Sources of viral samples
Cowpea plants with stunting and leaf-rolling symptoms were collected from Hefei city, Anhui province in 2017 and 2020, and samples were frozen in liquid nitrogen and stored at − 70 °C. N. benthamiana plants infected with tomato yellow leaf curl virus (TYLCV) were kindly provided by Dr. Xueping Zhou. N. benthamiana plants infected by rice stripe virus (RSV) were planted in our greenhouse. Some virus vectors such as turnip mosaic virus (TuMV) full length cDNA infectious clone vector: p35Tunos, tobacco mosaic virus (TMV) full length cDNA infectious clone vector: p35S-30B::GFP and porcine circovirus (PCV) full length cDNA clone vector: pEASY-T5-PCV3 were kindly provided by Dr. Fernando Ponz, Prof. Rongxiang Fang and Xiufang Yuan, respectively. Pepper mild mosaic virus (PMMoV) full length cDNA infectious clone vector: pCB-PMMoV plasmid, cucumber green mottle mosaic virus (CGMMV) full length cDNA infectious clone vector: pCB-CGMMV plasmid were construct by ourselves and kept in our laboratory. The virus samples and corresponding viral DNA or cDNA used in this study were all stored in our laboratory.
Sample preparation
Viral DNA was extracted from leaves of cowpea infected with MDV or N. benthamiana infected with TYLCV using an E.Z.N.A.® Plant DNA Kit (Omega Bio-tek, Inc. Norcross, GA).
The total RNA of N. benthamiana plants infected with RSV was extracted using TRIzol reagent (Thermo Fisher Scientific, US) according to the manufacturer’s protocol and the corresponding cDNA was synthesized from 1 μg of RNA using an oligo (dT) primer (21-nucleotide plus two anchoring nucleotides) and moloney murine leukemia virus reverse transcriptase (Takara Bio Inc., JP).
For the sensitivity detection, plasmid pEASY-T5-MDV which contains a 407 bp target segment in the pEASY™-T5 Zero Cloning Vector was constructed and sequenced. The purified positive control pEASY-T5-MDV plasmid (4362 bp) was used as the initial template with a DNA concentration of 31.7 ng/μL. The initial copy number was about 6.7× 109 copies/μL. The plasmids were then diluted with crude leaf extracts of healthy cowpea to provide templates of 106, 105, 104, 103, 102, 101, 100 copies/μL. The number of copies was estimated by mass concentration measured using a spectrophotometer with Avogadro constant NA = 6.022 × 1023 copies/mol, assuming that the molecular weight of 1 bp dsDNA is approximately 650 g/mol:
$$ \mathrm{number}\ \mathrm{of}\ \mathrm{copies}=\left(\mathrm{amount}\times 6.022\times {10}^{23}\right)/\left(\mathrm{length}\times 1\times {10}^9\times 650\right). $$
Crude sample extraction
MDV-infected cowpea plants together with healthy cowpea plants for controls were collected from Hefei city, Anhui province in 2017 and 2020. Approximately 200 mg fresh cowpea leaf tissue of each sample was homogenized in a mesh bag (Agdia, US) with 2 mL plant lysis buffer (Tiosbio, China). The homogenized crude extract was transferred from the mesh bag into a 2 mL Eppendorf tube and briefly centrifuged. 10 μL supernatant was then transferred and mixed with 90 μL ddH2O, and a dilution series was prepared by sequential transfers. 1 μL supernatant of the homogenized crude extracts or their corresponding dilutions (1:10, 1:100, 1:1000, 1:10000) were used as templates. Aliquots of crude extracts were either tested immediately or stored at − 20 °C until used.
Primers and probe design and optimizing for PCR and RPA
A pair of specific MDV primers suitable for both PCR and RPA were designed using DNAMAN Version 8 (CP1F: 5′-GTGAAGCGAATCTGACGGAA-3′ and CP1R: 5′- CATAACCTTCTTCATCTTATA − 3′) from the sequence of the MDV CP gene (GenBank accession: KY070245). These yield an amplicon size of 407 base pairs (bp). For PCR-LFS and RPA-LFS, the primers were first labeled with biotin or fluorescein isothiocyanate (FITC) at their 5′-ends, generating the forward primer CP1F-biotin and the reverse primer CP1R-FITC.
