A real-time RT-PCR for rapid detection and quantification of mosquito-borne alphaviruses

作者：Romeiro Marilia Farignoli^*; de Souza William Marciel; Tolardo Aline Lavado; Vieira Luiz Carlos; Henriques Dyana Alves; de Araujo Jansen; Hassegawa Siqueira Carlos Eduardo; Colombo Tatiana Elias; Aquino Victor Hugo; Lopes da Fonseca Benedito Antonio; de Morais Bronzoni Roberta Vieira; Nogueira Mauricio Lacerda; Durigon Edison Luiz; Moraes Figueiredo Luiz Tadeu

来源：Archives of Virology, 2016, 161(11): 3171-3177.

DOI：10.1007/s00705-016-3019-0

Mosquito-borne alphaviruses are widely distributed throughout the world, causing important human illnesses. Therefore, the development of methods to enable early diagnosis of infections by alphavirus is essential. We show here the development and evaluation of a quantitative real-time RT-PCR using genus-specific primers to the nsP1 viral gene of all mosquito-borne alphaviruses. The specificity and sensitivity of the assay were tested using seven alphaviruses and RNA transcribed from Venezuelan equine encephalitis virus. The detection limits of real-time RT-PCR ranged from 10 to 76 copies per ml. The melting temperature (T-M) values for amplification of the alphavirus genomes were 83.05 A degrees C and 85.28 A degrees C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with T-M peaks of 84.83 to 85.28 A degrees C and encephalitic alphaviruses of 83.34 A degrees C to 84.68 A degrees C could be discriminated both diseases. Real-time RT-PCR may prove very useful for the screening and preliminary diagnosis in outbreaks and surveillance studies as well as for measuring the viral load in pathogenesis studies.

• 出版日期2016-11