Development of a Calreticulin-Based Endogenous Control for Use in Culex restuans RT-qPCR Assays in Massachusetts

Hematophagous arthropods and associated pathogens are becoming more prevalent globally. With limited treatment options, prevention is the top priority to reduce disease. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has emerged as a standard detection method for many laboratories, but it has several drawbacks. Degraded sample quality, failed RNA preparations, and the presence of reaction inhibitors can lead to false negative results, introducing the need for a positive control. Existing control methods rely on adding exogenous nucleotides during amplification reactions, which may not reflect the quality of the sample. Incorporating assays to detect endogenous mosquito mRNA transcripts provides assurance that sample quality is sufficient for the downstream amplification of pathogen RNA. In the present study, we applied the Rapid Amplification of cDNA Ends (RACE) technique to obtain a full-length calreticulin cDNA sequence from the Culex restuans mosquito, collected in Massachusetts, USA. We then used the newly discovered cDNA sequence to design primers and probes spanning the first exon–exon junction of the calreticulin gene, which allowed for specific and accurate RNA transcript detection in an RT-qPCR assay. This assay detected the presence of calreticulin transcripts in extracted pools of mosquito RNA. This approach may be further developed to include other relevant mosquito species and, with extensive validation, may have potential for use as an endogenous control in arbovirus surveillance.