Quantitect probe kit

For initial primer evaluation, the QuantiTect Probe kit (Qiagen) for qPCR of DNA targets and the SuperScript III Platinum One-Step qRT-PCR Kit (ThermoFisher Scientific) for RNA targets, were used because they were the reagents commonly used in our laboratories.
To shorten assay time using fast-cycling, we evaluated the use of the following test kits and master mixes for qPCR:—the QuantiFast Probe (Qiagen); Kapa Probe Fast (Merck) ;or qPCRBIO Probe Mix Lo-Rox (PCR Biosystems); the EXPRESS One-Step SuperScript (ThermoFisher Scientific); and the qPCRBIO 1-Step Go Lo-Rox kit (PCR Biosystems), based on recommendations from PCR instrument manufacturers and colleagues.
To further increase assay sensitivity and reduce variability at low target concentrations, various concentrations of tRNA were added to the master-mix and optimized. A published “step-up” cycling protocol was employed to improve sensitivity and cross-subtype specificity³⁸ . “Step-up” cycling rescues PCR reactions where there are minor mismatches between the assay oligonucleotides and the target template species and serves to increase the sensitivity and precision for detection of HIV subtypes with minor sequence polymorphisms at low target concentrations.

Whole blood samples were collected and centrifuged in the laboratory to obtain serum. The sera were then aliquoted in small volumes and stored at − 80 °C until further analyses. All 1617 samples were analysed for HEV RNA. Water samples were concentrated using PEG/NaCl method in a final volume of 1 mL and tissues organs from rats euthanized according to the AVMA Guidelines³⁴ , were triturated in 2 mL of L-15 Medium (Leibovitz). Briefly, RNA was extracted from 140 µL of whole blood, serum samples, concentrated water samples or tissues organs using the QIAamp RNA Viral kit (Qiagen, Heiden, Germany) according to the manufacturer's recommendations. The extracted RNA were tested by real time RT-PCR using a previously described HEV assay targeting the ORF2 gene^{35 (link)} with the Quantitect probe kit (Qiagen, Heiden, Germany) according to the manufacturer's instructions. Subsequently, the reaction mix was performed in a final volume of 25 µL containing 5 µL of extracted RNA, 10 µL of buffer (2× QuantiTect Master mix), 1.25 µL of forward and reverse primer, 0.2 of TaqMan probe and 0.2 µL of enzyme mix. Experiments were performed on an ABI 7500 instrument (Applied Biosystem) using the following thermal cycling parameters: 10 min at 50 °C; 15 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

DNA template from individual shrimp was extracted using the DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA). Muscle tissue was homogenized using glass grinders followed by DNA extraction according to manufacturer’s instructions. The DNA was stored at −80 °C until use. Quantitative real-time PCR primers and standard were designed as described previously [26 (link)]. The primers used were WSSV forward primer: 5′-TGG TCC CGT CCT CAT CTC AG-3′; WSSV reverse primer: 5′-GCT GCC TTG CCG GAA ATT A-3′; and the probe: 5′-FAM-AGC CAT GAA GAA TGC CGT CTA TCA CAC A-TAMRA-3’. The qPCR reactions were performed using the Quantitect Probe kit (Qiagen) in 25 μL reactions containing 3 μL of DNA template, 12.5 μL of Master Mix, 0.5 μL of TaqMan probe, 1 μL of 20 μM WSSV forward primer, 1 μL of 20 μM WSSV reverse primer, and 7 μL of nuclease-free water. Each DNA sample was assayed in duplicate. Four different dilutions of standard were used to generate the standard curve. The reactions were run on a BioRad CFX96 real-time PCR machine (BioRad, Hercules, CA, USA) with cycling conditions including 15 min of preincubation at 95 °C followed by 35 cycles of 15 s at 95 °C and 1 min at 60 °C [16 (link)]. Virus copy number was calculated using CFX Manager software (BioRad) according to a standard curve.