CIMB, Vol. 44, Pages 5778-5787: Multiplex Detection of Pleurotus ostreatus Mycoviruses

Figure 1. Optimization of the annealing temperature on uniplex RT-PCR detection of (a) OMSV (672 bp); (b) OMIV (540 bp); (c) POSV (310 bp); (d) PoV1 (200 bp) and (e) multiplex RT-PCR assay. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000. Lanes 1–8, 60.0, 61.0, 62.3, 64.0, 66.3, 68.1, 69.3, 70.0 °C.

Figure 1. Optimization of the annealing temperature on uniplex RT-PCR detection of (a) OMSV (672 bp); (b) OMIV (540 bp); (c) POSV (310 bp); (d) PoV1 (200 bp) and (e) multiplex RT-PCR assay. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000. Lanes 1–8, 60.0, 61.0, 62.3, 64.0, 66.3, 68.1, 69.3, 70.0 °C.

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Figure 2. Detection limit based on ten-fold serial plasmid DNA dilutions of uniplex PCR and multiplex PCR detection. Uniplex PCR for (a) OMSV; (b) OMIV; (c) POSV; and (d) PoV1; (e) multiplex PCR. Lanes 1–8, 10 ng, 1 ng, 102 pg, 10 pg, 1 pg, 102 fg, 10 fg, and 1 fg of plasmids. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000. The amplified gene sizes were 672, 540, 310, and 200 bp for OMSV, OMIV, POSV, and PoV1, respectively.

Figure 2. Detection limit based on ten-fold serial plasmid DNA dilutions of uniplex PCR and multiplex PCR detection. Uniplex PCR for (a) OMSV; (b) OMIV; (c) POSV; and (d) PoV1; (e) multiplex PCR. Lanes 1–8, 10 ng, 1 ng, 102 pg, 10 pg, 1 pg, 102 fg, 10 fg, and 1 fg of plasmids. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000. The amplified gene sizes were 672, 540, 310, and 200 bp for OMSV, OMIV, POSV, and PoV1, respectively.

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Figure 3. Specificity testing of multiplex PCR with single or mixed templates. Lane 1 shows the empty vector. Lanes 2–16 indicate the plasmids containing genome sequences of the PoV1, POSV, OMIV, OMSV, POSV + PoV1, PoV1+ OMIV, PoV1 + OMSV, POSV + OMIV, POSV + OMSV, OMIV + OMSV, PoV1 + POSV + OMIV; PoV1+ POSV+ OMSV, PoV1 + OMIV + OMSV, POSV + OMIV + OMSV, PoV1 + POSV+ OMIV + OMSV as templates, respectively. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000.

Figure 3. Specificity testing of multiplex PCR with single or mixed templates. Lane 1 shows the empty vector. Lanes 2–16 indicate the plasmids containing genome sequences of the PoV1, POSV, OMIV, OMSV, POSV + PoV1, PoV1+ OMIV, PoV1 + OMSV, POSV + OMIV, POSV + OMSV, OMIV + OMSV, PoV1 + POSV + OMIV; PoV1+ POSV+ OMSV, PoV1 + OMIV + OMSV, POSV + OMIV + OMSV, PoV1 + POSV+ OMIV + OMSV as templates, respectively. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000.

Cimb 44 00392 g003 Figure 4. Viruses’ detection of P. ostreatus strains using the developed multiplex RT-PCR. Numbers 1–36 represent the 36 strains in Table 2. Lane M1 represents the mixed strains of PG-ZP20 and TD300. Lane M2, combined strains of PG-ZP20 and TD300 with the plasmid pTPoV1. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000. Figure 4. Viruses’ detection of P. ostreatus strains using the developed multiplex RT-PCR. Numbers 1–36 represent the 36 strains in Table 2. Lane M1 represents the mixed strains of PG-ZP20 and TD300. Lane M2, combined strains of PG-ZP20 and TD300 with the plasmid pTPoV1. N, negative control, the healthy P. ostreatus sample. Lane M: GL DNA Marker2000. Cimb 44 00392 g004

Table 1. The primers used for the multiplex detection of the four P. ostreatus mycoviruses.

Table 1. The primers used for the multiplex detection of the four P. ostreatus mycoviruses.

Primer NamePrimer Sequence (5′ to 3′)Target Virus aAmplicon Size (bp)OMSV-FACCCCCCCAGGATCTCAAGCTTCOMSV672OMSV-RGAGATGTAGACRTTGAAAGCOMIV-FAACATTGTTGATCACGCTCTOMIV540OMIV-RGGCTTCAGAATAAAGATTGTPOSV-FATCWCATGGCTATCAACCTAPOSV310POSV-RAGCTGAATTATCGTCACCCAPoV1-FAAACTCGAAGAGTTCCTTTCPoV1200PoV1-RGCGCGTGGGCCACGTTCGGG

Table 2. The names, sources, and presence of viruses in this study.

Table 2. The names, sources, and presence of viruses in this study.

NumberStrain NameSourcePresence of VirusOMSVOMIVPOSVPoV118129Yantai+−−−2DF5Yangzhou+−−−3DF5-2Liaocheng+−−−4969Liaocheng+−−−5Kang-2Liaocheng+−−−6Kang-3Liaocheng+−−−7P89Beijing+−−−8P99Beijing+−−−9PG-0122-1Yantai++−−10HeipingYantai++−−11PG-ZP20Yantai++−−12TD300Linyi−−+−13PG-2203Jinan−−−−14PG-2204Jinan−−−−15P2108Dezhou++−−16LD-0701Weifang+−−−17LD-0704Weifang−−−−18LD-0707Weifang−−+−19LD-0719Weifang−−−−20LD-1011Qingdao++−−21LD-1015Qingdao+−−−22PGH-1011Zibo−−+−23PGH-1012Zibo−−−−24PGH-1014Zibo++−−25PGZ-1020Weihai+−−−26PGZ-1021Weihai+−−−27PGZ-1022Weihai−−−−28HuimeiLiaocheng−−−−29Xianfeng-1Liaocheng−−−−30PG-0324Yantai−−−−31WPG-1107Yantai−−−−328105Yangzhou−−−−33Luping-0417-5Yantai−−−−34F803Linyi−−−−35PG-ZP17Yantai−−−−36LD-0328Yantai−−−−

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