2.1 Preparation of virus and tested samples
Samples containing Feline calicivirus (FCV), feline parvovirus (FPV), feline herpesvirus (FHV-1), canine parvovirus (CPV), and canine coronavirus (CCoV) were collected from pet hospitals in China and identified in our laboratory. A total of 100 clinical samples were obtained from pet hospitals in China between 2019 and 2020. Eight 3-month-old experimental cats weighing 1.5 kg were purchased from a market in Beijing. They were tested for FCV antigen and all antibody tests were negative. 0.5ml FCV was used to instill the eyes on the nose. 1-17 days after challenge, oral, nasal and eye swabs and anal swab were collected. FCV. All samples were kept at -80°C in our laboratory.
2.2 Design of Primers and TaqMan probes
Full-length sequences of FCV (n=41) were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/) and aligned using MEGA7.0. The most conservative regions were identified manually for the design of primers and probes. DNAMAN was used to evaluate the physical properties of the primers and TaqMan probes. FCV-BZP-F and FCV-BZP-R primers were expected to amplify a 624bp fragment. The Primer Express DNAMAN software was then used to prepare the FCV-F and FCV-R qRT-PCR primers and the FCV TaqMan-FAM probe (Table 1), which were predicted to generate a 129 bp fragment. The qRT-PCR probe, labeled at the 5′ end with FAM and with a dark quencher dye at its 3′ end, was designed to anneal an internal sequence of the amplified region.
2.3 RNA extraction and reverse transcription
1g stool samples were diluted with 1 mL of PBS (Gibco，CA). After three freeze-thaw cycles, the samples were then spun for 5 min at 3,000 g at 4 ◦C, with supernatants being collected. and stored at -80°C for later use. The viral RNA was extracted using the Qiagen Viral RNA kit (Qiagen, Hilden, Germany), in accordance with to the kit manufacturer’s instructions, after which the Reverse Transcription Kit (TIANGEN Beijing, China) was used to prepare cDNA based on provided guidelines. Extracted cDNA was stored at -20 ◦C. Every 20 μL reverse transcription reaction contained 4 μL 5×FastKing-RT SuperMix, 2 μL Total RNA, and 14 μL RNase-Free water. The PCR protocol for the reverse transcription was 15 min at 42°C, then 3 min at 95°C. The synthesized cDNA was stored at -20°C for later use.
2.4 Preparation of standard plasmids
The 624 bp target gene fragments were amplified via PCR in a reaction mixture containing 1 μL each of the forward and reverse primers (FCV-F and FCV-R, 10 μmol/L), 2 μL FCV cDNA template, 21 μL RNase-free water, and 25 μL Prime STAR Max Premix (TaKaRa Biotechnology，Dalian, China). The thermocycler settings were as follows: 98°C for 10 s, 55°C for 30 s, 30 cycles of 72°C for 10 s, and finally 72°C for 5 min. The amplified PCR products were incorporated into a pEASY-Blunt cloning vector (TransGen Bitech, Beijing, China), and then transformed into Trans1-T1 competent cells (TransGen Bitech, Beijing, China) according to the manufacturer’s guidelines. The bacteria containing positive recombinant plasmids were screened by PCR and sent to a sequencing facility for identification. Plasmids with correct sequences containing the FCV target fragment genes were the standard products. The plasmids were then purified using a commercial column and the plasmid concentration was quantified with a NanoDrop 1000 (ThermoFisher Scientific, USA). The number of plasmid copies in the sample was estimated based on the molecular weight of the plasmid standard and the plasmid concentration. (Calculation formula: copy number=6.02*1023 (copies/mol) × [standard concentration (ng/µL) × 10-9]/standard length (bp) × 660 (Dalton/bp)). Ten-fold serial dilutions of plasmids were prepared in RNase-free water(Solarbio, Beijing，China), and aliquots of each dilution were frozen at -80°C. Each aliquot was used only once for qRT-PCR. Dilutions of the standard plasmids were tested by qRT-PCR, and a standard curve was generated using Microsoft Excel(USA).
2.5 Optimization of real-time fluorescence quantitative PCR reaction conditions
Standard plasmids were amplified via qRT-PCR in an ABI QuantStuio 7 Flex Sequence Detection System (ThermoFisher Scientific, USA) using the Hieff Unicon® qPCR TaqMan Probe Master Mix (Hieff Unicon, China). Each 20 μL reaction mix contained 10 μL 2× Hieff Unicon® TaqMan multiplex qPCR master mix, 0.5 μL ROX reference dye as a passive reference, forward and reverse primers (FCV-F and FCV-R, 0.2-1.0 mol/L), probes (0.4-0.6 mol/L), standard plasmids (1-100 ng), and RNase-free water up to 20 μL. The thermocycler settings were as follows: 95°C for 1 min and 40 s, 95°C for 10 s, 30 cycles of 50°C for 35 s. The data were collected at the annealing step of each cycle and the threshold cycle (Ct) for each sample was calculated by determining the point at which the fluorescence exceeded the threshold limit.
2.6 qRT-PCR standard curve preparation
The standard plasmid was serially diluted 10-fold from 9.6×109 copies/µL to 9.6×100 copies/µL. The final standard curve was generated based on the Ct value and the logarithm of the standard copy number.
2.7 Specificity test
The specificity of the assay was evaluated using the above conditions with a range of different viruses (FCV, FPV, FHV-1, CPV, CCoV), together with the negative controls.
2.8 Sensitivity and reproducibility analyses
To determine the sensitivity of the assay, standard plasmids were serially diluted 10-fold with concentrations ranging from 9.6×109 to 9.6×100 copies/µL. The prepared standard plasmids were amplified with a qRT-PCR system to confirm the detection limit. The sensitivity of this qRT-PCR assay was assessed using a cRT-PCR assay. Intra-assay reliability was evaluated by simultaneously assessing three replicates of each of the three dilution samples. Inter-assay reliability was assessed by repeating three independent experiments. The inter- assay and intra-assay coefficients of variation (CVs) were determined by dividing the standard deviation of each tested sample by its mean prior to multiplying the result by 100.
2.9 Clinical sample analyses
In total, 100 clinical samples were collected from pet hospitals all over China and stored at -80°C in our laboratory. RT-qPCR and RT-PCR assay were performed to compare the detection rates of FCV of the two methods.
2.10 Detoxification test
The viral loads of the oral, nasal swabs and the anal swabs collected before and 17 days after the FCV infection were tested, and the law of FCV external detoxification was studied.