Ipomoea (Sweetpotato/Kumara) Post-Entry Quarantine Testing Manual

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Table 8: Generic cycling conditions for RNA real-time RT-PCR Step Temperature Time No. of cycles RT-Step 50ºC 30 min 1 Initial denaturation 95 o C 2 min 1 Denaturation 95 o C 10 sec 40 Annealing & elongation See Table 3 40 sec Table 9: Real-time PCR reaction componnets for DNA templates using Invitrogen Platinum ® qPCR SuperMix-UDG Reagent Volume per reaction (µl) Nuclease-free water 4.6 Platinum ® Quantitative PCR Supermix-UDG (Invitrogen 11730-017) 10.0 10 µg/µl Bovine Serum Albumin (BSA) (Sigma A7888) 0.6 5 µM Forward primer (300 nM) 1.2 5 µM Reverse primer (300 nM) 1.2 5 µM Dual-labelled fluorogenic probe (100 nM) 0.4 DNA 2.0 Total volume 20.0 Table 10: Generic cycling conditions for DNA real-time PCR Step Temperature Time No. of cycles UDG incubation hold (Invitrogen only) 50ºC 2 min 1 Initial denaturation 95ºC 2 min (Invitrogen) 5 min (Roche) 1 Denaturation 95ºC 10 sec 40 Annealing & elongation See Table 3 40 sec Interpretation of results for real-time PCR The real-time PCR or RT-PCR test will only be considered valid if: (a) the positive control produces an amplification curve with the pathogen-specific primers; and (b) no amplification curve is seen (i.e. cycle threshold [CT] value is 40) with the negative control (if used) and the no template control. If the COX internal control primers are also used, then the negative control (if used), positive control and each of the test samples must produce an amplification curve. Failure of the samples to produce an amplification plot with the internal control primers suggests that the nucleic acid extraction has failed or compounds inhibitory to PCR are present in the nucleic acid extract, or the nucleic acid has degraded. Virus positive controls for PCR Tobacco streak virus positive control controls may be obtained from the following sources: 1. American Type Culture Collection (ATCC; http://www.atcc.org): No. PV-276, PV-31, PV-352, PV-353, PV-360. 2. DSMZ Culture Collection (http://www.dsmz.de): PV-0309, PV-0612, PV-0738. 3. The commercial source listed in Table 2. Ipomoea Post-Entry Quarantine Testing Manual · November 2012 17
Positive control material, in the form of nucleic acid, for Sweetpotato chlorotic stunt virus and Sweetpotato leaf curl virus and Tobacco streak virus may be obtained from MPI (see the Contact Point, section 8). Positive control material for Sweetpotato vein mosaic virus and Sweetpotato mild speckling virus is currently unobtainable; however, an alternative Potyvirus may be used for the PCR. Potyvirus positive controls in the form of nucleic acid may also be obtained from the MPI. A charge may be imposed to recover costs. 7.1.3.2.1.1 Sweet potato chlorotic stunt virus Plants must be tested for Sweetpotato chlorotic stunt virus by real-time PCR using the primer pairs listed in Table 3. See section 7.1.3.2.1 for details of test methods and interpretation of results. Please note that SPCSV should be tested with both sets of primers listed in Table 3 in order to detect both East and West African strains. 7.1.3.2.1.2 Sweetpotato leaf curl virus Plants must be tested for Sweetpotato leaf curl virus by PCR or real-time PCR using the primer pairs listed in Table 3. See section 7.1.3.2.1 for details of test methods and interpretation of results. Please note the Li et al., (2004) PCR should be cycled as shown in Table 11 Table 11: Cycling conditions for SPLCV PCR Step Temperature Time No. of Cycles Initial denaturation 94 o C 2 min 1 Denaturation 94 o C 30 sec Annealing 58ºC 30 sec 40 Elongation 68 o C 90 sec Final elongation 68 o C 3 min 1 7.1.3.2.1.3 Sweetpotato mild speckling virus Plants can be tested for Sweetpotato mild speckling virus by RT-PCR using the primer pairs listed in Table 3. Please note that a suitable positive control is not available for Sweetpotato mild speckling virus, however, the PCR has been validated with other potyviruses. See section 7.1.3.2.1 for details of test methods and interpretation of results. 7.1.3.2.1.4 Sweetpotato vein mosaic virus Plants must be tested for Sweetpotato vein mosaic virus by RT-PCR using the primer pairs listed in Table 3. Please note that a suitable positive control is not available for Sweetpotato vein mosaic virus, however, the PCR has been validated with other potyviruses. See section 7.1.3.2.1 for details of test methods and interpretation of results. 7.1.3.2.1.5 Tobacco streak virus Plants must be tested for Tobacco streak virus by RT-PCR using the primer pair listed in Table 3. See section 7.1.3.2.1 for details of test methods and interpretation of results. Ipomoea Post-Entry Quarantine Testing Manual · November 2012 18
Page 1 and 2: Ipomoea (Sweetpotato/Kumara) Post-E
Page 3 and 4: 1.7 Dickeya chrysanthemi ..........
Page 5 and 6: The per capita income provided by s
Page 7 and 8: Viruses: Sweetpotato chlorotic flec
Page 9 and 10: 7. TESTING Each of the specific tes
Page 11 and 12: 7.1.1 Graft inoculation Each I. bat
Page 13 and 14: 7.1.2 Herbaceous indexing Each I. b
Page 15 and 16: Table 2: Source of antisera and pos
Page 17 and 18: 7.1.3.2.1 Virus reverse transcripti
Page 19: Recommended method for RNA viruses:
Page 23 and 24: The reaction and cycling conditions
Page 25 and 26: (a) Primers recAF/recAR (Waleron et
Page 27 and 28: 8. CONTACT POINT This manual was de
Page 29 and 30: Nassar, A; Darrasse, A; Lemattre, M
Page 31 and 32: . 1.5 Streptomyces ipomoea (a) (b)
Page 33 and 34: Appendix 2. Virus symptoms on graft
Page 35 and 36: Appendix 3. Protocols referenced in

Ipomoea (Sweetpotato/Kumara) Post-Entry Quarantine Testing Manual

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