Figures (6)  Tables (2)

    • Figure 1. 

      One-step RT-RPA and RT-PCR detection of ASGV. (a) One-step RT-RPA-LFD detection of ASGV. (b) One-step RT-RPA-gel detection of ASGV. (c) RT-PCR-gel detection of ASGV. In two gel images, lane M is a 100 bp DNA marker; lean '+' is positive; lean '−' is negative.

    • Figure 2. 

      Optimization of one-step RT-RPA reaction conditions for ASGV detection. (a) Optimization of ASGV detection for different reaction time (0, 10, 20, 30 and 40 min) at 40 °C. (b) Optimization of ASGV detection at various temperatures 37 °C, 38 °C, 39 °C, 40 °C and 41 °C for 30 min.

    • Figure 3. 

      Specificity of one- step RT-RPA-LFD assays. (a) RT-PCR detection of ACLSV. (b) RT-PCR detection of ASPV. (c) RT-PCR detection of ASGV. (d) One- step RT-RPA-LFD detection of ASGV. In all images, lane 'M' is 2000 bp DNA marker, lanes 1−3 are samples Yanfu 8-3, Yanfu 8-2 and GL-1 which was single-infected by ACLSV, ASPV and ASGV, respectively. '−' negative control, '+' positive control.

    • Figure 4. 

      One- step RT-RPA-LFD and RT-PCR amplicons at total RNA (500 ng/μL) dilutions levels ranging from 10−1 to 10−8 detect ASGV. (a) Detect ASGV by one- step RT-RPA-LFD. (b) Detect ASGV by RT-PCR. Lane M (b): 100 bp DNA marker.

    • Figure 5. 

      Rapid detection of ASGV with one-step RT-RPA-LFD using crude RNA.

    • Figure 6. 

      Primer and probe design position within the coat protein of ASGV.

    • Sample no.RT-PCROne-step
      RT-RPA-LFD
      ASGVASPVACLSVASGV
      Gala-1 (Positive control)+−−+
      Gala-2 (Negative control)−−−−
      Gala-3++−+
      Gala-4−−+−
      Gala-5++++
      Gala-6+−++
      Gala-7+−−+
      Gala-8+−−+
      Gala-9+−−+
      Gala-10+−++
      '+': ASGV positive; '−': ASGV negative.

      Table 1. 

      Comparison on the ASGV detection results between conventional RT-PCR protocol and One-step RT-RPA-LFD using crude RNA extracts.

    • Gene namePrimer sequenceLength (bp)Reference
      ASGVF: CCCGCTGTTGGATTTGATACACCTC524[37]
      R: CTGCAAGACCGCGACCAAGTTT
      ASPVF: ATGTCTGGAACCTCATGCTGCAA370[38]
      R: TTGGGATCAACTTTACTAAAAAG
      CATAA
      ACLSVF: GAGARTTTCAGTTTGCTMGA794[39]
      R: AGTCTACAGGCTATTTATTATAAGT
      nfo-ASGVF: ATCTGATAAGACCCAGTTTCCCGCT
      GTTGG      		264This study
      R: Biotin-AAGAAATTCCCGAGCCAAAT
      CAGCAAATGG
      P: FAM-GGTTCATTACAATCTGACAA
      CGACCCCAAAA(THF)GAAGGGGAGACTGAAG-C3 Spacer

      Table 2. 

      The primer used for RT-PCR in apple virus detection.