Establishment of RT-LAMP detection method for strawberry pallidosis-associated virus
Strawberry pallidosis-associated virus(SPaV)can cause strawberry pallidosis disease,and the symptoms will worse when SPaV coinfects with other viruses.This study established a reverse transcription-loop-mediated isothermal amplification(RT-LAMP)for SPaV detection.After evaluating and optimizing the conditions of the reaction system,the specificity and sensitivity of this RT-LAMP procedure were mensurated and field samples were tested.The results showed that the optimized reaction system contained 2.5 μL 10 × Isothermal Amplification Buffer,6 mmol/L Mg2+,1 mmol/L dNTPs,1 μmol/L SPaV-FIP and SPaV-BIP,0.1 μmol/L SPaV-F3 and SPaV-B3,0.3 μmol/L SPaV-LB,0.4 mol/L Betaine,0.256 U/μL WarmStart Bst 2.0 DNA Polymerase,0.12 U/μL WarmStart RTx Reverse Transcriptase,1 μL RNA,and incubated at 61℃ for 40 min.Only the sample containing the SPaV could be positively amplified by the optimized reaction system.The sensitivity of this RT-LAMP was 10 times higher than that of RT-PCR detection.In the field application,the RT-LAMP detection result was consistent with RT-PCR.The RT-LAMP method developed for SPaV detection in this study is simple,rapid and specific,and can be used in laboratory and field to identify virus-free seedlings and investigate virus diseases.
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