Evaluation of a TaqMan PCR assay to detect rabies virus RNA: influence of sequence variation and application to quantification of viral loads.
Published assays that use TaqMan PCR are consistently sensitive, rapid, and readily transferable. Here we describe a TaqMan PCR-based method for the detection of rabies virus (RV) RNA in tissue samples. We show that the method has an acceptable linear range, is both sensitive and specific, and, importantly, correlates with the concentration of infectious virus. In addition, the levels of RV-specific amplification are adjustable according to the levels of an endogenous control (beta-actin mRNA), allowing the calculation of comparable quantities. We tested the capacity of this assay to cope with target sequence variations. The number of sequence mismatches between gene-specific oligonucleotides and the target sequence significantly affects amplification (P < 0.001), and point mutations at the center of the probe can result in false-negative results through the prevention of probe binding and subsequent fluorescence. This study demonstrates that the genetic heterogeneity of RVs may prove a serious obstacle in the development of a diagnostic assay based on TaqMan PCR; however, the quantification of RV levels may prove to be a valuable application of this assay.
Hughes
G J
GJ
Rabies Section, Centers for Disease Control and Prevention, Atlanta, Georgia 30033, USA.
Smith
J S
JS
Hanlon
C A
CA
Rupprecht
C E
CE
D000818:Animals; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D011820:Rabies virus / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism; D019562:Viral Load
D012367:RNA, Viral; D019914:Taq Polymerase