Сельскохозяйственная биология, 2012, № 1, с. 110-116.

doi: 10.15389/agrobiology.2012.1.110eng

УДК 634.2:632.3:632.08

LARGE-SCALE TESTING OF LATERAL FLOW DEVICES FOR THE ON-SITE Plum pox virus DETECTION

S.N. Chirkov¹, N.A. Byzova², A.A. Sheveleva¹, I.V. Mitrofanova³, Yu.N. Prikhod’ko⁴, B.B. Dzantiev², I.G. Atabekov¹

For the first time Plum pox virus (PPV) genetic diversity has been studied in Russia. PPV isolates belonging to the D, M, C and W strains were found and identified by ELISA and immunocapture reverse-transcription polymerase chain reaction. The large-scale testing of lateral flow devices (test-strips) developed for the on-site PPV detection was carried out using samples infected with different PPV isolates. Test-strips have been shown to reveal the plants infected with the most prevalent in Russia D and M PPV strains. The time of immunochromatographic analysis does not exceed 10 min. The application of the test-strips for monitoring of sharka disease and by quarantine service can facilitate timely virus detection thus preventing this devastating pathogen dissemination through propagation of infected plant material.

Keywords: Plum pox virus, strain, ELISA, RT-PCR, immunochromatographic assay, lateral flow device.

Plum pox virus, the agent of sharka disease (PPV; the genus Potyvirus of family Potyviridae) is known as the most harmful pathogen of stone fruit (1, 2). The virus is a quarantine object in our country while being widespread in central and southern regions of European Russia (3). Usually, PPV is brought in a new region with introduction of infected plants and is transmitted through their vegetative propagation as well as by aphids in non-persistent manner. The spread of this virus can be significantly limited by timely detection and elimination of infection sources.
A laboratory diagnostics of PPV is performed by various immunochemical and molecular methods (4, 5). Usually, the leaves with symptoms of viral infection are collected for the analysis. However, in field conditions, this process is greatly complicated by a wide variation of disease symptoms. An on-site preliminary assessment of collected samples by non-instrumental rapid tests can improve the accuracy of sampling, the efficiency of monitoring and quarantine inspections.
Today, the problem of on-site tests is realized in lateral-flow devices (test-strips) – porous membranes for immunochromatographic analysis (ICA). Analysis of plant extracts using test-strips may take several minutes. Simplicity of the method allows its use in any conditions including the field without special training. The detection of PPV using ICA test-stripes  is almost as good as in the enzyme-linked immunosorbent assay (ELISA) (6-9). ICA-based diagnostic test kits are used in diagnostic protocols of the European and Mediterranean Plant Protection Organization (EPPO) for determination of quarantine viruses (10, 11) owing to the proven reliability of ICA method.
The authors have developed a test system for rapid diagnostics of PPV based on monoclonal antibodies to the virus and colloidal gold particles as a marker. The study of analytical characteristics of these test-strips has shown high sensitivity to the virus in extracts of leaves of stone fruit (12). However, these tests covered a very small number of samples not sufficient for reliable examination of diagnostic capabilities of the test-strips. Along with it, a more detailed study is needed owing to variability of the virus. Today, it has been described seven PPV strains - Dideron (D), Marcus (M), Cherry (C), El Amar (EA), Winona (W), Rec (D-M recombinant) and Turkish (T), which differ in nucleotide sequence of the viral RNA, antigen specificity, epidemiological properties, geographical spread and pathogenicity for different types of stone fruit (13, 14). The abovementioned test-strips contained monoclonal antibodies obtained by immunization of animals with a purified D-strain PPV preparation (12). In this context, it’s important to study the detection capability of these test-strips in respect to antigenically distinct strains of the virus.
The purpose of this work was to test the efficiency of domestically produced immunochromatographic test-trips on PPV isolates belonging to different strains derived from plant material samples of stone fruit.
Technique. Leaf samples with symptoms of PPV infection were collected in plantations of the All-Russia Research and Development Institute of Fruit Crops Genetics and Selection (VNIIGiSPR, Michurinsk), the Pyatigorsk Branch of the All-Russia Research and Development Institute of Plant Quarantine (VNIIKR, Pyatigorsk), N.V. Tsitsin Main Botanical Garden of RAS (GBS, Moscow), the Botanical Garden of M.V. Lomonosov Moscow State University (MGU, Moscow), in Nikitsky Botanical Gardens the Scientific Research Center of National Academy of Agrarian Sciences of Ukraine (NBS, Yalta) as well as in fruit orchards of Pyatigorsky district of Stavropol Krai and in private gardens of Moscow province.
The samples were analyzed using the kits ELISA Reagent set (Cat. ¹ 31 505, “Agdia”, USA) detecting all known PPV strains according to the manufacturer's protocol. The strains of PPV isolates were determined by non-direct sandwich ELISA using Agritest kits (Italy) based on monoclonal antibodies to PPV strains D, M or C, following the manufacturer’s guidelines. In addition, identification of strains was performed using immunocapture polymerase chain reaction with reverse transcription (IC-RT-PCR) with primers specific for the strains D, M, C, Rec and W as described (15-18). IgG for immunoadsorption was isolated from rabbit antiserum to PPV obtained from MSU Department of Virology  (the Faculty of Biology, M.V. Lomonosov Moscow State University). Positive control variants were the strains D, M and C from Agritest kits, as well as the isolate 1410 (strain W) derived from Prunus nigra (19). Negative control variants were leaf extracts of different stone fruit not infected with PPV, and the corresponding preparation from Agritest kits.
ICA was performed using the own developed test-strips produced as described earlier (12). The fragment of a fresh leaf (150-200 mg) was put in a plastic bag (size 10 ½ 10 cm) and added 3-4 ml extraction buffer. The leaf was mashed with fingers on a hard surface for 1 min; 0,3-0,4 ml leaf extract was poured into a microfuge tube 1,5 ml volume and a test strip was dipped in it for 10 min. After this time the strips were removed, visually inspected, and a digital image was obtained using the scanner HP ScanJet 5300C to calculate the brightness of analytical zone in the program TotalLab (12). PPV concentration in the same extracts was determined using ELISA Reagent set (“Agdia”, USA) from the calibration curve designed for a purified virus preparation diluted to a certain concentration with the leaf extract of healthy stone fruit plants. PPV isolate NAT BOC (D strain) was kindly provided by E. Mayss (Institute of Plant Diseases and Plant Protection, University of Hannover, Germany) was propagated in tobacco plants Nicotiana benthamiana and purified as described (20).
Results. In Russia, there’s no collection of PPV strains, so the first task was revealing the isolates belonging to different PPV strains among the samples collected during a monitoring of stone fruit plantations in European Russia and in the Crimea. The obtained samples were analyzed by ELISA and IC-RT-PCR according to the diagnostic protocol of EPPO (21) to confirm the virus presence in a sample and to determine its strain. Most of the collected samples were infected with PPV, while the rest were free from this virus despite the obvious symptoms.
The analysis of isolates has shown the presence of PPV strains D, M, C and W (Table); Rec strain was not detected. Determination of PPV strains EA and T endemic to, resp., the Nile delta and the vicinity of Ankara (Turkey) (14) wasn’t conducted.

