Fourteen days post-infection, mosquitoes were screened for infection rate (IR), transmission rate (TR) and transmission efficiency (TE), as described previously [32 (link)]. Briefly, the IR is defined as the number of virus-positive mosquito bodies per number of fed females, TR as the number of virus-positive saliva per the number of virus-positive bodies and TE is calculated as the number of virus-positive saliva per the total number of fed and analyzed specimens. Detection and quantification of RNA was done by quantitative real-time PCR assay (RealStar Chikungunya RT-PCR Kit 2.0; RealStar Zika Virus RT-PCR Kit 1.0; RealStar WNV RT-PCR Kit 1.0; altona Diagnostics, Hamburg, Germany). A salivation assay was performed as previously described, detecting viable virus particles in the saliva by incubation of saliva solution on Vero cells (Chlorocebus sabaeus; CVCL_0059, obtained from ATCC, Cat# CCL-81) [35 (link)]. The R program [36 ] was used for all data analysis and visualizations, including the readxl [37 ], stringr [38 ], dplyr [39 ], plyr [40 (link)] and ggplot2 [41 ] packages.
Realstar wnv rt pcr kit 1
Manufactured by Altona Diagnostics
Sourced in Germany
The RealStar WNV RT-PCR Kit 1.0 is a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the qualitative detection of West Nile virus RNA. It is designed for use with compatible real-time PCR instruments.
Automatically generated - may contain errors
2 protocols using realstar wnv rt pcr kit 1
1
Mosquito Vector Competence Evaluation
2
Viral RNA Extraction from Mosquito and Bird Organs
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RNA extraction from mosquito and bird organs: the mosquito pools (5-30 individuals) and 0.5 g of bird organ (0.1 g of brain, lung, heart, kidney, and lungs) were placed in a 2 ml Eppendorf tube containing three to ve steel beads (7 mm, Qiagen, Hilden, Germany), frozen on liquid nitrogen and directly pulverized in a Tissue-Lyser LT (Qiagen, Hilden, Germany) with a -20°C pre-cooled rotor at 50 Hz for 2-5 min. Repeated freezing and lysis was necessary in some cases. The samples were then re-suspended in 300-500 µl PBS (depending on tissue size) containing 500 IU/ml penicillin, 10% fetal calf serum, and 500 µg/ml amphotericin, and centrifuged at 2,000 × g. Viral RNA extraction was done with QIAamp ® Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
RT-qPCR: 25 µl RNA of each sample extractions were tested using RealStar ® WNV RT-PCR Kit 1.0, (Altona Diagnostics, Hamburg, Germany) for the detection of West Nile virus (WNV) RNA, according to the manufacturer's instructions.
RT-qPCR: 25 µl RNA of each sample extractions were tested using RealStar ® WNV RT-PCR Kit 1.0, (Altona Diagnostics, Hamburg, Germany) for the detection of West Nile virus (WNV) RNA, according to the manufacturer's instructions.
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