2012 from Bolivia, one isolate of G. artemisiae from Russia, one isolate of Punctodera punctata (Thorne, 1928) Mulvey & Stone, 1976 from USA, two isolates of Punctodera stonei Brzeski, 1998 from Russia and Canada, one isolate of Punctodera chalcoensis Stone, Sosa, Sosa Moss & Mulvey, 1976 from Mexico, one isolate of Cactodera rosae Cid del Prado Vera & Miranda, 2008 from Mexico, one isolate of Cactodera solani Escobar-Avila, Subbotin & Tovar-Soto, 2021 from Mexico, one isolate of Heterodera avenae Wollenweber, 1924 from Turkey and one isolate of Heterodera humuli Filipjev, 1934 (Table 1). All nematodes were identified by molecular method using the ITS rRNA gene sequences (Subbotin et al., 2020).
Sequence analysis. Alignment of the ITS rRNA
sequences for Globodera and other cyst nematodes were created using ClustalX 1.83 (Chenna et al., 2003) with default parameters using sequences deposited in the GenBank and unpublished sequences (Subbotin et al., 2011, 2020).
RPA-LFA assay. The RPA-LFA assay conducted with the G. rostochiensis-specific RPA-LFA primers: GrF4 (5'-CTG TGT ATG GGC TGG CAC ATT GAC CAA CA-3'); GrR4 (5'-[Biotin]TAC GGC ACG TAC AAC ATG GAG TAG CAG CTA C-3'); and probe: GrP (5'-[Fam]CGG AGG AAG CAC GCC CAC AGG GCA CCC TAA CG[THF] CTG TGC TGG CGT CTG T[C3-spacer]-3') (Wang et al., 2022), which were synthetised in Biosearch Technologies (CA, USA). The RPA-LFA assay was done using AmplifyRP® Acceler8® Discovery Kit (Agdia, IN, USA). The reaction mixture for each RPA assay was prepared according to the manufacturer's instructions: the lyophilised reaction pellet was suspended with a mixture containing 6 ^l of the
rehydration buffer, 2 ^l of distilled water, 0.45 ^l of each forward and reverse primers (10 ^M), 0.15 ^l of the probe (10 ^M), 0.5 ^l of magnesium acetate. One ^l of the DNA template was added in a reaction tube. The reaction tubes were incubated at 39°C in a MyBlock Mini Dry Bath (Benchmark Scientific, Edison, NJ, USA) for 20 min. For visual analysis with Milenia® Genline Hybridetect-1 strips (Milenia Biotec GmbH, Giessen, Germany), 120 ^l of HybriDetect assay buffer was added to a reaction tube and then a dipstick was placed in this mixture. Visual results were observed within 3-5 min. The amplification product was indicated by the development of a coloured test line (lower), and/or a separate control line (upper) to confirm that the system worked properly.
RESULTS AND DISCUSSION
Lateral flow detection of RPA products obtained using AmplifyRP® Acceler8® Discovery Kit (Agdia, IN, USA) showed positive strong or moderate intensive test lines on the strips with all studied DNA samples of G. rostochiensis, G. pallida, G. mexicana, G. tabacum, G. ellingtonae, G. artemisiae, Punctodera punctata, P. stonei and P. chalcoensis. No test lines were detected with DNA samples of Cactodera rosae, C. solani, Heterodera avenae, H. humuli and control without DNA (Fig. 1A & B). Control lines were visualised on all strips. Thus, ITS rRNA gene primers and probe proposed by Wang et al. (2022) for the golden potato cyst nematode RPA-LFA detection assay gave positive reactions not only with G. rostochiensis, but also with other Globodera species from solanaceous plants, G. artemisiae, and representatives of the genus Punctodera from grasses.
The success of molecular identification depends strictly on the choice of the target gene fragment accounting for possible interspecific sequence variation to avoid strict overlapping with fragments of non-target species. For this aim, the ITS rRNA gene is used widely for diagnostics of cyst-forming nematodes (Subbotin et al., 2011). The ITS region was selected as target gene fragment for diagnostics of G. rostochiensis with conventional PCR (Mulholland et al., 1996; Bulman & Marshall, 1997; Zouhar et al., 2000; Trayanov et al., 2020), realtime PCR (Madani et al., 2008; Toyota et al., 2008) and loop-mediated isothermal amplification (Ahuja et al., 2021) with species-specific primers. However, use of this fragment for development of these diagnostic methods is problematic, because of a high sequence identity between species. For example,
Fig. 1. RPA-LFA specificity assay with examples of lateral flow strips. DNA of cyst-forming nematodes: A. Strips:
1 - Globodera pallida (CD2555a); 2 - G. pallida (CD2554b); 3 - G. tabacum (CD2684a); 4 - Punctodera chalcoensis (CD2813); 5 - P. stonei (CD2994a); 6 - G. rostochiensis (CD2617a); 7 - Cactodera rosae (CD3562); 8 - C. solani (CD3586a); 9 - Heterodera humuli (CD3144b); 10, 11 - NC (control without DNA). B. Strips: 1 - G. artemisia (CD2178a); 2 - G. tabacum (CD2689b); 3 - P. punctata (CD2715a); 4 - P. stonei (CD3088a); 5 - G. mexicana (CD3563a); 6 - G. rostochiensis (CD2200a); 7 - G. tabacum (CD2689a); 8 - H. avenae (CD2006b); 9 - G. pallida (CD2553b); 10, 11 - NC (control without DNA).
