Detección y cuantificación de virus dengue 2 en lisado celular y plasma de niños por qPCR en tiempo real usando un estuche comercial y el equipo EcoTM System-Illumina

Abstract

Methods  for  Dengue  virus  (DENV)  diagnosis  in  endemic areas  are  greatly  needed.  One of  them  is  the  real-time polymerase  chain  reaction  (qPCR)  that  also  enables  to quantitate  the  viral  genome.  Kits of  qPCR  for  DENV  are expensive and restrict their use to a small number of qPCR devices, which limits the application of the technique. Here, we evaluated the performance of a commercial kit of qPCR to DENV-2 detection  on  a  locally  available  qPCR  device (Eco^TM System, Illumina), not cited by the kit manufacturer.VERO-76 cells lysate and plasma from children, both with confirmed ongoing DENV-2 infection, were evaluated. As specificity control, cell lysates and plasma  from  children infected  with  DENV-1,  and  uninfected  lysate,  were  also included. The reactions were simultaneously evaluated in an Applied Bio systems  7300  device.  The standard  curve generated  by  Eco^TM  was  robust  (R2=  0.99)  with  low variability in the replicates (<10%). The reaction efficiency was high (88.8%) and signal was only obtained in lysates and plasma infected  with  DENV-2.  There was  a  strong positive  correlation  (R2=  1.0,  P=  0.0028)  between  the number of copies of viral RNA in the samples detected by both  qPCR  devices.  Thus, the use  of  the  evaluate  kit  for detection of DENV-2 here tested can be extended to Eco^TM. With  this  work,  technological  capacity  for  DENV  study  in an endemic zone is greatly strengthened.