ISSN 1003-8280 CN 10-1522/R 中国疾病预防控制中心 主办
Chinese Journal of Vector Biology and Control >
Development of CODEHOP 4-step program Real-time PCR to detect Arenaviruses in rodents
Received date: 2015-12-27
Online published: 2016-06-20
Supported by
Supported by the Natural Science Foundation of Ningbo City (No. 2013A610240)
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.
HU Qun, ZOU Chun-ying, MA Si-jie . Development of CODEHOP 4-step program Real-time PCR to detect Arenaviruses in rodents[J]. Chinese Journal of Vector Biology and Control, 2016 , 27(3) : 248 -252 . DOI: 10.11853/j.issn.1003.8280.2016.03.009
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