TY - JOUR
T1 - A real-time RT-PCR for rapid detection and quantification of mosquito-borne alphaviruses
AU - Romeiro, Marilia Farignoli
AU - de Souza, William Marciel
AU - Tolardo, Aline Lavado
AU - Vieira, Luiz Carlos
AU - Henriques, Dyana Alves
AU - de Araujo, Jansen
AU - Siqueira, Carlos Eduardo Hassegawa
AU - Colombo, Tatiana Elias
AU - Aquino, Victor Hugo
AU - da Fonseca, Benedito Antonio Lopes
AU - de Morais Bronzoni, Roberta Vieira
AU - Nogueira, Maurício Lacerda
AU - Durigon, Edison Luiz
AU - Figueiredo, Luiz Tadeu Moraes
N1 - Publisher Copyright:
© 2016, Springer-Verlag Wien.
PY - 2016/11/1
Y1 - 2016/11/1
N2 - 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 (TM) values for amplification of the alphavirus genomes were 83.05 °C and 85.28 °C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with TM peaks of 84.83 to 85.28 °C and encephalitic alphaviruses of 83.34 °C to 84.68 °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.
AB - 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 (TM) values for amplification of the alphavirus genomes were 83.05 °C and 85.28 °C. Interestingly, the assay suggested the possibility the arthritogenic alphaviruses with TM peaks of 84.83 to 85.28 °C and encephalitic alphaviruses of 83.34 °C to 84.68 °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.
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U2 - 10.1007/s00705-016-3019-0
DO - 10.1007/s00705-016-3019-0
M3 - Article
C2 - 27558120
AN - SCOPUS:84983433825
SN - 0304-8608
VL - 161
SP - 3171
EP - 3177
JO - Archives of virology
JF - Archives of virology
IS - 11
ER -