Globodera rostochiensis (EU517119) Globodera ellingtonae (OQ339228) Globodera pallida (FJ212165) Globodera mexicana (OQ318153) Globodera tabacum (HQ260402) Punctodera stonei (JF895515) Punctodera punctata <OL314 545) Punctodera chalcoensis (CD2813) Globodera artemisiae (AF274415) Cactodera solani (MN994489) Cactodera rosae (HQ260413) Heterodera humuli (MT804361) Heterodera avenae (AY148366)
Globodera rostochiensis (EU517119) Globodera ellingtonae (OQ339228} Globodera pallida (FJ212165) Globodera mexicana (OQ318153) Globodera tabacum (HQ260402) Punctodera stonei (JF895515) Punctodera punctata <QL314545) Punctodera chalcoensis (CD2813) Globodera artemisiae (AF274415) Cactodera solani (MN994489) Cactodera rosae (HQ260413) Heterodera humuli (MT804361) Heterodera avenae (AY148366)
Globodera rostochiensis (EU517119) Globodera ellingtonae (OQ339228) Globodera pallida (FJ212165) Globodera mexicana (OQ318153) Globodera tabacum (HQ26Q4Q2) Punctodera stonei (JF895515) Punctodera punctata <OL314 545) Punctodera chalcoensis (CD2813) Globodera artemisiae (AF274415) Cactodera solani (MN994489) Cactodera rosae (HQ260413) Heterodera humuli (MT804361) Heterodera avenae (AY148366)
Fig. 2. The fragment of sequence alignment of the ITS rRNA gene for several cyst nematodes with the positions of RPA-LFA primers (GrF4 and GrR4, in green) (Wang et al., 2022) and probe (GrP, in blue) (Wang et al., 2022) used in this study. Coloured areas indicated sequences of species that generated a positive reaction in this assay.
using in silico analysis, Subbotin et al. (2011) noticed that the G. rostochiensis specific primer PITSr3 proposed by Bulman and Marshall (1997) was well matched with the corresponding sequence regions for the ITS rRNA gene of G. artemisiae, G. millefolii and Globodera sp. from Portugal, as well as a Globodera sp. from South Africa, differing only in two or three nucleotides and potentially giving false-positive results with these samples.
In Figure 2, the fragment of sequence alignment of the ITS rRNA gene with positions of specific primers and probe in this assay for several tested cyst nematode species is given. Analysis showed that sequences of GrF4 and GrR4 primers have from one to four, and from two to five mismatches, respectively, with gene sequence fragments of species, which give positive reactions in this assay. For GrP probe, mismatches varied from one to six nucleotides. Only one nucleotide mismatch in the 3' end was found between GrF4 and the corresponding gene fragment for G. ellingtonae.
It has been known that complementarity between primers and template is often crucial for DNA amplification. Base mismatches located in the 3'-end region affect target amplification much more than mismatches towards the 5'-end, since 3'-end mismatches can disrupt the nearby polymerase active site (Stadhouders et al., 2010). Stadhouders et al. (2010) showed that primer mismatches induces a variety of effects on PCR results, they are strongly dependent on mismatch types, their position and the chemicals used. Some studies on this topic have been published for PCR, whereas no data on mismatch behaviour are available for RPA. We believe that lower reaction temperature during RPA might also lead to less specific DNA hybridisation and may have a significant effect in the occurring of false positive results despite mismatches.
Thus, the Globodera rostochiensis RPA-LFA detection assay developed by Wang et al. (2022) does not give specific detection of this species. Gene fragments containing sufficient nucleotide sequence polymorphism should be selected as diagnostics targets for RPA to obtain a reliable detection of G. rostochiensis.
ACKNOWLEDGEMENTS
The author thanks Drs V.N. Chizhov, I.H. Elek^ioglu, Z. Handoo, Q. Yu, J. Franco, and other colleagues for providing cyst nematode samples. This work is sponsored by the USDA APHIS FarmBill grant: AP22PPQS&T00C175. Author thanks Dr R.N. Inserra for critical reading and correcting the manuscript.
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С.А. Субботин. О надежности рекомбиназной полимеразной амплификации, интегрированной с анализом латерального потока с использованием праймеров и пробы гена ITS рРНК как нового метода обнаружения золотистой картофельной нематоды Globodera rostochiensis. Резюме. Рекомбиназная полимеразная амплификация (РПА) представляет собой метод изотермической амплификации нуклеиновых кислот in vitro, который был введен в качестве новой молекулярной технологии для простой, надежной, быстрой и малозатратной диагностики нематод и других вредителей. Недавно РПА, интегрированная с анализом латерального потока (ЛП) с использованием праймеров и пробы гена ITS рРНК, была предложена Wang et al. (2022) в качестве нового метода обнаружения Globodera rostochiensis. Надежность любого диагностического метода должна быть проверена на широком диапазоне популяций разных видов из разных географических мест, а также на родственных таксонах, чтобы избежать ошибочной идентификации с ложноположительными результатами. В нашем исследовании для подтверждения аналитической специфичности упомянутого выше метода было использовано в общей сложности 24 изолята 13 видов цистообразующих нематод. Результаты испытаний показали, что праймеры и проба гена ITS рРНК не дают с помощью РПА-ЛП специфического определения для золотистой картофельной нематоды и дают положительные реакции не только с G. rostochiensis, но и с другими глободерами, паразитирующими на пасленовых растениях, G. artemisiae и представителями рода Punctodera, паразитирующими на злаках.
