关键词: 沙粒病毒, 一致-简并杂合寡核苷酸引物, 实时荧光PCR

Abstract:

Objective To develop a consensus-degenerate hybrid oligonucleotide primer (CODEHOP) 4-step program Real-time PCR method for the detection of Arenaviruses in rodents. Methods Designed a pair of CODEHOP primers for Arenaviruses, analyzed the amplification curve and melting curve of Real-time PCR. According to the melting temperatures (Tm) of specific amplicons and primer-dimers (PDs), CODEHOP 4-step program Real-time PCR was developed by adding a 5 s holding at the optimal temperature between Tm of PDs and amplicons for reading fluorescence signal after extension step of conventional 3-step Real-time PCR. The sensitivity, specificity and repeatability of the method were evaluated. Results Tm of PDs was between 75.32-76.86℃ and Tm of specific amplicons was 86.84℃. Adding the step which fluorescence signal reading temperature was 84℃, can effectively eliminated the artifact of PDs in CODEHOP Real-time PCR. The specificity of the method was 100% and the minimum detection to Arenaviruses RNA was 59.6 pg. The method had fine repeatability with CV value less than 5%. Twelve blind rodent lung samples were detected successfully. Conclusion The developed CODEHOP 4-step program Real-time PCR method has a high level of specificity and sensitivity, that can be effectively used for detection of different Arenaviruses carried by rodents.

Key words: Arenavirus, Consensus-degenerate hybrid oligonucleotide primer, Real-time PCR