Real-time qPCR assay
Real-time qPCR was performed in an BIORADCFX96 apparatus (BIORAD, USA) in a 20 μL reaction containing 10 μL KOD SYBR qPCR Mix, 80pM of each primer (CP1F and CP1R) and 0.8 μL DNA solution of the template. To determine the sensitivity of qPCR detection, 0.8 μL of the pEASY-T5-MDV plasmid template and its series of diluents were used, and for evaluating RPA with qPCR in field detection, 0.8 μL crude leaf extracts of each sample were used. The reaction conditions were: Pre-denaturation at 98 °C for 2 min, then 40 cycles each of 98 °C for 10s, 50 °C for 30s and 68 °C for 30s, and finally 95 °C for 15 s, 60 °C for 1 min and 99 °C for 15 s.
MDV PCR- LFS assay
PCR was carried out in a 50 μL reaction containing 25 μL 2 × PCR buffer for KOD FX, 0.4 mM of each dNTP, 80pM of each primer, 2 μL DNA solution of pEASY-T5-MDV and 1 μL KOD FX. After the PCR reaction, 25 μL of the PCR amplification product was transferred to a new Eppendorf tube for duplicate testing, and the latex microsphere test strips (Tiosbio, China) were then directly inserted into the reaction tube. Lateral chromatography was performed for 2–4 min and the results were observed and recorded within 10 min. Positive detection of MDV was indicated by the presence of a color test line; a separate control line confirmed that the system was working properly. The ability of the LFS assay to detect MDV-PCR products from a dilution series (106, 105, 104, 103, 102, 101, 100 copies/μL) was also explored. Reactions with crude leaf extracts of healthy cowpea were included as negative controls.
MDV RPA- LFS assay and comparison of different incubation methods
RPA was carried out using a Twist Amp™ Basic kit (Twist Amp, Cambridge, UK) in accordance with the manufacturer’s protocols. 1 μL DNA solution of pEASY-T5-MDV plasmid was used as template and added into a 23.75μLreaction mixture containing 14.75 μL rehydration buffer, 14 mM MgAc2 and 0.5 μM of each primer. Reactions with crude leaf extracts of healthy cowpea were included as negative controls. The tubes were then incubated at 37 °C for 30 min in a PCR instrument, water bath or oven. The sensitivity of detection was also tested using a dilution series as before. After the reactions were completed, 1 μL of each amplification product was diluted 4000 times and latex microsphere test strips were then inserted into 40 μL of the diluted sample before incubation and examination as described above.
Determination of specificity and sensitivity
The specificity of the primer pair was tested using the plasmid pEASY-T5-MDV and various non-target DNAs or cDNA of viruses. The templates used were: DNA of N. benthamiana plants infected by TYLCV, cDNA of N. benthamiana plants infected by RSV and the control virus plasmids including pEASY-T5-PCV3, pCB-PMMoV, pCB-CGMMV, pGR-TuMV-GFP and p35S-30B::GFP. Among these viruses, PCV is a virus with a somewhat similar structure to MDV [1, 16], and TYLCV is a geminivirus which, like MDV, can also infect tobacco [17]. TuMV, PMMoV and TMV can all infect tobacco, while CGMMV and RSV are viruses unrelated to MDV.
To explore the sensitivity of the primer pair, pEASY-T5-MDV plasmid DNA (4362 bp) with the concentration of 31.7 ng/μL was used as the initial plasmid, and ten-fold serial dilutions of pEASY-T5-MDV plasmid DNA (106, 105, 104, 103, 102, 101, 100 copies/μL) with crude leaf extracts of healthy cowpea, that was also as a negative control, were used as templates for both PCR and RPA. Three technical repeats were made.
After the PCR reaction, 5 μL of the amplicon of each PCR product was used for agarose gel electrophoresis. After the RPA reaction, 25 μL of the amplicon of each product was mixed with 100 μL tris saturated phenol and centrifuged before using 5 μL of the purified RPA for agarose gel electrophoresis.