Thus, this study for the first time presents the data on strain diversity of PPV isolates in Russia. The detection of W strain is of a particular interest. Until recently, only two isolates of W strain had been described: 3174 in Canada (22) and 44 191 in the USA (23). Both these isolates were derived from plum P. domestica imported into these countries from the Ukraine. The later reports about W-like isolates – the sample 1410 on Canada plum in Moscow (19) and LV-145bt on blackthorn in Latvia (24) – suggest that the spread area of W strain in the former Soviet Union is wider than it is commonly believed. The authors’ finding of W strain in different regions of European Russia (Table) apparently confirms this assumption.
All virus-positive and a part of the virus-negative samples were used in testing ICA test-strips (Table). PPV concentration in the analysed leaf extracts ranged from 0,05 to 0,5 ug/ml or more. Under this concentration range, the test-strips provided a visual detection of PPV strains D and M in samples of different stone fruit. The extracts of leaves collected from different parts of a tree crown of one plant were analyzed (samples RD1, RD2, P10, P3, M23, P13); it was found that the test-strips detect the virus in any leaves with symptoms of infection. The analysis of digital images of test-strips has revealed a somewhat correlation between PPV content and the brightness of analytical zone in respect to isolates of D strain. In a number of samples infected with D strain the virus wasn’t revealed by ICA test-strips, which false-negative results amounted to 10% compared to ELISA.
The results of ICA detection of the virus are specific, because negative controls didn’t show any coloration in analytical zone of the test-strips (Table). Along with it, there wasn’t any false-positive reactions in more than 30 samples of stone fruit with symptoms of infection but free from PPV as found by ELISA (the data not shown in Table). This fact emphasizes that selection by symptoms is actually unreliable. The time of a test is less than 10 minutes, and much faster at high concentrations of the virus (analytical zone gets colored in 2-3 min).
Thus, the domestically developed test-strips allow the detection of PPV isolates related to strains D and M. Possibly, they are also capable to detect Rec strain, as the capsule proteins of PPV strains M and Rec are almost identical (25). PPV strains D, M and Rec are known as the most widespread in the world (25, 26), and the large majority of PPV isolates described in Russia are related to strains D and M (27).
At the same time, these test-strips does not detect C and W strains even at a relatively high content of the virus in samples (Table). The most probable reason for that is a limited specificity of monoclonal antibodies of these test-stripes. However, the total proportion of these two PPV strains in Europe is about 1% (24), and a similar fact can be expected in Russia, even though there’s no any data on a spread area of these strains in our country.
So, a wide range of different stone fruit samples was tested by immunochromatographic test-strips proved to be suitable for rapid detection of plum pox virus (PPV, or sharka) isolates belonging to the most common strains. Such rapid tests are quite important during the monitoring of viral infection and phyto-quarantine, because they ensure early detection of infected individuals in stone fruit plantations and prevent the spread of this damaging disease with infected planting material.

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