10.2. Strand-specific RT-qPCR

One of the most popular methods in bee virology for detecting virus replication is the specific detection of negative strand viral RNA, using strand-specific Reverse Transcriptase-PCR (Peng et al., 2012; DiPrisco et al., 2011; Boncristiani et al., 2009; Dainat et al., 2009; Eyer et al., 2009; Gisder et al., 2009; Celle et al., 2008), following its first application in bee virology by Yue and Genersch (2005). The procedure is illustrated in Fig. 5, including the most common cause of false-positive results (non-specific cDNA synthesis) and how best to avoid this (tagged-cDNA primer followed by tag-specific PCR). 

Theoretically, strand-specificity can be achieved by performing the reverse transcription reaction in the presence of only one primer specifically annealing with a unique region of the viral negative strand before amplifying the obtained cDNA by adding the second primer or a specific primer pair for PCR amplification. Unfortunately, strand-specific RT-PCR is highly susceptible to false positive results (Gunji et al., 1994; Lanford et al., 1995; Lanford et al., 1994; McGuiness et al., 1994; Craggs et al., 2001; Peyrefitte et al., 2003; Boncristiani et al., 2009) due to:

  • False-priming of the incorrect strand by the cDNA primer.
  • Self-priming of positive-strand RNA in areas of complex secondary structures.
  • Random priming by contaminating cellular nucleic acids.
  • Incomplete inactivation of the reverse transcriptase, leaving residual activity during PCR amplification (which contains both negative and positive strand primers).

To overcome these drawbacks and improve the specificity of the assays, certain effective techniques have been developed that enhance strand-specificity. These include:

  • Thermostable reverse transcriptases.
  • Tagged-cDNA primers.
  • Inactivation/removal of residual tagged-cDNA primers prior to PCR.
  • Chemical blocking of free 3’ ends before or after reverse transcription.


Fig. 5. Outline of the procedure for strand-specific RT-PCR amplification of negative-strand viral RNA, using tagged-cDNA primers to avoid amplification of non-target cDNAs. Only cDNA produced with tagged-cDNA primers, and amplified with the tag and a virus-specific primer will be amplified. RdRp refers to the viral RNA-dependent RNA polymerase.  Image © JR de Miranda.

12116PN revised Fig 5

 

10.2.9. Controls