Invisorb spin virus rna mini kit

To check for correctness of the HA sequences in the recombinant viruses, RNA was isolated from cleared cell culture supernatants with Invisorb Spin Virus RNA Mini Kit (Stratec, Birkenfeld, Germany) followed by reverse-transcription and polymerase chain reaction (RT-PCR) using HA specific primers and the OneStep RT-PCR kit (Qiagen, Hilden, Germany). Before sequencing, PCR products were treated with the ExoSAP-IT Cleanup Kit (Affymetrix, Halbergmoos, Germany) to remove excess of primers and desoxinucleotide triphosphates (dNTPs), which might hinder the sequencing reaction. Two µL ExoSAP-IT reagent was added to 5 µL PCR product, and incubated at 37 °C for 15 min and heat-inactivated for 15 min at 80 °C. Sequencing was performed by GATC Biotech (Konstanz, Germany).

RNA from ECE homogenates was extracted using Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Berlin, Germany) according to manufacturer’s instructions. ECE homogenates which were found positive by Pan-BTV RT-qPCR were further assessed by RT-PCR for the typing of serotypes 2, 3, 4, 5, 8, 12, 15, 16, 24 and 28 (all with in house developed primer pairs), which are known to be present in Israel and neighboring countries, using One-Step RT-PCR kit (Qiagen, Hilden, Germany). Identification of BTV-6 isolates was performed with the following pair of primers (developed in house): 6VP2-124F 5′- TGTAACCCAAATTCCCACGAA-3′ and 6VP2-1030R 5′-CAGAGGCGGCTATCATA-3′. Amplified fragments were subsequently sequenced.

All mosquito samples were ground in 1× phosphate buffered saline (PBS) and centrifuged at 11,000× g for 10 min. The supernatant was used for viral RNA extraction by using a viral RNA extraction kit, Invisorb^® Spin Virus RNA Mini kit (STRATEC molecular GmbH, Berlin, Germany) following the manufacturer’s instructions. RNA concentration and purity were quantified by a Nano Drop 2000c spectrophotometer (Thermo Fisher Scientific, MA, USA). The extracted RNA samples were used for ZIKV detection immediately and the rest of samples were stored at −80 °C.

The swab specimens were suspended in 2 ml PBS, incubated for 1 h at room temperature and then clarified by centrifugation at 1000 rpm for 10 min. The supernatants were recovered for extraction and were stored at −80 °C until analysis. Total nucleic acid was extracted from 200 μl swab samples using Invisorb Spin virus RNA mini kit (STRATEC, molecular GmbH, Berlin, Germany), according to the manufacturer's instructions. Extracted RNA was tested for the presence of MERS-CoV RNA by real-time reverse transcription-quantitative polymerase chain reaction (qrt RT-PCR) hydrolysis probe assay using Bio Rad CFX 96 Real Time detection system (Bio Rad, Hercules, CA, USA). The primers and probe encompassed upstream the envelope gene (UpE) [9 ].

Viral RNA was extracted from individual mosquitoes as follows. First, the mosquito was ground in 1X phosphate-buffered saline (PBS) and centrifuged at 11,000× g for 5 min. The supernatant was processed for viral RNA extraction using a viral RNA extraction kit, Invisorb^® Spin Virus RNA Mini kit (STRATEC Molecular GmbH, Berlin, Germany), according to the manufacturer’s instructions. The RNA concentration was quantified by a Nano Drop 2000c spectrophotometer (Thermo Fisher Scientific, MA, USA). The extracted RNA samples were used immediately for CHIKV detection, and the samples were maintained for long-term storage at −80 °C.

All sand fly samples were ground in 200 μl of 1× PBS and then centrifuged at 11,000×g for 5 min. Subsequently, 100 μl of supernatant with 300 μl of minimum essential medium (MEM) was used for virus culture. The pellet and some of the supernatant with lysis buffer were processed for viral RNA extraction using the Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Germany) following the manufacturer’s instructions. The extracted RNA samples were immediately used for detection of virus families, and the remaining samples were stored at −80 °C. The RNA samples were amplified for the detection of the Orbivirus [22 (link)], Phlebovirus [23 (link)], and Flavivirus [24 (link)] genera and family Rhabdoviridae [25 (link)]. All the methods for viral RNA detection were briefly described in Phumee et al. [18 (link)]. PCR amplicon sizes were determined by 1.5% ultrapure low-melting-point (LMP) agarose (Gibco, USA) gels, and gels were stained with ethidium bromide and visualized with Quantity One quantification analysis version 4.5.2 software (Gel Doc EQ System; Bio-Rad, USA). The estimated sizes for the Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae were 188, 244, 270, and 460 base pairs (bp), respectively. A synthesized plasmid was used as a positive control to prevent sample contamination.