Increasing the Sensitivity of RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis.

OBJECTIVE

RNA virus was detected the first time in the New World species. Recent studies were also showed the presence of RNA virus 2 (LRV2) in Old Word species including Turkish and isolates. This study aimed to increase the sensitivity of qPCR with a modification in the denaturation step of cDNA preparation protocol.

METHODS

In this study, LRV2+ three , two strains and control strain (MHOM/SU/73/5-ASKH) were included. Total RNA isolation was done using different numbers of promastigotes (10, 10 and 10). Before cDNA synthesis, samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR was undertaken using 0.5 mM primers (LRV F-HR/LRV R-HR) diluted in SYBR Green Master mix.

RESULTS

We observed lower Ct values in amplicons with the modified version than with the classical kit protocol for cDNA synthesis, in all of the strains used in the study. The addition of pre-denaturation step at 95 °C showed lower Ct values meaning the sensitivity increased. Different parasite dilutions showed similar results.

CONCLUSION

It is important to increase the sensitivity especially with the aim for detecting LRV in clinical samples obtained from patients probably have less number of parasites. The presence and burden of the virus can help to understand the relationship between the clinical findings and the pathogenicity of the parasite which may lead to changes in the course of treatment.