Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus.
In this study, a rapid and specific TaqMan-based, one-step real-time quantitative reverse transcription PCR (qRT-PCR) has been described for the detection of peste des petits ruminants virus (PPRV). Primers and probe were designed based on the nucleocapsid protein gene sequence. The real-time qRT-PCR assay was able to detect PPRV isolates from very distinct geographical areas (Africa, Middle East and Asia). The specificity of the assay was assessed by including rinderpest virus and other morbillivirus RNAs but none of these tested positive in the assay. The analytical sensitivity of the real-time qRT-PCR assay was achieved through the construction of an in-house PPRV cRNA for the generation of a standard curve. The detection limit of the assay was found to be 8.1 RNA copies per reaction mixture. The assay had excellent intra- and inter-assay reproducibility. In total 30 field samples were screened for the presence of PPRV by conventional RT-PCR in parallel with qRT-PCR. The detection rate increased from 46.7% to 73.3% by use of the real-time qRT-PCR. The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PPRV in tissue samples from field cases.
Bao
Jingyue
J
China Animal Health and Epidemiology Center, Qingdao, Shandong 266032, China.
Li
Lin
L
Wang
Zhiliang
Z
Barrett
Tom
T
Suo
Longciren
L
Zhao
Wenji
W
Liu
Yutian
Y
Liu
Chunju
C
Li
Jinming
J
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D015511:Goat Diseases / Q000175:diagnosis* / Q000821:virology; D006041:Goats; D019590:Nucleocapsid Proteins / Q000235:genetics; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis* / Q000821:virology; D018107:Peste-des-petits-ruminants virus / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000821:virology; D018609:Tibet
D017931:DNA Primers; D019590:Nucleocapsid Proteins